Apparently complex yet relatively simple procedure in orthodontics is palatal expansion. Its versatility is unique for despite the many controversies surrounding it, desirable results are achieved when used in the appropriate situation by a skilled clinician.
Expansion of the palate was first achieved by Emerson C. Angell in 1860. Palatal expansion can be carried out in different ways which are broadly classified as rapid & slow.
RAPID MAXILLARY EXPANSION (R.M.E.)
Rapid maxillary expansion is also known by the terms rapid palatal expansion or split palate. It is a skeletal type of expansion that involves the separation of the mid - palatal suture and movement of the maxillary shelves away from each other.
Emerson C. Angell is considered the father of rapid maxillary expansion. Angell, for the first time in 1860, used a jack screw type of device between the maxillary premolars in a 14 year old girl and achieved an increase in arch width by 1/4 inch in 14 days (fig 1).
Walter Coffin in 1877 introduced a spring called Coffin spring for the purpose of expanding the arch. These efforts however were not accepted by the orthodontic community at that time.
It was the oral surgeons and E.N.T. surgeons who popularized this technique during the early part of this century. E.N.T. surgeons used this technique in treatment of nasal insufficiency and constricted naso-maxillary complex with great success.
Korhkaus and Andrew Hass during the 1950's reintroduced rapid maxillary expansion to the orthodontic community. They popularized the concept with excellent research publications on animals and humans using a variety of techniques and methods.
APPLIED ANATOMY
The maxilla together with the palatine bone forms the hard palate, floor and greater part of the lateral walls of the nasal cavity. The maxilla is a paired bone that articulates with its opposite member and various other bones including frontal, ethmoid, nasal, lacrimal, vomer, zygomatic and the palatine bones. Most of the sutural attachments of the maxilla to the adjoining bones are at its posterior and superior aspects leaving the anterior and inferior aspects free, which makes it vulnerable for lateral displacement.
The inter-maxillary and the inter-palatine sutures are collectively called the mid-palatal suture. Rapid maxillary expansion should be initiated prior to the ossification of the mid - palatal suture. Various studies have been done to ascertain the age at which the mid - palatal suture ossifies. Melsen reports that the transverse growth of the mid - palatal suture continued up to 16 years in girls and 18 years in boys. Most studies report a broad range of association timetable i.e. between 15 - 27 years. The clinician should hence ascertain that. The suture is not ossified by using appropriate diagnostic aids to be described later in this chApter.
The sphenoid and the zygomatic bones have a buttressing effect resisting mid - palatal suture opening.
INDICATION FOR R.M.E.
Rapid maxillary expansion has been carried out for dental as well as medical purposes. The following are some of the indications for rapid maxillary expansion:
(1) Posterior crossbite (fig 2)
associated with real or relative maxillary deficiencies. A real maxillary deficiency is associated with an undersized / narrow maxilla. Relative maxillary deficiency is characterized by normal maxilla but oversized mandible.
(2) Class III malocclusion of dental or skeletal cause. Improvement is seen in both anterior as well as posterior crossbites.
(3) Cleft palate patients with collapsed maxillary
arch.
(4) In cases requiring face mask therapy, R.M.E. is used along with face mask to loosen the maxillary sutural attachments so as to facilitate protraction.
(5) The medical indications for rapid maxillary expansion include nasal stenosis, poor nasal airway, septal deformities, recurrent ear and nasal infection, allergic rhinitis, D.N.S., e.t.c.,.
DIAGNOSTIC AIDS
The routine diagnostic aids such as case history, clinical examination and study models are useful in diagnosis. The mid - palatal suture can be visualized in a maxillary occlusal view radiograph. These radiographs are also useful during treatment to check for mid - palatal split and also to estimate the amount of maxillary expansion achieved. P.A. cephalogram is another valuable diagnostic aid in rapid maxillary expansion procedures to estimate the amount of expansion that has taken place.
THE EFFECTS OF R.M.E.
Though R.M.E. is essentially a dento-facial orthopaedic appliance used by orthodontists, it finds application in other fields such as oral surgery, E.N.T. and plastic surgery.
Maxillary skeletal effect: The maxillary posterior teeth are used as handles to apply a transverse reciprocal force so as to open the mid - palatal suture. Since the force employed for the procedure is very high, not much of orthodontic changes can be observed. The appliance on activation compresses the periodontal ligament and bends the alveolar process bucally and slowly opens the mid - palatal suture. The opening of the mid - palatal suture is fan-shaped or tzriangular with maximum opening at the incisor region and gradually diminishing towards the posterior part of palate (fig 3 a).
This can be appreciated in a post R.M.E. occlusal radiograph. Similar fan shaped or non-parallel opening is also seen in the superio-inferior direction. The maximum opening is towards the oral cavity with progressively less opening towards the nasal aspect (fig 3 b).
According to Krebs, the two halves of the maxilla rotate in the sagittal and coronal planes. In the coronal plane the two halves of the maxilla rotate away from each other. The point at which the rotation takes place is around the frontomaxillary suture. In the sagittal plane, the maxilla is found to rotate in a downward and forward direction.
Amount of expansion achieved: An increase in maxillary width of upto 1 Omm can be achieved by rapid maxillary expansion. The rate of expansion is about 0.2 to 0.5mm per day.
Effect on alveolar bone: The alveolar bone in the area adjacent to the anchor teeth bends slightly. This is due to the resilient nature of the alveolar bone.
Effect on maxillary anterior teeth: The appearance of a midline spacing between the two maxillary central incisors is the most reliable clinical evidence of the maxillary separation. The incisor separation is about half of the distance the screw is opened. By three to five months, the midline diastema closes as a result of the transseptal fiber traction.
Effect on maxillary posterior teeth: The maxillary posterior teeth are used as anchors during rapid maxillary expansion. These teeth show buccal tipping (fig 4)
and are also believed to extrude to a limited extent.
Effect on mandible: Most authors have observed a downward and backward rotation of the mandible following rapid expansion. This is accompanied by a slight increase in the mandibular plane angle. The reason attributed for the mandibular rotation is the extrusion and buccal tipping of the maxillary molars.
EFFECT ON ADJACENT CRANIAL BONES AND SUTURES:
Rapid maxillary expansion not only results in opening of the mid - palatal suture but also has far reaching effects on adjacent cranial
Fig 4 (A) Normal axial inclination of the anchor molars (B) Buccally tipped anchor molars
Structures. In addition to the effects on those bones directly articulating with the maxilla, bones of the cranium such as parietal and occipital were also found to be displaced.
Effects of R.M.E. on nasal cavity: Following rapid maxillary expansion an increase in intranasal space occurs due to the outer walls of nasal cavity moving apart. This increase in nasal cavity width is maximum in the inferior region of the nasal cavity and gradually decreases towards the superior aspect. Similar gradient is also found in an anterio-posterior direction with the greatest increase being in the anterior region.
Air flow resistance is believed to reduce by 45 - 60 %, thereby improving nasal breathing.
Numerous appliances have been used for rapid maxillary expansion. Broadly they can be classified as:
1. Removable appliances
2. Fixed Appliances
a. Tooth borne
b. Tooth and tissue borne
REMOVABLE APPLIANCES
The reliability of these appliances in producing skeletal expansion is highly questionable. Although it is possible to split the sutures using removable plates, it nevertheless is unpredictable. Treatment during the deciduous or early mixed dentition is considered more favorable in producing appreciable skeletal effects.
A removable type of rapid maxillary expansion device consists of a split acrylic plate with a midline screw. The appliance is retained using clasps on the posterior teeth. The disadvantages of a removable rapid expansion appliance is the need for patient co-operation
and the difficulty in retaining the plate inside the mouth.
FIXED APPLIANCES
Appliances that are fixed onto the teeth are more reliable and found to produce consistent skeletal effects. These fixed rapid expanders can be classified into tooth and tissue borne appliances and tooth borne appliances. Two of the commonly used tooth and tissue borne appliances are :
1. Derichsweiler type
2. Hass type
I Examples of tooth borne appliances
Include:
1. Isaacson type 2. Hyrax type
Derlchsweller type
The first premolars and the first molars are banded. Wire tags are soldered onto the palatal aspect of the bands. These wire tags get inserted into a split palatal acrylic plate incorporating a screw at its center (fig 6.a).
HASS TYPE
The first premolar and molar of either side are banded. A thick stainless steel wire of 1.2 mm diameter is soldered on the buccal and lingual aspects connecting the premolar and molar bands. The lingual wire is kept longer so as to extend past the bands both anteriorly and posteriorly. These extensions are bent palatally to get embedded in the palatal acrylic. The split palatal acrylic has a midline screw. The plate does not extend over the rugae area (fig 6.b).
ISAACSON TYPE
This is a tooth borne appliance without any acrylic palatal covering. This design makes use of a spring loaded screw called a MINNE expander (developed at the University of Minnesota, Dental School) .
The first premolars and molars are banded. Metal flanges are soldered onto the bands on the buccal and lingual sides. The expander consists of a coil spring having a nut which can compress the spring. This coil spring is made to extend between the lingual metal flanges that have been soldered. The expander is activated by closing the nut so that the spring gets compressed (fig 6.c).
HYRAX TYPE
This type of appliance makes use of a special type of screw called HYRAX (Hygienic Rapid Expander). The screws have heavy gauge wire ex. tensions that are adapted to follow the palatal contour and are soldered to bands on premolar; and molars (fig 6.d).
BONDED R.M.E
Most of the rapid maxillary expansion appliances described earlier are banded appliances. They incorporate bands on the first premolars and molars. An alternative design of the appliance would be to have a splint covering variable number of teeth on either side to which the jack screw is attached. Splints can be of two types:
1 . Cast Cap Splints 2. Acrylic Splints
The cast cap splints are made of silvercopper alloy. The acrylic splints are made of polymethyl-methacrylate. A wire framework may be adapted around the teeth to reinforce the acrylic. These splints are bonded to teeth using either glass ionomer or other bonding adhesives, after adequate etching.
DESCRIPTION OF A TYPICAL EXPANSION SCREW
A typical expansion screw consists of an oblong body divided into two halves. Each half has a threaded inner side that receives one end of a double ended screw. The screw has a central bossing with four holes. These holes receive a key which is used to turn the screw (fig 8).
The turning of the screw by 90 degree (i.e. one turn) brings about a linear movement of 0.18 mm. The pattern of threading on either side is of opposite direction. Thus turning the screw withdraws it from both sides simultaneously.
ACTIVATION SCHEDULE
Various authors have advocated different activation schedules to achieve the desired results.
Schedule by Timms
For patients of upto 15 years of age, 90° rotation in the morning and evening. In patients over 15 years, Timms recommends 45° activation 4 times a day.
Schedule by Zimring and Isaacson
In young growing patients, they recommend two turns each day for 4 - 5 days and later one turn per day till the desired expansion is achieved. In case of non growing adult patients, they recommend two turns each day for first two days, one turn per day for the next 5- 7 days and one turn every alternate day till desired expansion is achieved.
TREATMENT EVALUATION DURING R.M.E.
Clinically, the most noticeable feature during rapid maxillary expansion is the appearance of 0 midline diastema. Studies by various authors show that the amount of incisor separation is roughly half the amount of jack screw separation. But the amount of diastema should not be taken as 0 reliable factor in estimating the amount of expansion. Maxillary occlusal radiograph and
P.A. cephalogram are more reliable in estimating the amount of maxillary expansion.
CONTRAINDICATIONS OF R.M.E.
Some cases where R.M.E. is contraindicated
are:
1. Single tooth crossbites.
2. In patients who are un-cooperative, R.M.E. is contraindicated as the appliance requires frequent activation and maintenance of good oral hygiene.
3. Rapid maxillary expansion is not carried out after ossification of the mid - palatal suture unless it is accompanied by adjunctive surgical procedures.
4. Skeletal asymmetry of maxilla and mandible and adult cases with severe antero-posterior skeletal discrepancies.
5. Vertical growers with steep mandibular plane angle are usually a contra-indication.
6. As the posterior teeth are used as anchors to move the bones apart, the procedure is not
indicated in a periodontally weak dentition.
RETENTION FOLLOWING R.M.E.
Failure to retain the expansion results in relapse. Most authors recommend a retention period of not less than 3 - 6 months. Isaacson recommends the use of the R.M.E. appliance itself for the purpose of retention. The screw shou Id be immobilized using cold cure acrylic (fig 9).
Alternatively, either a removable or fixed retainer (e.g. TPA) can be used.
SURGERY AS AN ADJUNCT
Patients who exhibit unusual resistance to separation of the palatine bones may require surgical intervention. This usually occurs in female patients over 16 years of age and male patients over 18 years of age in whom the mid-palatal suture has ossified. Surgical separation may also be required in patients exhibiting increased circum-maxillary rigidity as a result of aging.
Maxillary expansion can be brought about by surgery alone or by surgery along with a rapid expansion appliance. The surgical procedures usually carried out are:
a. Palatal osteotomy
b. Lateral maxillary osteotomy
c. Anterior maxillary osteotomy
CLINICAL TIPS FOR R.M.E.
1. Oral hygiene instructions should be given to the patient and reinforced during the procedure.
2. Orthodontic movement of the anchor teeth should be avoided prior to rapid maxillary expansion, as mobile teeth do not offer adequate anchorage for palatal split. Recently moved teeth tend to tip.
3. The patient should be trained to use the key. The key should be tied to a string and the free end should be secured around the patient's wrist to avoid accidental swallowing.
4. Maxillary occlusal radiographs should be taken at regular intervals to monitor the
expansion.
5. The possible immediate effects of premature appliance removal include dizziness, pressure at the bridge of nose, pressure under eyes, blanching of soft tissues under the eyes, etc.,. These symptoms may occur on removal of the appliance for repair or recementation. The patients should therefore be kept seated and asked not to stand immediately after appliance removal.
SLOW EXPANSION
According to the proponents of slow expansion, the results are more stable when the maxillary arch is expanded slowly at a rate of 0.5- 1 mm per week. The forces generated by such procedures is much lower i.e. 2-4 pounds as against 10-20 pounds generated during rapid maxillary expansion. Unlike in rapid maxillary expansion where the treatment is completed in 1 -2 weeks, slow expansion may take a$ much as 2-5 months.
Slow expansion has traditionally been termed dento-alveolar expansion, although some skeletal changes can be observed. The slower expansion techniques have also been associated with a more physiologic adjustment to the maxillary expansion, producing greater stability and less relapse potential than in rapid expansion procedures.
APPLIANCES USED FOR SLOW EXPANSION
Jack screws
The various jack screws incorporated in the appliances described for rapid expansion can be used for slow expansion (fig 1 0),
but with a more spread out activation schedule. The screws used for slow expansion have a smaller pitch than those used in R.M.E.
Coffin spring
This appliance was designed by Walter Coffin around the beginning of this century (fig 11 ).
It is a removable appliance capable of slow dentoalveolar expansion. The appliance consists of an omega shaped wire of 1.25 mm thickness, placed in the mid-palatal region. The free ends of the omega wire are embedded in acrylic covering the slopes of the palate. The spring is activated by pulling the two sides apart manually. It can also be activated by using three prong pliers. Coffin spring is believed to bring about a
Table 1 comparison between slow and rapid expansion.
Feature Slow expansion Rapid expansion
Type of expansion Mostly dental Skeletal
Rate of expansion Slow Rapid
Type of tissue reaction Force used More physiologic More traumatic
Force Used Milder force Forces Greater
Frequency of activation Less Frequent More frequent
Duration of treatment Long Short
Type of appliance Either fixed or removable Mostly fixed appliance
Age Any Age Before fusion of midpalatal suture
Retention Lesser chance of relapse More chance of relapse
dento-alveolar expansion. However use of this appliance in younger patients is believed to bring about some amount of skeletal expansion.
Quad helix
One of the appliances used to expand a narrow maxilla is the quad helix (fig 12).
It is said to have evolved from the original Coffin loop. The quad helix incorporates four helices that increase the wire length. Therefore the flexibility and range olaction of this appliance is more. The appliance is constructed using 0.038 inch wire and is soldered to bands on the first molars.
The quad helix consists of a pair of anterior helices and a pair of posterior helices. The portion of wire between the two anterior helices is called the anterior bridge. The wire between the anterior and posterior helices is called the palatal bridge. The free wire ends adjacent to the posterior helices are called outer arms. They rest against the lingual surface of the buccal teeth and are soldered on to the lingual aspect of the molar bands.
The quad helix can be used to expand a narrow arch as well as to bring about rotation of molars. It can be pre-activated by stretching the two molar bands apart prior to cementation or by using three prong pliers after cementation (fig 13).
The quad helix brings about a slow dento-alveolar expansion. But when it is used in children during the deciduous and early mixed dentition periods, a skeletal mid-palatal splitting can be achieved.
ARCH EXPANSION USING FIXED APPLIANCES
Arch expansion can be achieved in a patient who is undergoing fixed mechanotherapy. Mild expansion can be brought about by using expanded arch wires. In addition appliances such as the quad helix or the transpalatal arch can be used along with fixed mechotherapy.
TREATMENT OF TRANSVERSE MAXILLARY CONSTRICTION:-
Skeletal maxillary constriction is distinguished by a narrow palatal vault. It can be corrected by opening the mid palatal suture, which widens the roof of the mouth and the floor of the nose. This transverse expansion corrects the posterior crossbite, sometimes moves the maxilla forward, increases space in the arch, and repositions underlying permanent tooth buds, it can be done at any time prior to the end of the adolescent growth spurt. The major reasons for doing it sooner are to eliminate functional problems and mandibular shifts on closure, and to provide more space for the Erupting maxillary teeth.
Several methods of arch expansion are possible, but to obtain skeletal effects, it is necessary to place force directly across the suture. In preadolescent children. Three methods can be used for palatal expansion:
1. A split removable arch with a jackscrew or heavy midline spring.
2. A lingual arch often of the w arch or quad helix design.
3. A fixed attached to bands or incorporated into a bonded appliance. Removable palates and lingual arches produce slow expansion. The fixed expander can be activated for either rapid (0.5 mm or more per day)( semi-rapiD (0.25mm/day) or methods appropriate questions. Are does it achieve the expansion ? does it have iatrogenic side effects? And is the expansion stable?
PALATAL EXPANSION IN THE PRIMARY AND EARLY MIXED DENTITION:
Because less force is needed to open the suture in you8nger children, it is relatively easy to obtain palatal expansion. In the early mixed dentition, all three types of expansion appliances produce both skeletal and dental changes.
With a removable appliance, the rate of expansion ust be quite slow, and the force employed during the process must be low, because faster expansion produces higher forces that create problem with retention of the appliance, multiple clasps that are well adjusted are mandatory. Because of the instability of the teeth during the expansion process, failure to wear the appliance even for 1 day requires adjustment of the jackscrew, usually bye the practitioner, to cAn resumed. Compliance in activation and wear time are always issue with these appliances. Successful expansion with a removable appliance can take so much time that it is not cost effective.
Ligual arches of the W-arch and helix designs have been demonstrated to open the midpalatal suture in young patients. These appliances generally deliver a few handred grams of force and provide slow expansion they relatively clean and reasonably effective, producing a mix of skeletal and dental change.
Fixed jackscrew appliances attached to bands or bonded splints also can be used in the early treatment of maxillary second molars is relatively simple, but banding primary first molars can be challenging. Using a bonded appliance in the mixed dentition is relatively straightforward. This appliance can deliver a variety of forces and can extinguish habits by virtue of its bulk. In young children, in comparison with a lingual arch, there are two major disadvantages. First , the fixed jackscrew appliance is more bulky than an expansion lingual arch and more difficult to place and remove. The patient inevitably has problems in cleaning it and either the patient or parent must activate the appliance, second a fixed appliance of this type can be activated rapidly which in young children is a disadvantage, not an advantage, rapid expansion should not be done in a young child. There is a risk of distortion of facial structures with rapid expansion movement and high forces produce better or more stable expansion.
Many functional appliances incorporate some components to expand the maxillary arch either intrinsic force generating mechanism like springs and jackscrews or buccal shields to relieve buccal soft tissue pressure. When arch expansion occurs during functional appliance treatment. It is possible that some opening of the midpalatal suture contributes to it. But the precise mix of skeletal and dental change is not well documented.
On balance, therefore, slow expansion with an active lingual arch is the preferred approach to maxillary constriction in young children in the primary and early mixed dentitions. A fixed jackscrew appliance is an acceptable alternative if activated carefully and slowly.
PALATAL EXPANSION IN THE LATE MIXED DENTITION
With increasing age, the midpalatal suture becomes more and more tightly inter digited, but in most individuals, it remains possible to obtain significant increments in maxillary width up to age 15 to 18.expansion in adolescents is discussed.
Even in the late mixed dentition, satural expansion requires placing a relatively heavy force directed across the suture to move the halves of the maxilla apart. A fixed jackscrew appliance (either banded or bonded 0 is required.
as many teeth as possible should be included in the anchorage unit. In the late mixed dentition. Root resorption of primary molars may have reached the point that these teeth offer little resistance, and it may be wise to wait for eruption of the first premolars before beginning expansion.
Although Some Studies Have reported increases in vertical facial height with maxillary expansion. Long term evidence in dictes this change is transitory. A bonded appliance that covers the occlusal surface of he posterior teeth may be a better choice for a child with a long face tendency by producing less mandibular rotation than a banded appliance but this is not totally clear. Perhaps the best summary is that the older the patient when maxillary expansion is done. The less likely it is that vertical changes will be recovered by subsequent growth.
RAPID OR SLOW EXPANSION:
In the late mixed dentition, either rapid or slow expansion is clinically acceptable. It now appear that slower activation of the expansion appliance (i.e.at the rate of about 1 mm/week) provides approximately the same ultimate result over a 10 to 12 week period as rapid expansion with less trauma to the teeth and bones.
Rapid expansion typically is done with two turns daily of the jackscrew (0.5 mm activation ) this creates 10 to 20 pounds of pressure across the suture enough to create microfractures of interdigitating bone spicules. When a screw is the activating device the force is transmitted immediately to the teeth and then to the suture. Sometimes a large coil spring is incorporated along with the screw which modulates the amount of force, depending on the length and stiffness of the spring.
the suture opens wider and faster anteriorly because closure begins in the posterior area of the midpalatal suture and there is a buttressing effect area of the midpalatal suture and there is a buttressing effect of the other maxillary structures in the posterior regions with rapic or semi rapid expansion a diastema usually appears between the central incisors as the bones separate in this area.
expansion usually is continued until the maxillary lingual cusps occlude with the lingual inclines of the buCCal cusps of the mandibular molars. When expansion has been completed a 3 month period of retention with the appliance in place is recommenced. After the 3 month retention period, the fixed appliance can be removed, but a removable retainer that covers the palate is often needed as further insurance against carly relapse
A relatively heavy expanded maxillary archwire provides retention if further treatment is being accomplished immediately.
The theory behind rapid activation was that force on the teeth would be transmitted to the bone, and the two halves of the maxilla would separate before significant tooth movement could occur. In other words rapid activation was conceived as a way to maximize skeletal hange and minimize dental change. It was not realized initially that during the time it takes for bone to fill in the space that was created between the left and right halves of the maxilla, skeletal relapse begins to occur almost immediately, even though the teeth are held in position. The central diastema closes from a combination of skeletal relapse and tooth movement created by stretched gingival fibers. The net treatment effect therefore is approximately equal skeletal and dental expansion.
Slow activation of the expansion appliance at the rate of 1 mm /week which produces about 2 pounds of pressure in a mixed dentition child, opens the suture at a rate that is close to the maximum speed of bone formation. The suture is not obviously pulled apart on radiographs, and no midline diastema appears but both skeletal and dental changes occur. After 10 to 12 weeks approximately the same roughly equal amounts of skeletal and dental expansion are present that were sEen at the same time with rapid expansion. When bonded slow and rapid palatal expanders in early adolescents were compared, the major difference was greater expansion across the canines in the rapid expansion group. This translated into a predicted greeted arch perimeter change but similar opening of the suture posterioly. So by using slow palatal expansion (one turn) every other day in a typical fixed expansion appliance or by using a spring to produce about 2 pounds of force, effective expansion with minimal disruption of the suture can be achieved for a late mixed dentition child.
CLINICLA MANAGEMENT OF PALATAL EXPANSION DEVICES
Most traditional palate expansion devices use bands for retention on first premolars and permanent first molars if possible. During the late mixed dentition years the first premolars often are not fully erupted and are difficult to band. If the primary second molars are firm they can be banded along with the permanent first molars. Alternatively cotacting the other posterior primary and erupting permanent teeth near their gingival margins.
The bands are stabilized in an impression while it is poured, so they are retained in the completed working model. A soldered wire framework and palatal portions, if desired are added during appliance fabrication.
After crossbite correction is completed, band removal can be difficult because the teeth are mobile and sensitive. In those cases, sectioning the bands is appropriate.
An alternative approach is to use a bonded palatal expander during fabrication of the working casts, plastic is generally extended over the occlusal and facial and lingual surfaces of the posterior teeth. When the appliance is returned from the laboratory, because of poor dimensional stability and distortion of the plastic portion, it may be necessary to relieve the acrylic where it seats on the maxillary teeth, reline this area with additional plastic, and refit the appliance in the mouth. By removing the appliance before final polymerization, it can be trimmed and further adjusted without complication. Generally, a composite resin is used to retain the appliance, with only the facial and lingual surfaces is not recommended bonding there is unnecessary for retention and can greatly complicate appliance removal.
Removal of the appliance is accomplished with a band remover engaged under a facial or lingual plastic margin and is facilitated by including loops of wire extending from the facial surfaces the appliance can be sectioned but this is time consuming and usually unnecessary. Complete resin removal can be sectioned but this is time consuming and usually unnecessary. Complete resin removal can be laborious, so using only an adequate amount of resin is crucial. There is a delicate balance. Inadequate resin will lead to excessive leakage onto the no bonded surfaces, which can result in decalcification, or appliance loss. Too much resin, on the other hand, can make tooth and appliance cleaning, as well as appliance removal, difficult. For these reasons, some clinicians use glass ionomer cement for retention. The strength of the material usually is adequate but bonding failure may occur. Fluoride release from these cements may prove advantageous in the short term.
ARCH EXPANSION COMPONENTS:-
Plastic buccal shields and lip pads, both of which are incorporated into the frankel appliance hold the soft tissues away from the teeth.
The effect is to disrupt the tongue ckeck equilibrium, and this in turn leads to disrupt the tongue ckeck equilibrium, and this is in rurn lead to facial movement of the teeth and arch expansion. A buccal shield is more effective in producing buccal expansion than wires to hold the cheeks away from the teeth, lip pads positioned low in the vestibule force the lip musculature to stretch during function.
A combination of lip pads and buccal shields will result in an increase in ardh circumference as well buccal shields and lip pads are an integral part of the frankel appliance, but can be added to any appliance. They add to the potential for soft tissue irritation that can inhibit patient compliance, and must be monitored to prevent this.
Expansion screws and springs can be used to actively increase the transverse dimension of the arches or to modify the anteroposterior dimension of the appliance.
They generate toothmoving forces within the applicance, beyond those generated by the patients soft tissues and function, which almost always is undesirable when the goal is growth modification.
APPLIANCES FOR BILATRAL MAXILLARY CONSTRICTION:
The preferred appliance for modest expansion of the maxillary arch to correct a posterior crossbite in a preadolescent child is an adjustable lingual arch that requires little patient cooperation. Both the W-arch and the quad helix are reliable and easy to use. The w-arch is a fixed appliance constructed of 36 mil steel wire soldered to molar bands. It is activated simply by opening the apices of the W and is easily adjusted to provide more anterior than posterior expansion, or vice versa, if this is desired. The appliance delivers proper force levels when opened 4-5 mm wider than the passive width and should be adjusted to this dimension before being inserted. It is not uncommon for the teeth an maxilla tomove more on one side than the other, so precise bilateral expansion is the exception rather than the rule, but acceptalble correction and tooth position are almost always achieved.
The quad helix is a more flexible version of the W-arch. The helices in the anterior palate are bulky, which can effectively serve as a reminder to aid in stopping a finger habit. The combination of a posterior crossbite and a finger sucking habit is the best indication for this appliance. The extra wire incorporated in this appliance gives it forces are equivalent. Attention to soft tissue irritation is also recommended with this appliance. Both the W-arch and the quad helix leave an imprint on the tongue, about which the parents and child should be warned. This will disappear when the appliance is removed.
With both types of expansion lingual arches, some opening of the midpalatal suture can be expected in a young child so the expansion is not solely dental. This is of no consequence and will require no difference in either treatment or retention. Expansion should continue at the rate of no consequence and will require no difference in either treatment or retention. Expansion should continue at eh rate of 2 mm per month (1 mm tooth movement on each side) until the corssbite is slightly overcorrected. In other words, the lingual cusps of the maxillary teeth should occlude on the lingual inclines of the buccal cusps of the mandibular molars at the adjustment is possible but may lead to unexpected changes. For this reason removal and recementation are recommended at each active treatment visit. Most posterior cross bites require 2 to 3 months of active treatment and 3 months of retention (during which the lingual arch is left passively in place)
Some children do have true unilateral crossbites due to unilateral maxillary constriction of the upper arch.
in these cases the ideal treatment is to move selected teeth on the constricted side. To a limited extent, this goal can be achieved by using different length arms on a W-arch or quad helix.
but some bilateral expansion must be expected, an alternative is to use a mandibular lingual arch to stabilize the lower teeth and attach cross elastics to the maxillary teeth that are at fault. This is more complicated and requires cooperation to be successful, but is more unilateral in its effect. A third alternative is to use a removable appliance similar to the one but secioned asymmettically. This has the effect of pitting more teeth against fewer teeth and results in asymmetric movement. Of course, this appliance has the same restrictions as all removable appliances; its success depends on both the quality of its retentive clasps and the patients cooperation.
Monday, April 5, 2010
project for mca
ABSTRACT
Seed leaves and flower tissue were extracted in P.V.P. the extract were screen qualitatively dot-blot agar plate technique for amylase inhibitor were further analyzed for quantitative estimation of Amylase inhibitor activity by DNSA method. Endogenous amylase and amylase activator also present within the tissue. these are detected by developing specific protocol for each.
The inhibitors were separated in polyacrylamide gel containing 0.5% soluble starch by electrophoresis and visualized by incubation of the gel in salivary amylase solution and staining with iodine. Starch in the gel is hydrolise by amylase during incubation but starch in vicinity of amylase inhibitor is protected from hydrolysis and appear as the blue band after staining.
Keywords: - Amylase inhibitors, Enzyme, Endogenous amylase, Amylase activator, Electrophoresis,
INTRODUCTION
α-Amylase inhibitors:-
Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. These inhibitors can be grouped into six classes based on their tertiary structures. Lectin-like, knottin-like, cereal-type, kunitz-like, γ-purothionin like and thaumatin like inhibitors, some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites.
In animals, α-amylase inhibitors reduce the glucose peaks that can occur after a meal, slowing the speed with which α-amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extra cellular glucose from being cleared quickly from the blood. Therefore, diabetics tend to have low α-amylase levels in order to keep glucose levels under control, except after taking insulin, which causes a rise in pancreatic α-amylase.
Plants also use α-amylase inhibitors as a defense strategy. These inhibitors impede the digestive action of α-amylases and protinases in the insect gut, thereby acting as insect anti-feedants. As a result, α-amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes.( Marshal JJ, Lauda)
What do they do?
Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other, secondary, enzymes. They are claimed to be useful for weight loss. But when they were first developed years ago, research did not find them very effective for limiting carbohydrate absorption. Later, however, highly concentrated versions of amylase inhibitors did show potential for reducing carbohydrate absorption in humans.
Purified starch blocker extracts. When given with a starchy meal, have also been shown to reduce the subsequent rise in blood sugar levels of both healthy people and diabetics. The is effect could be helpful in the treatment of blood sugar disorders.( Choudhary A, Maeda)
Where are they found?
Amylase inhibitors can be extracted from several types of plants, especially those in the legume family. Currently available amylase inhibitors are extracted from either white kidney bean or wheat.
The relative inefficacy of alpha-amylase inhibitors in affecting human digestion of starch has been highlighted by reacent scientific and public controversy over the commercial sales of so-called starch-blockers or slimming pillis alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes obesity and hyperlipaemia studies of the structures of the numerous enzyme inhibitors found in cereal grains have led to the recognition of a super family of homologous proteins which includes inhibitors of alha-amylase, proteinase and bifunctional inhibitors of alpha-amylase, proteinase and bifunctional inhibitors active against two or more classes of enzymes.(Alam and Gourinath, 2001, Octavio and Rigden 2002 Richardson, 1991)
The first alpha-amylase inhibitor determined was that of the monomeric 13 kD known as 0.31 form, from wheat (kashlan and Richardson, 1981) other dimeric 0.19, 0.23, 0.28, 0.53 form of weat inhibitors of exogenous alpha-amylase were later shown (oneda et al 2004, kondo and ida1995, Roy and Gupta 2000, Richardson, 1991, Octavio and Rigden 2002)
The favored hyupotheses about physiological roles fo the enzyme inhibitors in seeds is that they act as storage or reserve proteins as regulators of endogenous enzyme or as defensive agents against the attacks of animal predators and insect or microbial pests. It seems likely that in certain species these proteins may fulfill a combination of these proteins may fulfill a combination of these function(Octavia and Rigden 2002, Octivio and Rigden 2000, Richardson 1991) Also plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plant against pests (Octavio and Rigden2002)
Nutritional and metabolic effects of enzyme inhibitors certainly some of inhibitors certainly some of inhibitors found in cereal and legume seeds can inactive the salivary and pancereatic enzymes of human (pick and wober) and their sucseotibility to be rather variable (singhand bbkundel, 2001) many are destroyed by cooking but some retain inhibitory activity even after baking (Richardson 1991)
Amylase inhibitors present in seeds currently used as food present few nutritional problem for healthy people but may have some toxicological significance in the diets if infants who have a lower production of pancreatic alpha-amylase than adults and for patients with impaired peptic or gastric function (brietender and rauduer ,2004. richrdson ,1991 shewry etal2001) also one inhibitor of insect alpha-amylase isolated from barley flour is the major allergen associated with bakers asthma disease (barber et al 1989) the inhibition is strictly competitive and in the1:1 complexes aloof the activities of the enzyme are completely abolished .
Crystallographic nuclear, magnetic resonance (NMR) and mechanistic studies all indicates that the inhibitors act as highly specific substrate for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond .
The reaction mechanics involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood (silan 1986) but there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the mechanism or that the inhibitor may induce confermational changes in the enzyme molecule.
Materials:-
Different part of the plants including leaves, flower, seeds were collected from local region. Some sample were collected from Nanded ,Rahuri,Pathardi total 146 sample were collected.
Start was from qualgen fine chemical company Bombay India
Polyvinylpolypurrolidone (P.V.P) was from Sigma
Human saliva was diluted and used as salivary amylase all other chemicals were of the highest purity available
LEAVES:
Sr. no Common name Botonical name
1 Sitaphal Annona sequmusa
2 Subhabhul Leueaene lapisiliqua
3 Errand Risinus comnuis
4 Naginiche pan Piper bettne
5 Bhabhul
6 Ghaneri Lantana camera
7 Gawti chaha Cymbopogan flexuosus
8 Aalu Calocasia esculanta
9 Rui Calotropis procera
10 Kadhipatta Murraya kotnigii
11 Gahu Triticum aestivum
12 Papai Papaya indica
13 Amba Mangifera indica
14 Mohair Brassia juncea
15 Naral Coccus nucifera
16 Jambul Syzium cumini
17 Aalu Leaves Calocasia esculanta
18 Harbara Cicer aeriantum
19 Dhol Amba
20 Nimbu Citrus cemani
21 Palak Spinach
22 Kadulimb Azadirecta indica
23 Tobacco Nicotiane tobacum
24 Tulas Ocimum Americanum
25 Tarwad Cassia auriculata
26 Kardali Canna indica
27 Wel
28 Nishigandha Polianthus tuberosa
29 Manjiri Ocimum Americanum
30 Tomato Lycopersicon escunentum
31 Beshram Ipomea
32 Sunflower Helianthus annus
33 Chandan Santalum album
34 Maka Zea mays
35 Halad Curcuma aromatica
36 Sadaba Runca chalepensis
37 Ratali Ipomeabatatas
38 Bartodi Morinda, citrifolia
39 Apte Bahunia
40 Palas Bulea monosperma
41 Biba Semecarpus anacardium
42 Rohini
43 Acasia Chandra
44 Agav Americana
45 Glyrisidium capium
46 Ipomea
47 Carissa carandus
48 Sag Tectona granvis
49 Maniplant
50 Cactus
51 Tur Cajanus cajan
52 Pimple
FLOWER:
1 Dhotra Datura alba
2 Jaswand Hibicus rosasinensis
3
4 Tarwad Cassia auriculata
5
6
7 Kaneri Nerium indicum
8 Zendu Tagehun erecta
9 Rose Red
10
11 Rui Calatropis procera
12 Chafa Michella champaka
13
14
15 Jai Jasminium auricylamn
16 Allamnda
17 Shewga Moringa olifera
18 Gandhar
19 Boganwel Boganvillia spectibilis
20 Kunda
21 Nishigandh Polyanthus tuberose
22
23 Gulbakshi Mirabilis jalapa
24 kardali Canna indica
25
26 Ghosali Lutta aegeyptia
27 Danger
28 Gerbera
29 Aboli Crossendra undilifolia
30 Sadafully Catharathus roses
31 Biliat
32 Tur Cajanus cajan
33 Shevari Sesbania sesban
34 Ghevda Psophocarpus tetiagomolobes
35
36
37 Palas Butea monosporne
38 Sunflower Tridax procambers
39
40 Khajawe
41 Sweet potato Ipomebatata
42 Kandhar
43 Lakh Acacia
44 Sag Tectona Gran
45 Chirisidium caprsium
SEED:
1 Mug Phaseomus aureus
2 Mahsoor Lens esculenta
3 Macca Zea maize
4 Chawali Vigna sinensis
5 Udit Phaseolus mango
6 Tur Cajanus cajan
7 watana Pisum Sativum
8 Hulga
9 Jawari Sorghum valgare
10 Tag
11 Methi Trigonella toenum
12 Ubal bee
13 Math
14 Soyabeen Glycine max
15 Karale
16 Kabuli Harbala Cicer grientum
17 Chinchuka Tamarindus indica
18 Airnda Ricimus communis
19 Tandul Orizha Sathiva
20 Bhagar
21 Dudhi Bhopla
22 Kharbuz Cacumis melo
23 Gokarn Clitonia kleio
24 Rale
25 Wange Solalum righhi
26 Khas khas
27 Shengadane Arachis hypogea
28 Coffee Caffia Arabica
29 Danger
30 Mohari Brassica kmphesis
31 Rajgira Amaranthus
32 Mire Piper nigrum
33 Rajma
34 Elayachi Elettoria cordomomum
35 Tulas Ocium santum
36 Hawari
37 Jira Caminum cyminum
38 Dhane Corriander sativa
39 Mirchi Capsium annum
40 Apte Bahunia racemose
41 Badishop Foenicumum valgari
42 Jawas Alhagi pseudalhagi
43 Bajari Pennisenem typhoids
44 Ghosale Laffer cylindrical
45 Sabja Ocimum bascilium
46 God babhul Accacia
47 Gahu Triticum aestivam
METHOD
Extraction of amylase inhibitors:-
extraction if amylase inhibitors leaves, flower, seed are done by same procedure.
1. Decorticated seeds of all sample were dried and ground in blender to obtain fine flour.
2. Leaves are crused making the fine as possible as in small amount of acetone.
3. Same procedure was used for the flower sample.
4. After removing pigments all the sample are kept in hexane for overnight to remove the fatty material.
5. This procedure was repeated 2 to 3 times.
6. Then samples are carefully dried and packed.
7. Proper method for numbering and identification was used
8. The defatted seed powder was stirred with 1% PVP in 1:5 ratio for 48 hour.
9. Inclusion of PVP helpful in removal of phenolics from extract.
10. The suspension was centrifuged on cooling centrifuged at 10000 rpm for 20 min at 4oC
11. addition of PVP and centrifugation was repeated twice for same sample.
12. The clear supernatant contained amylase inhibitors was stored in freeze and used for analysis.
(Here after mentioned as partially purified extract.)
DOT BLOT INHIBITOR ASSAY:-
1. Extracted sample was tested on agar-starch plat in different concentration for screnning.
2. Using the enzyme (Amylase) Buffer (6.8PH) inhibitor at different proportion dot-blot assay was done.
3. For that 2% agar and 1% starch was used.
4. These are screening method due to this reaction sample which show inhibition are repeated once again and confirm that they show inhibition for amylase.
5. Among the 146 sample 58 sample shown inhibitions.
6. In that these are further categorized in strong inhibitor, medium inhibitor, weak inhibitor.
7. In 146 samples 27 samples are strongly inhibitor, 22 samples are inhibitor, and 9 samples are weak inhibitor.
8. After the screening of 146 samples the extracts which show strong inhibition and medium inhibition was taken for their amylase inhibitor assay by DNSA.
AMYLASE INHIBITOR ASSY :
Amylase activity was assayed by measuring liberated maltose. amylase inhibitory ativity was assyed by measuring reduction in maltose liberated by salivary amylase using dinitrosalisylic acid reagent.
One amylase activity unit is defined as activity resulting in to liberation of 1 mg of maltose from starch at ph 6.9 at 37o C in 3 min.
one amylase inhibitor unit is one amylase unit inhibited under the given assay condition.
In this assay same extract showing the exact inhibition but here again same extract does not show inhibition.
The samples which show inhibition are again tested for the endogenous amylase and amylase activator.
ENDOGENOUS AMYLASE:-
1. Presence of amylases in biological sample particularly from plant case a major problem in detection of amylase inhibitor. The detection of endogenous amylase is possible with DNSA method.
2. In that tow test are taken in one reaction was arrested by addition of DNSA and in anther the reaction are carried out complete and then DNSA was added.
AMYLASE ACTIVATOR:-
Amylase activator also present within the extract there can be detected by DNSA.
In one test tube enzyme, buffer and substrate are added and in another activator was added with enzyme, buffer substrate. Incubate the both test tube and add DNSA activeter was add in first test tube after incubation the difference in OD indicated the amylase activator.
ELECTROPHORETIC SEPRATION OF AMYLASE INHIBITOR :-
Amylase inhibitors in the partially purified extract were analyzed on a vertical slab gel electrophoresis system in 7% polyacrylamide gels containing 0.5% soluble starch without stacking gels containing 0.5% soluble starch without stacking gel. For that tris-glycine (PH 8.9) both in gel and electrode tank was used.
VISUALIZATION OF AMYLASE INHIBITORS:-
After electrophoresis gels are placed in 20mm phosphate buffer (PH 6.9) containing 6.7mm NaCl for 5-10 mm for equilibration and incubated in salivary amylase in phosphate buffer (PH 6.9) for 30 min at 37 C. after incubation the gels are wash with D/W and placed in iodine solution. (10 MM iodine in 14mm KI) for 4 to 5 min.
Exact band of amylase inhibiter way not found.
RESULT AND DISCUSSION:-
Amylase inhibitors are detected by using three different concentrations the strong inhibitor show the inhibitor at all three concentration. Where the weak inhibitor show inhibition only at low concentration of amylase by using this technique determination of amylase activator is also possible. Same extract were help to the activation. amylase hydrolyzes the starch present in the agar. After staining with iodine. It show white area indicate that it is not inhibitor. When these area are broad than the control, then it is activator
DOT-BLOT ASSAY :-
TEST ENZYME BUFFER INHIBITOR
CONTROL 40 40 -
STRONG 40 28 12
MEDIUM 28 24 28
WEAK 12 28 40
SAMPLE NO TYPE OF INHIBITION
Seed- 1,4,7,8,11,9,23,24,26,27,42,43,15,19 * * *
25,30,31,28,43,3,5, * *
6,12,13,15,16,19,20 *
Flower-
1,2,8,10,20,23,27,30 * * *
5,6,7,11,43 * *
9 *
Leaves-
25,29,32,35,49, * * *
4,18,31,33,36,41,46,47,48,49 * *
26 *
*** = Strong Inhibitor
** = Medium Inhibitor
* = Weak Inhibitor
The extracts which show inhibitions for amylase are tested by DNSA assay for each sample two test are done. In first test extract were added after incubation and another, extract added before incubation. amylase inhibitory activity was assayed by measuring reeducation in maltose liberated by salivary amylase using DNSA reagent
AMYLASE INHIBITOR (DNSA)
TEST ENZ EXTRACT BUFF SUB EXTRACT DNSA
(ML) DILU
(ML) O.D
Control - - 1.0 0.5 Incubation for twenty min - 1 Bwb for 10 min 2 -
Test-1 0.4 - 0.5 0.5 0.1 1 2 -
Test-2 0.4 0.1 0.5 0.5 - 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
1 S2 0.53 0.62 L31 0.77 0.87
2 S3 0.49 0.39 L32 0.79 0.85
3 S5 0.44 0.50 L33 0.78 0.78
4 S7 0.84 0.81 L35 0.80 0.77
5 S8 0.97 1.02 L36 0.76 0.83
6 S9 0.85 0.85 L41 0.89 0.90
7 S10 0.83 0.81 L46 0.93 0.86
8 S12 1.15 1.19 L47 0.97 1.01
9 S13 0.86 0.84 L49 0.88 0.85
10 S16 0.84 0.84 F1 1.38 1.42
11 S19 0.85 0.85 F2 1.40 1.30
12 S24 0.62 0.55 F6 1.38 1.35
13 S26 0.75 0.58 F8 1.41 1.40
14 S27 0.56 0.43 F9 1.31 1.38
15 S28 0.96 0.78 F10 1.40 1.38
16 S30 1.01 0.97 F23 1.11 0.98
17 S31 0.65 0.59 F26 1.32 1.38
18 S4 0.76 0.73 F29 1.07 1.14
19 S42 0.63 0.53 F30 1.19 0.93
20 S43 0.58 0.67 F39 1.28 1.36
21 L4 0.80 0.74 F42 0.98 1.03
22 L18 0.78 0.79 F43 1.17 0.97
23 L26 0.77 0.74
24 L29 0.78 0.75
Amylase Inhibitor:-
S3, S24, S26, S27, S28, S31, S4, S42, L4, L26, L46, L49, F2, F23, F30, F43.
Presence of amylases in biological sample particularly from plant is a major problem in detection of amylase inhibitors. The detection of amylase inhibitors in sample containing endogenous amylases is possible. Endogenous amylase in sample can be detected. In one test tube DNSA are added before incubation and reaction are stopped. In another reaction are carried out. Difference between the O.D. indicate the concentration of endogenous amylase.
ENDOGENOUS AMYLASE :
TEST EXTRACT
(ML) BUFF.
(ML) DNSA
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 0.5 1 2 -
T1 0.1 0.5 1 0.5 - 2 -
T2 0.1 0.5 0.5 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
S3 0.80 0.93 L35 0.63 0.58
S24 0.90 0.94 L49 0.51 0.52
S26 1.26 1.29 L33 0.60 0.63
S27 0.81 1.31 L10 1.06 0.98
S30 1.40 1.49 F2 0.78 0.81
S43 1.40 1.53 F8 0.93 0.95
S5 0.89 1.20 F10 1.10 1.17
S42 0.51 0.84 F23 1.03 1.07
L4 0.41 0.80 F30 1.40 1.51
L26 0.50 0.58 F43 0.84 0.97
Amylase activator is also present within the extract. This can also detect. In one test tube extract were added after addition of DNSA and in another test tube extract were added before incubation. Differences between those indicate the presence or absence of amylase activator.
AMYLASE ACTIVATOR
TEST ENZ
(AMY) BUFF.
(ML) ACT.
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML) ACT.
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 - 0.5 1 - 2 -
T1 0.4 0.5 - 0.5 - 0.05 2 -
T2 0.4 0.5 0.05 0.5 1 - 2 -
SAMPLE T1 T2
L31 0.42 0.50
L32 0.52 0.54
L19 0.56 0.70
L25 0.60 0.58
S43 0.57 0.72
S12 0.98 1.09
S8 1.01 1.10
F9 1.29 1.41
Salivary amylase inhibitors in extract also separated by using starch polyacrylamide gel. But in many attempt the amylase inhibitor bands were not found clearly. The gels are incubate in the amylase solution, amylase hydrolyze the starch as it enters the gel. However, starch in the vicinity of amylase inhibitor band is protected from hydrolysis due to inhibition band is protected from hydrolysis due to inhibition of amylase and appears as blue band after staining with iodine. The size and intensity of blue bands correspond to the extent of inhibition of amylase which depends upon the concentration and activity of amylase inhibitor protein in gel.
This method is sensitive entire procedure takes about one hour after electrophoresis run. In the particle purification of samples by ammonium sulfate fractionation. Increase the possibility of band formation by enriching amylase inhibitory activity. Most of the amylase inhibitor is temp sensitive. So storage at cooling condition is necessary.
Albumins in the seed extract also inhibit starch-iodine complex formation by sequestering iodine and may be confuse for amylase activity bands. In the gel same time starch having the tendency to form clumps and precipitate during polymerization of gels. Slow polymerization and overnight keeping of gels prior to run help in even distribution of starch in the gel.
However, our attempts on visualizing amylase inhibitor band in starch poly acryl amide gas were unsuccessful.
REFRANCES
1. Alam n. And S, Gourinath, 2001. Substratre-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase / trypsin inhibitor (RATT) and various N-terminal fragments. Biochem, apr. 10:40: 4229-33
2. Barbar D, Sanchez, R. Mong, I. Gomez and G. Salcedo 1989, FEBSKett, 248:119-122.
3. Brieteneder, H and C.A. Radauer, 2004, Classification of plant food allergens. J. Allergy and Clin. Immunol. May 113:821-830.
4. K. Murayama R, Dimango EP, Character of a wheat amylase inhibitor preparation and effects on fasting human pancreaticobliliary secretions and hormones. Gastroenterology 1996:111:1313-20
5. M.M Pichare and M.S. Kachole. J. Biochem. Biophys. Methods 28 (1994) 215-224
6. CM, Purification and properties of phaseolamin , an inhibitor of alpha-amylase, from the kidney bean, phaseolus vulgaris. J Biol Chem 1975:250:8030-7
7. Octavio, L. and D. Rigden, 2002. Plant amylase inhibitos and their interaction with amylases. Eur. J. Biochem. 269:397-412
8. Octiva L. And D. Rigden, 2000. Activity of wheat amylase inhibitors towards bruchid. Amylase and structural explanation of observed specificities. Eur. J. Biochem, 267: 2166-2173
9. Richardson, M. 1991, Methods in Biochemistry, volume 5, Academic press, pp:259-
Seed leaves and flower tissue were extracted in P.V.P. the extract were screen qualitatively dot-blot agar plate technique for amylase inhibitor were further analyzed for quantitative estimation of Amylase inhibitor activity by DNSA method. Endogenous amylase and amylase activator also present within the tissue. these are detected by developing specific protocol for each.
The inhibitors were separated in polyacrylamide gel containing 0.5% soluble starch by electrophoresis and visualized by incubation of the gel in salivary amylase solution and staining with iodine. Starch in the gel is hydrolise by amylase during incubation but starch in vicinity of amylase inhibitor is protected from hydrolysis and appear as the blue band after staining.
Keywords: - Amylase inhibitors, Enzyme, Endogenous amylase, Amylase activator, Electrophoresis,
INTRODUCTION
α-Amylase inhibitors:-
Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. These inhibitors can be grouped into six classes based on their tertiary structures. Lectin-like, knottin-like, cereal-type, kunitz-like, γ-purothionin like and thaumatin like inhibitors, some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites.
In animals, α-amylase inhibitors reduce the glucose peaks that can occur after a meal, slowing the speed with which α-amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extra cellular glucose from being cleared quickly from the blood. Therefore, diabetics tend to have low α-amylase levels in order to keep glucose levels under control, except after taking insulin, which causes a rise in pancreatic α-amylase.
Plants also use α-amylase inhibitors as a defense strategy. These inhibitors impede the digestive action of α-amylases and protinases in the insect gut, thereby acting as insect anti-feedants. As a result, α-amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes.( Marshal JJ, Lauda)
What do they do?
Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other, secondary, enzymes. They are claimed to be useful for weight loss. But when they were first developed years ago, research did not find them very effective for limiting carbohydrate absorption. Later, however, highly concentrated versions of amylase inhibitors did show potential for reducing carbohydrate absorption in humans.
Purified starch blocker extracts. When given with a starchy meal, have also been shown to reduce the subsequent rise in blood sugar levels of both healthy people and diabetics. The is effect could be helpful in the treatment of blood sugar disorders.( Choudhary A, Maeda)
Where are they found?
Amylase inhibitors can be extracted from several types of plants, especially those in the legume family. Currently available amylase inhibitors are extracted from either white kidney bean or wheat.
The relative inefficacy of alpha-amylase inhibitors in affecting human digestion of starch has been highlighted by reacent scientific and public controversy over the commercial sales of so-called starch-blockers or slimming pillis alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes obesity and hyperlipaemia studies of the structures of the numerous enzyme inhibitors found in cereal grains have led to the recognition of a super family of homologous proteins which includes inhibitors of alha-amylase, proteinase and bifunctional inhibitors of alpha-amylase, proteinase and bifunctional inhibitors active against two or more classes of enzymes.(Alam and Gourinath, 2001, Octavio and Rigden 2002 Richardson, 1991)
The first alpha-amylase inhibitor determined was that of the monomeric 13 kD known as 0.31 form, from wheat (kashlan and Richardson, 1981) other dimeric 0.19, 0.23, 0.28, 0.53 form of weat inhibitors of exogenous alpha-amylase were later shown (oneda et al 2004, kondo and ida1995, Roy and Gupta 2000, Richardson, 1991, Octavio and Rigden 2002)
The favored hyupotheses about physiological roles fo the enzyme inhibitors in seeds is that they act as storage or reserve proteins as regulators of endogenous enzyme or as defensive agents against the attacks of animal predators and insect or microbial pests. It seems likely that in certain species these proteins may fulfill a combination of these proteins may fulfill a combination of these function(Octavia and Rigden 2002, Octivio and Rigden 2000, Richardson 1991) Also plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plant against pests (Octavio and Rigden2002)
Nutritional and metabolic effects of enzyme inhibitors certainly some of inhibitors certainly some of inhibitors found in cereal and legume seeds can inactive the salivary and pancereatic enzymes of human (pick and wober) and their sucseotibility to be rather variable (singhand bbkundel, 2001) many are destroyed by cooking but some retain inhibitory activity even after baking (Richardson 1991)
Amylase inhibitors present in seeds currently used as food present few nutritional problem for healthy people but may have some toxicological significance in the diets if infants who have a lower production of pancreatic alpha-amylase than adults and for patients with impaired peptic or gastric function (brietender and rauduer ,2004. richrdson ,1991 shewry etal2001) also one inhibitor of insect alpha-amylase isolated from barley flour is the major allergen associated with bakers asthma disease (barber et al 1989) the inhibition is strictly competitive and in the1:1 complexes aloof the activities of the enzyme are completely abolished .
Crystallographic nuclear, magnetic resonance (NMR) and mechanistic studies all indicates that the inhibitors act as highly specific substrate for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond .
The reaction mechanics involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood (silan 1986) but there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the mechanism or that the inhibitor may induce confermational changes in the enzyme molecule.
Materials:-
Different part of the plants including leaves, flower, seeds were collected from local region. Some sample were collected from Nanded ,Rahuri,Pathardi total 146 sample were collected.
Start was from qualgen fine chemical company Bombay India
Polyvinylpolypurrolidone (P.V.P) was from Sigma
Human saliva was diluted and used as salivary amylase all other chemicals were of the highest purity available
LEAVES:
Sr. no Common name Botonical name
1 Sitaphal Annona sequmusa
2 Subhabhul Leueaene lapisiliqua
3 Errand Risinus comnuis
4 Naginiche pan Piper bettne
5 Bhabhul
6 Ghaneri Lantana camera
7 Gawti chaha Cymbopogan flexuosus
8 Aalu Calocasia esculanta
9 Rui Calotropis procera
10 Kadhipatta Murraya kotnigii
11 Gahu Triticum aestivum
12 Papai Papaya indica
13 Amba Mangifera indica
14 Mohair Brassia juncea
15 Naral Coccus nucifera
16 Jambul Syzium cumini
17 Aalu Leaves Calocasia esculanta
18 Harbara Cicer aeriantum
19 Dhol Amba
20 Nimbu Citrus cemani
21 Palak Spinach
22 Kadulimb Azadirecta indica
23 Tobacco Nicotiane tobacum
24 Tulas Ocimum Americanum
25 Tarwad Cassia auriculata
26 Kardali Canna indica
27 Wel
28 Nishigandha Polianthus tuberosa
29 Manjiri Ocimum Americanum
30 Tomato Lycopersicon escunentum
31 Beshram Ipomea
32 Sunflower Helianthus annus
33 Chandan Santalum album
34 Maka Zea mays
35 Halad Curcuma aromatica
36 Sadaba Runca chalepensis
37 Ratali Ipomeabatatas
38 Bartodi Morinda, citrifolia
39 Apte Bahunia
40 Palas Bulea monosperma
41 Biba Semecarpus anacardium
42 Rohini
43 Acasia Chandra
44 Agav Americana
45 Glyrisidium capium
46 Ipomea
47 Carissa carandus
48 Sag Tectona granvis
49 Maniplant
50 Cactus
51 Tur Cajanus cajan
52 Pimple
FLOWER:
1 Dhotra Datura alba
2 Jaswand Hibicus rosasinensis
3
4 Tarwad Cassia auriculata
5
6
7 Kaneri Nerium indicum
8 Zendu Tagehun erecta
9 Rose Red
10
11 Rui Calatropis procera
12 Chafa Michella champaka
13
14
15 Jai Jasminium auricylamn
16 Allamnda
17 Shewga Moringa olifera
18 Gandhar
19 Boganwel Boganvillia spectibilis
20 Kunda
21 Nishigandh Polyanthus tuberose
22
23 Gulbakshi Mirabilis jalapa
24 kardali Canna indica
25
26 Ghosali Lutta aegeyptia
27 Danger
28 Gerbera
29 Aboli Crossendra undilifolia
30 Sadafully Catharathus roses
31 Biliat
32 Tur Cajanus cajan
33 Shevari Sesbania sesban
34 Ghevda Psophocarpus tetiagomolobes
35
36
37 Palas Butea monosporne
38 Sunflower Tridax procambers
39
40 Khajawe
41 Sweet potato Ipomebatata
42 Kandhar
43 Lakh Acacia
44 Sag Tectona Gran
45 Chirisidium caprsium
SEED:
1 Mug Phaseomus aureus
2 Mahsoor Lens esculenta
3 Macca Zea maize
4 Chawali Vigna sinensis
5 Udit Phaseolus mango
6 Tur Cajanus cajan
7 watana Pisum Sativum
8 Hulga
9 Jawari Sorghum valgare
10 Tag
11 Methi Trigonella toenum
12 Ubal bee
13 Math
14 Soyabeen Glycine max
15 Karale
16 Kabuli Harbala Cicer grientum
17 Chinchuka Tamarindus indica
18 Airnda Ricimus communis
19 Tandul Orizha Sathiva
20 Bhagar
21 Dudhi Bhopla
22 Kharbuz Cacumis melo
23 Gokarn Clitonia kleio
24 Rale
25 Wange Solalum righhi
26 Khas khas
27 Shengadane Arachis hypogea
28 Coffee Caffia Arabica
29 Danger
30 Mohari Brassica kmphesis
31 Rajgira Amaranthus
32 Mire Piper nigrum
33 Rajma
34 Elayachi Elettoria cordomomum
35 Tulas Ocium santum
36 Hawari
37 Jira Caminum cyminum
38 Dhane Corriander sativa
39 Mirchi Capsium annum
40 Apte Bahunia racemose
41 Badishop Foenicumum valgari
42 Jawas Alhagi pseudalhagi
43 Bajari Pennisenem typhoids
44 Ghosale Laffer cylindrical
45 Sabja Ocimum bascilium
46 God babhul Accacia
47 Gahu Triticum aestivam
METHOD
Extraction of amylase inhibitors:-
extraction if amylase inhibitors leaves, flower, seed are done by same procedure.
1. Decorticated seeds of all sample were dried and ground in blender to obtain fine flour.
2. Leaves are crused making the fine as possible as in small amount of acetone.
3. Same procedure was used for the flower sample.
4. After removing pigments all the sample are kept in hexane for overnight to remove the fatty material.
5. This procedure was repeated 2 to 3 times.
6. Then samples are carefully dried and packed.
7. Proper method for numbering and identification was used
8. The defatted seed powder was stirred with 1% PVP in 1:5 ratio for 48 hour.
9. Inclusion of PVP helpful in removal of phenolics from extract.
10. The suspension was centrifuged on cooling centrifuged at 10000 rpm for 20 min at 4oC
11. addition of PVP and centrifugation was repeated twice for same sample.
12. The clear supernatant contained amylase inhibitors was stored in freeze and used for analysis.
(Here after mentioned as partially purified extract.)
DOT BLOT INHIBITOR ASSAY:-
1. Extracted sample was tested on agar-starch plat in different concentration for screnning.
2. Using the enzyme (Amylase) Buffer (6.8PH) inhibitor at different proportion dot-blot assay was done.
3. For that 2% agar and 1% starch was used.
4. These are screening method due to this reaction sample which show inhibition are repeated once again and confirm that they show inhibition for amylase.
5. Among the 146 sample 58 sample shown inhibitions.
6. In that these are further categorized in strong inhibitor, medium inhibitor, weak inhibitor.
7. In 146 samples 27 samples are strongly inhibitor, 22 samples are inhibitor, and 9 samples are weak inhibitor.
8. After the screening of 146 samples the extracts which show strong inhibition and medium inhibition was taken for their amylase inhibitor assay by DNSA.
AMYLASE INHIBITOR ASSY :
Amylase activity was assayed by measuring liberated maltose. amylase inhibitory ativity was assyed by measuring reduction in maltose liberated by salivary amylase using dinitrosalisylic acid reagent.
One amylase activity unit is defined as activity resulting in to liberation of 1 mg of maltose from starch at ph 6.9 at 37o C in 3 min.
one amylase inhibitor unit is one amylase unit inhibited under the given assay condition.
In this assay same extract showing the exact inhibition but here again same extract does not show inhibition.
The samples which show inhibition are again tested for the endogenous amylase and amylase activator.
ENDOGENOUS AMYLASE:-
1. Presence of amylases in biological sample particularly from plant case a major problem in detection of amylase inhibitor. The detection of endogenous amylase is possible with DNSA method.
2. In that tow test are taken in one reaction was arrested by addition of DNSA and in anther the reaction are carried out complete and then DNSA was added.
AMYLASE ACTIVATOR:-
Amylase activator also present within the extract there can be detected by DNSA.
In one test tube enzyme, buffer and substrate are added and in another activator was added with enzyme, buffer substrate. Incubate the both test tube and add DNSA activeter was add in first test tube after incubation the difference in OD indicated the amylase activator.
ELECTROPHORETIC SEPRATION OF AMYLASE INHIBITOR :-
Amylase inhibitors in the partially purified extract were analyzed on a vertical slab gel electrophoresis system in 7% polyacrylamide gels containing 0.5% soluble starch without stacking gels containing 0.5% soluble starch without stacking gel. For that tris-glycine (PH 8.9) both in gel and electrode tank was used.
VISUALIZATION OF AMYLASE INHIBITORS:-
After electrophoresis gels are placed in 20mm phosphate buffer (PH 6.9) containing 6.7mm NaCl for 5-10 mm for equilibration and incubated in salivary amylase in phosphate buffer (PH 6.9) for 30 min at 37 C. after incubation the gels are wash with D/W and placed in iodine solution. (10 MM iodine in 14mm KI) for 4 to 5 min.
Exact band of amylase inhibiter way not found.
RESULT AND DISCUSSION:-
Amylase inhibitors are detected by using three different concentrations the strong inhibitor show the inhibitor at all three concentration. Where the weak inhibitor show inhibition only at low concentration of amylase by using this technique determination of amylase activator is also possible. Same extract were help to the activation. amylase hydrolyzes the starch present in the agar. After staining with iodine. It show white area indicate that it is not inhibitor. When these area are broad than the control, then it is activator
DOT-BLOT ASSAY :-
TEST ENZYME BUFFER INHIBITOR
CONTROL 40 40 -
STRONG 40 28 12
MEDIUM 28 24 28
WEAK 12 28 40
SAMPLE NO TYPE OF INHIBITION
Seed- 1,4,7,8,11,9,23,24,26,27,42,43,15,19 * * *
25,30,31,28,43,3,5, * *
6,12,13,15,16,19,20 *
Flower-
1,2,8,10,20,23,27,30 * * *
5,6,7,11,43 * *
9 *
Leaves-
25,29,32,35,49, * * *
4,18,31,33,36,41,46,47,48,49 * *
26 *
*** = Strong Inhibitor
** = Medium Inhibitor
* = Weak Inhibitor
The extracts which show inhibitions for amylase are tested by DNSA assay for each sample two test are done. In first test extract were added after incubation and another, extract added before incubation. amylase inhibitory activity was assayed by measuring reeducation in maltose liberated by salivary amylase using DNSA reagent
AMYLASE INHIBITOR (DNSA)
TEST ENZ EXTRACT BUFF SUB EXTRACT DNSA
(ML) DILU
(ML) O.D
Control - - 1.0 0.5 Incubation for twenty min - 1 Bwb for 10 min 2 -
Test-1 0.4 - 0.5 0.5 0.1 1 2 -
Test-2 0.4 0.1 0.5 0.5 - 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
1 S2 0.53 0.62 L31 0.77 0.87
2 S3 0.49 0.39 L32 0.79 0.85
3 S5 0.44 0.50 L33 0.78 0.78
4 S7 0.84 0.81 L35 0.80 0.77
5 S8 0.97 1.02 L36 0.76 0.83
6 S9 0.85 0.85 L41 0.89 0.90
7 S10 0.83 0.81 L46 0.93 0.86
8 S12 1.15 1.19 L47 0.97 1.01
9 S13 0.86 0.84 L49 0.88 0.85
10 S16 0.84 0.84 F1 1.38 1.42
11 S19 0.85 0.85 F2 1.40 1.30
12 S24 0.62 0.55 F6 1.38 1.35
13 S26 0.75 0.58 F8 1.41 1.40
14 S27 0.56 0.43 F9 1.31 1.38
15 S28 0.96 0.78 F10 1.40 1.38
16 S30 1.01 0.97 F23 1.11 0.98
17 S31 0.65 0.59 F26 1.32 1.38
18 S4 0.76 0.73 F29 1.07 1.14
19 S42 0.63 0.53 F30 1.19 0.93
20 S43 0.58 0.67 F39 1.28 1.36
21 L4 0.80 0.74 F42 0.98 1.03
22 L18 0.78 0.79 F43 1.17 0.97
23 L26 0.77 0.74
24 L29 0.78 0.75
Amylase Inhibitor:-
S3, S24, S26, S27, S28, S31, S4, S42, L4, L26, L46, L49, F2, F23, F30, F43.
Presence of amylases in biological sample particularly from plant is a major problem in detection of amylase inhibitors. The detection of amylase inhibitors in sample containing endogenous amylases is possible. Endogenous amylase in sample can be detected. In one test tube DNSA are added before incubation and reaction are stopped. In another reaction are carried out. Difference between the O.D. indicate the concentration of endogenous amylase.
ENDOGENOUS AMYLASE :
TEST EXTRACT
(ML) BUFF.
(ML) DNSA
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 0.5 1 2 -
T1 0.1 0.5 1 0.5 - 2 -
T2 0.1 0.5 0.5 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
S3 0.80 0.93 L35 0.63 0.58
S24 0.90 0.94 L49 0.51 0.52
S26 1.26 1.29 L33 0.60 0.63
S27 0.81 1.31 L10 1.06 0.98
S30 1.40 1.49 F2 0.78 0.81
S43 1.40 1.53 F8 0.93 0.95
S5 0.89 1.20 F10 1.10 1.17
S42 0.51 0.84 F23 1.03 1.07
L4 0.41 0.80 F30 1.40 1.51
L26 0.50 0.58 F43 0.84 0.97
Amylase activator is also present within the extract. This can also detect. In one test tube extract were added after addition of DNSA and in another test tube extract were added before incubation. Differences between those indicate the presence or absence of amylase activator.
AMYLASE ACTIVATOR
TEST ENZ
(AMY) BUFF.
(ML) ACT.
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML) ACT.
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 - 0.5 1 - 2 -
T1 0.4 0.5 - 0.5 - 0.05 2 -
T2 0.4 0.5 0.05 0.5 1 - 2 -
SAMPLE T1 T2
L31 0.42 0.50
L32 0.52 0.54
L19 0.56 0.70
L25 0.60 0.58
S43 0.57 0.72
S12 0.98 1.09
S8 1.01 1.10
F9 1.29 1.41
Salivary amylase inhibitors in extract also separated by using starch polyacrylamide gel. But in many attempt the amylase inhibitor bands were not found clearly. The gels are incubate in the amylase solution, amylase hydrolyze the starch as it enters the gel. However, starch in the vicinity of amylase inhibitor band is protected from hydrolysis due to inhibition band is protected from hydrolysis due to inhibition of amylase and appears as blue band after staining with iodine. The size and intensity of blue bands correspond to the extent of inhibition of amylase which depends upon the concentration and activity of amylase inhibitor protein in gel.
This method is sensitive entire procedure takes about one hour after electrophoresis run. In the particle purification of samples by ammonium sulfate fractionation. Increase the possibility of band formation by enriching amylase inhibitory activity. Most of the amylase inhibitor is temp sensitive. So storage at cooling condition is necessary.
Albumins in the seed extract also inhibit starch-iodine complex formation by sequestering iodine and may be confuse for amylase activity bands. In the gel same time starch having the tendency to form clumps and precipitate during polymerization of gels. Slow polymerization and overnight keeping of gels prior to run help in even distribution of starch in the gel.
However, our attempts on visualizing amylase inhibitor band in starch poly acryl amide gas were unsuccessful.
REFRANCES
1. Alam n. And S, Gourinath, 2001. Substratre-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase / trypsin inhibitor (RATT) and various N-terminal fragments. Biochem, apr. 10:40: 4229-33
2. Barbar D, Sanchez, R. Mong, I. Gomez and G. Salcedo 1989, FEBSKett, 248:119-122.
3. Brieteneder, H and C.A. Radauer, 2004, Classification of plant food allergens. J. Allergy and Clin. Immunol. May 113:821-830.
4. K. Murayama R, Dimango EP, Character of a wheat amylase inhibitor preparation and effects on fasting human pancreaticobliliary secretions and hormones. Gastroenterology 1996:111:1313-20
5. M.M Pichare and M.S. Kachole. J. Biochem. Biophys. Methods 28 (1994) 215-224
6. CM, Purification and properties of phaseolamin , an inhibitor of alpha-amylase, from the kidney bean, phaseolus vulgaris. J Biol Chem 1975:250:8030-7
7. Octavio, L. and D. Rigden, 2002. Plant amylase inhibitos and their interaction with amylases. Eur. J. Biochem. 269:397-412
8. Octiva L. And D. Rigden, 2000. Activity of wheat amylase inhibitors towards bruchid. Amylase and structural explanation of observed specificities. Eur. J. Biochem, 267: 2166-2173
9. Richardson, M. 1991, Methods in Biochemistry, volume 5, Academic press, pp:259-
Project on Enzyme
I take this opportunity to express my deep sense of gratitude to my project guide Dr. Padul M.V. under whose guidance the present dissertation was completed. I shall ever remain grateful to him for his scholastic guidance, helpful and constrictive criticism during the entire period of there investigation.
I fee highly indebted to Dr. A.D. Chaugale, Dr. Baladhye and miss. Shaikh R.R. for her Valuable suggestion and instruction.
I must place on record my sincere and grateful thanks to Proff. M.T.Patil and Prof. Sable for their valueable contribution in naming and identification of plants.
I wish to express my profound gratitude to Prof. H.M. Humbe H.O.D. Chemistry for providing necessary facility during the entire course of this project.
I also express my sinsiour thanks to the teaching and non teaching staff of bio chemistry, micro biology, botony for their valuable suggestion and mindful help. Extended to me for getting the work done.
I would remiss if do not acknowledge my debit to my friend and class mate for their wonderful contribution and rendered to me during the experimental work.
No words and thanks to enough to express my heartfelt filling of love and indebtness to parent and brother for there sincere efforts in successfully keeping the best selling through most stable envoirment for their sacrifice in moulding me to a learn citizen
Mr. G.R. Sambare
ABSTRACT
Seed leaves and flower tissue were extracted in P.V.P. the extract were screen qualitatively dot-blot agar plate technique for amylase inhibitor were further analyzed for quantitative estimation of Amylase inhibitor activity by DNSA method. Endogenous amylase and amylase activator also present within the tissue. these are detected by developing specific protocol for each.
The inhibitors were separated in polyacrylamide gel containing 0.5% soluble starch by electrophoresis and visualized by incubation of the gel in salivary amylase solution and staining with iodine. Starch in the gel is hydrolise by amylase during incubation but starch in vicinity of amylase inhibitor is protected from hydrolysis and appear as the blue band after staining.
Keywords: - Amylase inhibitors, Enzyme, Endogenous amylase, Amylase activator, Electrophoresis,
INTRODUCTION
α-Amylase inhibitors:-
Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. These inhibitors can be grouped into six classes based on their tertiary structures. Lectin-like, knottin-like, cereal-type, kunitz-like, γ-purothionin like and thaumatin like inhibitors, some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites.
In animals, α-amylase inhibitors reduce the glucose peaks that can occur after a meal, slowing the speed with which α-amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extra cellular glucose from being cleared quickly from the blood. Therefore, diabetics tend to have low α-amylase levels in order to keep glucose levels under control, except after taking insulin, which causes a rise in pancreatic α-amylase.
Plants also use α-amylase inhibitors as a defense strategy. These inhibitors impede the digestive action of α-amylases and protinases in the insect gut, thereby acting as insect anti-feedants. As a result, α-amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes.( Marshal JJ, Lauda)
What do they do?
Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other, secondary, enzymes. They are claimed to be useful for weight loss. But when they were first developed years ago, research did not find them very effective for limiting carbohydrate absorption. Later, however, highly concentrated versions of amylase inhibitors did show potential for reducing carbohydrate absorption in humans.
Purified starch blocker extracts. When given with a starchy meal, have also been shown to reduce the subsequent rise in blood sugar levels of both healthy people and diabetics. The is effect could be helpful in the treatment of blood sugar disorders.( Choudhary A, Maeda)
Where are they found?
Amylase inhibitors can be extracted from several types of plants, especially those in the legume family. Currently available amylase inhibitors are extracted from either white kidney bean or wheat.
The relative inefficacy of alpha-amylase inhibitors in affecting human digestion of starch has been highlighted by reacent scientific and public controversy over the commercial sales of so-called starch-blockers or slimming pillis alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes obesity and hyperlipaemia studies of the structures of the numerous enzyme inhibitors found in cereal grains have led to the recognition of a super family of homologous proteins which includes inhibitors of alha-amylase, proteinase and bifunctional inhibitors of alpha-amylase, proteinase and bifunctional inhibitors active against two or more classes of enzymes.(Alam and Gourinath, 2001, Octavio and Rigden 2002 Richardson, 1991)
The first alpha-amylase inhibitor determined was that of the monomeric 13 kD known as 0.31 form, from wheat (kashlan and Richardson, 1981) other dimeric 0.19, 0.23, 0.28, 0.53 form of weat inhibitors of exogenous alpha-amylase were later shown (oneda et al 2004, kondo and ida1995, Roy and Gupta 2000, Richardson, 1991, Octavio and Rigden 2002)
The favored hyupotheses about physiological roles fo the enzyme inhibitors in seeds is that they act as storage or reserve proteins as regulators of endogenous enzyme or as defensive agents against the attacks of animal predators and insect or microbial pests. It seems likely that in certain species these proteins may fulfill a combination of these proteins may fulfill a combination of these function(Octavia and Rigden 2002, Octivio and Rigden 2000, Richardson 1991) Also plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plant against pests (Octavio and Rigden2002)
Nutritional and metabolic effects of enzyme inhibitors certainly some of inhibitors certainly some of inhibitors found in cereal and legume seeds can inactive the salivary and pancereatic enzymes of human (pick and wober) and their sucseotibility to be rather variable (singhand bbkundel, 2001) many are destroyed by cooking but some retain inhibitory activity even after baking (Richardson 1991)
Amylase inhibitors present in seeds currently used as food present few nutritional problem for healthy people but may have some toxicological significance in the diets if infants who have a lower production of pancreatic alpha-amylase than adults and for patients with impaired peptic or gastric function (brietender and rauduer ,2004. richrdson ,1991 shewry etal2001) also one inhibitor of insect alpha-amylase isolated from barley flour is the major allergen associated with bakers asthma disease (barber et al 1989) the inhibition is strictly competitive and in the1:1 complexes aloof the activities of the enzyme are completely abolished .
Crystallographic nuclear, magnetic resonance (NMR) and mechanistic studies all indicates that the inhibitors act as highly specific substrate for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond .
The reaction mechanics involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood (silan 1986) but there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the mechanism or that the inhibitor may induce confermational changes in the enzyme molecule.
Materials:-
Different part of the plants including leaves, flower, seeds were collected from local region. Some sample were collected from Nanded ,Rahuri,Pathardi total 146 sample were collected.
Start was from qualgen fine chemical company Bombay India
Polyvinylpolypurrolidone (P.V.P) was from Sigma
Human saliva was diluted and used as salivary amylase all other chemicals were of the highest purity available
LEAVES:
Sr. no Common name Botonical name
1 Sitaphal Annona sequmusa
2 Subhabhul Leueaene lapisiliqua
3 Errand Risinus comnuis
4 Naginiche pan Piper bettne
5 Bhabhul
6 Ghaneri Lantana camera
7 Gawti chaha Cymbopogan flexuosus
8 Aalu Calocasia esculanta
9 Rui Calotropis procera
10 Kadhipatta Murraya kotnigii
11 Gahu Triticum aestivum
12 Papai Papaya indica
13 Amba Mangifera indica
14 Mohair Brassia juncea
15 Naral Coccus nucifera
16 Jambul Syzium cumini
17 Aalu Leaves Calocasia esculanta
18 Harbara Cicer aeriantum
19 Dhol Amba
20 Nimbu Citrus cemani
21 Palak Spinach
22 Kadulimb Azadirecta indica
23 Tobacco Nicotiane tobacum
24 Tulas Ocimum Americanum
25 Tarwad Cassia auriculata
26 Kardali Canna indica
27 Wel
28 Nishigandha Polianthus tuberosa
29 Manjiri Ocimum Americanum
30 Tomato Lycopersicon escunentum
31 Beshram Ipomea
32 Sunflower Helianthus annus
33 Chandan Santalum album
34 Maka Zea mays
35 Halad Curcuma aromatica
36 Sadaba Runca chalepensis
37 Ratali Ipomeabatatas
38 Bartodi Morinda, citrifolia
39 Apte Bahunia
40 Palas Bulea monosperma
41 Biba Semecarpus anacardium
42 Rohini
43 Acasia Chandra
44 Agav Americana
45 Glyrisidium capium
46 Ipomea
47 Carissa carandus
48 Sag Tectona granvis
49 Maniplant
50 Cactus
51 Tur Cajanus cajan
52 Pimple
FLOWER:
1 Dhotra Datura alba
2 Jaswand Hibicus rosasinensis
3
4 Tarwad Cassia auriculata
5
6
7 Kaneri Nerium indicum
8 Zendu Tagehun erecta
9 Rose Red
10
11 Rui Calatropis procera
12 Chafa Michella champaka
13
14
15 Jai Jasminium auricylamn
16 Allamnda
17 Shewga Moringa olifera
18 Gandhar
19 Boganwel Boganvillia spectibilis
20 Kunda
21 Nishigandh Polyanthus tuberose
22
23 Gulbakshi Mirabilis jalapa
24 kardali Canna indica
25
26 Ghosali Lutta aegeyptia
27 Danger
28 Gerbera
29 Aboli Crossendra undilifolia
30 Sadafully Catharathus roses
31 Biliat
32 Tur Cajanus cajan
33 Shevari Sesbania sesban
34 Ghevda Psophocarpus tetiagomolobes
35
36
37 Palas Butea monosporne
38 Sunflower Tridax procambers
39
40 Khajawe
41 Sweet potato Ipomebatata
42 Kandhar
43 Lakh Acacia
44 Sag Tectona Gran
45 Chirisidium caprsium
SEED:
1 Mug Phaseomus aureus
2 Mahsoor Lens esculenta
3 Macca Zea maize
4 Chawali Vigna sinensis
5 Udit Phaseolus mango
6 Tur Cajanus cajan
7 watana Pisum Sativum
8 Hulga
9 Jawari Sorghum valgare
10 Tag
11 Methi Trigonella toenum
12 Ubal bee
13 Math
14 Soyabeen Glycine max
15 Karale
16 Kabuli Harbala Cicer grientum
17 Chinchuka Tamarindus indica
18 Airnda Ricimus communis
19 Tandul Orizha Sathiva
20 Bhagar
21 Dudhi Bhopla
22 Kharbuz Cacumis melo
23 Gokarn Clitonia kleio
24 Rale
25 Wange Solalum righhi
26 Khas khas
27 Shengadane Arachis hypogea
28 Coffee Caffia Arabica
29 Danger
30 Mohari Brassica kmphesis
31 Rajgira Amaranthus
32 Mire Piper nigrum
33 Rajma
34 Elayachi Elettoria cordomomum
35 Tulas Ocium santum
36 Hawari
37 Jira Caminum cyminum
38 Dhane Corriander sativa
39 Mirchi Capsium annum
40 Apte Bahunia racemose
41 Badishop Foenicumum valgari
42 Jawas Alhagi pseudalhagi
43 Bajari Pennisenem typhoids
44 Ghosale Laffer cylindrical
45 Sabja Ocimum bascilium
46 God babhul Accacia
47 Gahu Triticum aestivam
METHOD
Extraction of amylase inhibitors:-
extraction if amylase inhibitors leaves, flower, seed are done by same procedure.
1. Decorticated seeds of all sample were dried and ground in blender to obtain fine flour.
2. Leaves are crused making the fine as possible as in small amount of acetone.
3. Same procedure was used for the flower sample.
4. After removing pigments all the sample are kept in hexane for overnight to remove the fatty material.
5. This procedure was repeated 2 to 3 times.
6. Then samples are carefully dried and packed.
7. Proper method for numbering and identification was used
8. The defatted seed powder was stirred with 1% PVP in 1:5 ratio for 48 hour.
9. Inclusion of PVP helpful in removal of phenolics from extract.
10. The suspension was centrifuged on cooling centrifuged at 10000 rpm for 20 min at 4oC
11. addition of PVP and centrifugation was repeated twice for same sample.
12. The clear supernatant contained amylase inhibitors was stored in freeze and used for analysis.
(Here after mentioned as partially purified extract.)
DOT BLOT INHIBITOR ASSAY:-
1. Extracted sample was tested on agar-starch plat in different concentration for screnning.
2. Using the enzyme (Amylase) Buffer (6.8PH) inhibitor at different proportion dot-blot assay was done.
3. For that 2% agar and 1% starch was used.
4. These are screening method due to this reaction sample which show inhibition are repeated once again and confirm that they show inhibition for amylase.
5. Among the 146 sample 58 sample shown inhibitions.
6. In that these are further categorized in strong inhibitor, medium inhibitor, weak inhibitor.
7. In 146 samples 27 samples are strongly inhibitor, 22 samples are inhibitor, and 9 samples are weak inhibitor.
8. After the screening of 146 samples the extracts which show strong inhibition and medium inhibition was taken for their amylase inhibitor assay by DNSA.
AMYLASE INHIBITOR ASSY :
Amylase activity was assayed by measuring liberated maltose. amylase inhibitory ativity was assyed by measuring reduction in maltose liberated by salivary amylase using dinitrosalisylic acid reagent.
One amylase activity unit is defined as activity resulting in to liberation of 1 mg of maltose from starch at ph 6.9 at 37o C in 3 min.
one amylase inhibitor unit is one amylase unit inhibited under the given assay condition.
In this assay same extract showing the exact inhibition but here again same extract does not show inhibition.
The samples which show inhibition are again tested for the endogenous amylase and amylase activator.
ENDOGENOUS AMYLASE:-
1. Presence of amylases in biological sample particularly from plant case a major problem in detection of amylase inhibitor. The detection of endogenous amylase is possible with DNSA method.
2. In that tow test are taken in one reaction was arrested by addition of DNSA and in anther the reaction are carried out complete and then DNSA was added.
AMYLASE ACTIVATOR:-
Amylase activator also present within the extract there can be detected by DNSA.
In one test tube enzyme, buffer and substrate are added and in another activator was added with enzyme, buffer substrate. Incubate the both test tube and add DNSA activeter was add in first test tube after incubation the difference in OD indicated the amylase activator.
ELECTROPHORETIC SEPRATION OF AMYLASE INHIBITOR :-
Amylase inhibitors in the partially purified extract were analyzed on a vertical slab gel electrophoresis system in 7% polyacrylamide gels containing 0.5% soluble starch without stacking gels containing 0.5% soluble starch without stacking gel. For that tris-glycine (PH 8.9) both in gel and electrode tank was used.
VISUALIZATION OF AMYLASE INHIBITORS:-
After electrophoresis gels are placed in 20mm phosphate buffer (PH 6.9) containing 6.7mm NaCl for 5-10 mm for equilibration and incubated in salivary amylase in phosphate buffer (PH 6.9) for 30 min at 37 C. after incubation the gels are wash with D/W and placed in iodine solution. (10 MM iodine in 14mm KI) for 4 to 5 min.
Exact band of amylase inhibiter way not found.
RESULT AND DISCUSSION:-
Amylase inhibitors are detected by using three different concentrations the strong inhibitor show the inhibitor at all three concentration. Where the weak inhibitor show inhibition only at low concentration of amylase by using this technique determination of amylase activator is also possible. Same extract were help to the activation. amylase hydrolyzes the starch present in the agar. After staining with iodine. It show white area indicate that it is not inhibitor. When these area are broad than the control, then it is activator
DOT-BLOT ASSAY :-
TEST ENZYME BUFFER INHIBITOR
CONTROL 40 40 -
STRONG 40 28 12
MEDIUM 28 24 28
WEAK 12 28 40
SAMPLE NO TYPE OF INHIBITION
Seed- 1,4,7,8,11,9,23,24,26,27,42,43,15,19 * * *
25,30,31,28,43,3,5, * *
6,12,13,15,16,19,20 *
Flower-
1,2,8,10,20,23,27,30 * * *
5,6,7,11,43 * *
9 *
Leaves-
25,29,32,35,49, * * *
4,18,31,33,36,41,46,47,48,49 * *
26 *
*** = Strong Inhibitor
** = Medium Inhibitor
* = Weak Inhibitor
The extracts which show inhibitions for amylase are tested by DNSA assay for each sample two test are done. In first test extract were added after incubation and another, extract added before incubation. amylase inhibitory activity was assayed by measuring reeducation in maltose liberated by salivary amylase using DNSA reagent
AMYLASE INHIBITOR (DNSA)
TEST ENZ EXTRACT BUFF SUB EXTRACT DNSA
(ML) DILU
(ML) O.D
Control - - 1.0 0.5 Incubation for twenty min - 1 Bwb for 10 min 2 -
Test-1 0.4 - 0.5 0.5 0.1 1 2 -
Test-2 0.4 0.1 0.5 0.5 - 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
1 S2 0.53 0.62 L31 0.77 0.87
2 S3 0.49 0.39 L32 0.79 0.85
3 S5 0.44 0.50 L33 0.78 0.78
4 S7 0.84 0.81 L35 0.80 0.77
5 S8 0.97 1.02 L36 0.76 0.83
6 S9 0.85 0.85 L41 0.89 0.90
7 S10 0.83 0.81 L46 0.93 0.86
8 S12 1.15 1.19 L47 0.97 1.01
9 S13 0.86 0.84 L49 0.88 0.85
10 S16 0.84 0.84 F1 1.38 1.42
11 S19 0.85 0.85 F2 1.40 1.30
12 S24 0.62 0.55 F6 1.38 1.35
13 S26 0.75 0.58 F8 1.41 1.40
14 S27 0.56 0.43 F9 1.31 1.38
15 S28 0.96 0.78 F10 1.40 1.38
16 S30 1.01 0.97 F23 1.11 0.98
17 S31 0.65 0.59 F26 1.32 1.38
18 S4 0.76 0.73 F29 1.07 1.14
19 S42 0.63 0.53 F30 1.19 0.93
20 S43 0.58 0.67 F39 1.28 1.36
21 L4 0.80 0.74 F42 0.98 1.03
22 L18 0.78 0.79 F43 1.17 0.97
23 L26 0.77 0.74
24 L29 0.78 0.75
Amylase Inhibitor:-
S3, S24, S26, S27, S28, S31, S4, S42, L4, L26, L46, L49, F2, F23, F30, F43.
Presence of amylases in biological sample particularly from plant is a major problem in detection of amylase inhibitors. The detection of amylase inhibitors in sample containing endogenous amylases is possible. Endogenous amylase in sample can be detected. In one test tube DNSA are added before incubation and reaction are stopped. In another reaction are carried out. Difference between the O.D. indicate the concentration of endogenous amylase.
ENDOGENOUS AMYLASE :
TEST EXTRACT
(ML) BUFF.
(ML) DNSA
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 0.5 1 2 -
T1 0.1 0.5 1 0.5 - 2 -
T2 0.1 0.5 0.5 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
S3 0.80 0.93 L35 0.63 0.58
S24 0.90 0.94 L49 0.51 0.52
S26 1.26 1.29 L33 0.60 0.63
S27 0.81 1.31 L10 1.06 0.98
S30 1.40 1.49 F2 0.78 0.81
S43 1.40 1.53 F8 0.93 0.95
S5 0.89 1.20 F10 1.10 1.17
S42 0.51 0.84 F23 1.03 1.07
L4 0.41 0.80 F30 1.40 1.51
L26 0.50 0.58 F43 0.84 0.97
Amylase activator is also present within the extract. This can also detect. In one test tube extract were added after addition of DNSA and in another test tube extract were added before incubation. Differences between those indicate the presence or absence of amylase activator.
AMYLASE ACTIVATOR
TEST ENZ
(AMY) BUFF.
(ML) ACT.
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML) ACT.
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 - 0.5 1 - 2 -
T1 0.4 0.5 - 0.5 - 0.05 2 -
T2 0.4 0.5 0.05 0.5 1 - 2 -
SAMPLE T1 T2
L31 0.42 0.50
L32 0.52 0.54
L19 0.56 0.70
L25 0.60 0.58
S43 0.57 0.72
S12 0.98 1.09
S8 1.01 1.10
F9 1.29 1.41
Salivary amylase inhibitors in extract also separated by using starch polyacrylamide gel. But in many attempt the amylase inhibitor bands were not found clearly. The gels are incubate in the amylase solution, amylase hydrolyze the starch as it enters the gel. However, starch in the vicinity of amylase inhibitor band is protected from hydrolysis due to inhibition band is protected from hydrolysis due to inhibition of amylase and appears as blue band after staining with iodine. The size and intensity of blue bands correspond to the extent of inhibition of amylase which depends upon the concentration and activity of amylase inhibitor protein in gel.
This method is sensitive entire procedure takes about one hour after electrophoresis run. In the particle purification of samples by ammonium sulfate fractionation. Increase the possibility of band formation by enriching amylase inhibitory activity. Most of the amylase inhibitor is temp sensitive. So storage at cooling condition is necessary.
Albumins in the seed extract also inhibit starch-iodine complex formation by sequestering iodine and may be confuse for amylase activity bands. In the gel same time starch having the tendency to form clumps and precipitate during polymerization of gels. Slow polymerization and overnight keeping of gels prior to run help in even distribution of starch in the gel.
However, our attempts on visualizing amylase inhibitor band in starch poly acryl amide gas were unsuccessful.
REFRANCES
1. Alam n. And S, Gourinath, 2001. Substratre-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase / trypsin inhibitor (RATT) and various N-terminal fragments. Biochem, apr. 10:40: 4229-33
2. Barbar D, Sanchez, R. Mong, I. Gomez and G. Salcedo 1989, FEBSKett, 248:119-122.
3. Brieteneder, H and C.A. Radauer, 2004, Classification of plant food allergens. J. Allergy and Clin. Immunol. May 113:821-830.
4. K. Murayama R, Dimango EP, Character of a wheat amylase inhibitor preparation and effects on fasting human pancreaticobliliary secretions and hormones. Gastroenterology 1996:111:1313-20
5. M.M Pichare and M.S. Kachole. J. Biochem. Biophys. Methods 28 (1994) 215-224
6. CM, Purification and properties of phaseolamin , an inhibitor of alpha-amylase, from the kidney bean, phaseolus vulgaris. J Biol Chem 1975:250:8030-7
7. Octavio, L. and D. Rigden, 2002. Plant amylase inhibitos and their interaction with amylases. Eur. J. Biochem. 269:397-412
8. Octiva L. And D. Rigden, 2000. Activity of wheat amylase inhibitors towards bruchid. Amylase and structural explanation of observed specificities. Eur. J. Biochem, 267: 2166-2173
9. Richardson, M. 1991, Methods in Biochemistry, volume 5, Academic press, pp:259-
I fee highly indebted to Dr. A.D. Chaugale, Dr. Baladhye and miss. Shaikh R.R. for her Valuable suggestion and instruction.
I must place on record my sincere and grateful thanks to Proff. M.T.Patil and Prof. Sable for their valueable contribution in naming and identification of plants.
I wish to express my profound gratitude to Prof. H.M. Humbe H.O.D. Chemistry for providing necessary facility during the entire course of this project.
I also express my sinsiour thanks to the teaching and non teaching staff of bio chemistry, micro biology, botony for their valuable suggestion and mindful help. Extended to me for getting the work done.
I would remiss if do not acknowledge my debit to my friend and class mate for their wonderful contribution and rendered to me during the experimental work.
No words and thanks to enough to express my heartfelt filling of love and indebtness to parent and brother for there sincere efforts in successfully keeping the best selling through most stable envoirment for their sacrifice in moulding me to a learn citizen
Mr. G.R. Sambare
ABSTRACT
Seed leaves and flower tissue were extracted in P.V.P. the extract were screen qualitatively dot-blot agar plate technique for amylase inhibitor were further analyzed for quantitative estimation of Amylase inhibitor activity by DNSA method. Endogenous amylase and amylase activator also present within the tissue. these are detected by developing specific protocol for each.
The inhibitors were separated in polyacrylamide gel containing 0.5% soluble starch by electrophoresis and visualized by incubation of the gel in salivary amylase solution and staining with iodine. Starch in the gel is hydrolise by amylase during incubation but starch in vicinity of amylase inhibitor is protected from hydrolysis and appear as the blue band after staining.
Keywords: - Amylase inhibitors, Enzyme, Endogenous amylase, Amylase activator, Electrophoresis,
INTRODUCTION
α-Amylase inhibitors:-
Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. These inhibitors can be grouped into six classes based on their tertiary structures. Lectin-like, knottin-like, cereal-type, kunitz-like, γ-purothionin like and thaumatin like inhibitors, some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites.
In animals, α-amylase inhibitors reduce the glucose peaks that can occur after a meal, slowing the speed with which α-amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extra cellular glucose from being cleared quickly from the blood. Therefore, diabetics tend to have low α-amylase levels in order to keep glucose levels under control, except after taking insulin, which causes a rise in pancreatic α-amylase.
Plants also use α-amylase inhibitors as a defense strategy. These inhibitors impede the digestive action of α-amylases and protinases in the insect gut, thereby acting as insect anti-feedants. As a result, α-amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes.( Marshal JJ, Lauda)
What do they do?
Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other, secondary, enzymes. They are claimed to be useful for weight loss. But when they were first developed years ago, research did not find them very effective for limiting carbohydrate absorption. Later, however, highly concentrated versions of amylase inhibitors did show potential for reducing carbohydrate absorption in humans.
Purified starch blocker extracts. When given with a starchy meal, have also been shown to reduce the subsequent rise in blood sugar levels of both healthy people and diabetics. The is effect could be helpful in the treatment of blood sugar disorders.( Choudhary A, Maeda)
Where are they found?
Amylase inhibitors can be extracted from several types of plants, especially those in the legume family. Currently available amylase inhibitors are extracted from either white kidney bean or wheat.
The relative inefficacy of alpha-amylase inhibitors in affecting human digestion of starch has been highlighted by reacent scientific and public controversy over the commercial sales of so-called starch-blockers or slimming pillis alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes obesity and hyperlipaemia studies of the structures of the numerous enzyme inhibitors found in cereal grains have led to the recognition of a super family of homologous proteins which includes inhibitors of alha-amylase, proteinase and bifunctional inhibitors of alpha-amylase, proteinase and bifunctional inhibitors active against two or more classes of enzymes.(Alam and Gourinath, 2001, Octavio and Rigden 2002 Richardson, 1991)
The first alpha-amylase inhibitor determined was that of the monomeric 13 kD known as 0.31 form, from wheat (kashlan and Richardson, 1981) other dimeric 0.19, 0.23, 0.28, 0.53 form of weat inhibitors of exogenous alpha-amylase were later shown (oneda et al 2004, kondo and ida1995, Roy and Gupta 2000, Richardson, 1991, Octavio and Rigden 2002)
The favored hyupotheses about physiological roles fo the enzyme inhibitors in seeds is that they act as storage or reserve proteins as regulators of endogenous enzyme or as defensive agents against the attacks of animal predators and insect or microbial pests. It seems likely that in certain species these proteins may fulfill a combination of these proteins may fulfill a combination of these function(Octavia and Rigden 2002, Octivio and Rigden 2000, Richardson 1991) Also plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plant against pests (Octavio and Rigden2002)
Nutritional and metabolic effects of enzyme inhibitors certainly some of inhibitors certainly some of inhibitors found in cereal and legume seeds can inactive the salivary and pancereatic enzymes of human (pick and wober) and their sucseotibility to be rather variable (singhand bbkundel, 2001) many are destroyed by cooking but some retain inhibitory activity even after baking (Richardson 1991)
Amylase inhibitors present in seeds currently used as food present few nutritional problem for healthy people but may have some toxicological significance in the diets if infants who have a lower production of pancreatic alpha-amylase than adults and for patients with impaired peptic or gastric function (brietender and rauduer ,2004. richrdson ,1991 shewry etal2001) also one inhibitor of insect alpha-amylase isolated from barley flour is the major allergen associated with bakers asthma disease (barber et al 1989) the inhibition is strictly competitive and in the1:1 complexes aloof the activities of the enzyme are completely abolished .
Crystallographic nuclear, magnetic resonance (NMR) and mechanistic studies all indicates that the inhibitors act as highly specific substrate for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond .
The reaction mechanics involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood (silan 1986) but there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the mechanism or that the inhibitor may induce confermational changes in the enzyme molecule.
Materials:-
Different part of the plants including leaves, flower, seeds were collected from local region. Some sample were collected from Nanded ,Rahuri,Pathardi total 146 sample were collected.
Start was from qualgen fine chemical company Bombay India
Polyvinylpolypurrolidone (P.V.P) was from Sigma
Human saliva was diluted and used as salivary amylase all other chemicals were of the highest purity available
LEAVES:
Sr. no Common name Botonical name
1 Sitaphal Annona sequmusa
2 Subhabhul Leueaene lapisiliqua
3 Errand Risinus comnuis
4 Naginiche pan Piper bettne
5 Bhabhul
6 Ghaneri Lantana camera
7 Gawti chaha Cymbopogan flexuosus
8 Aalu Calocasia esculanta
9 Rui Calotropis procera
10 Kadhipatta Murraya kotnigii
11 Gahu Triticum aestivum
12 Papai Papaya indica
13 Amba Mangifera indica
14 Mohair Brassia juncea
15 Naral Coccus nucifera
16 Jambul Syzium cumini
17 Aalu Leaves Calocasia esculanta
18 Harbara Cicer aeriantum
19 Dhol Amba
20 Nimbu Citrus cemani
21 Palak Spinach
22 Kadulimb Azadirecta indica
23 Tobacco Nicotiane tobacum
24 Tulas Ocimum Americanum
25 Tarwad Cassia auriculata
26 Kardali Canna indica
27 Wel
28 Nishigandha Polianthus tuberosa
29 Manjiri Ocimum Americanum
30 Tomato Lycopersicon escunentum
31 Beshram Ipomea
32 Sunflower Helianthus annus
33 Chandan Santalum album
34 Maka Zea mays
35 Halad Curcuma aromatica
36 Sadaba Runca chalepensis
37 Ratali Ipomeabatatas
38 Bartodi Morinda, citrifolia
39 Apte Bahunia
40 Palas Bulea monosperma
41 Biba Semecarpus anacardium
42 Rohini
43 Acasia Chandra
44 Agav Americana
45 Glyrisidium capium
46 Ipomea
47 Carissa carandus
48 Sag Tectona granvis
49 Maniplant
50 Cactus
51 Tur Cajanus cajan
52 Pimple
FLOWER:
1 Dhotra Datura alba
2 Jaswand Hibicus rosasinensis
3
4 Tarwad Cassia auriculata
5
6
7 Kaneri Nerium indicum
8 Zendu Tagehun erecta
9 Rose Red
10
11 Rui Calatropis procera
12 Chafa Michella champaka
13
14
15 Jai Jasminium auricylamn
16 Allamnda
17 Shewga Moringa olifera
18 Gandhar
19 Boganwel Boganvillia spectibilis
20 Kunda
21 Nishigandh Polyanthus tuberose
22
23 Gulbakshi Mirabilis jalapa
24 kardali Canna indica
25
26 Ghosali Lutta aegeyptia
27 Danger
28 Gerbera
29 Aboli Crossendra undilifolia
30 Sadafully Catharathus roses
31 Biliat
32 Tur Cajanus cajan
33 Shevari Sesbania sesban
34 Ghevda Psophocarpus tetiagomolobes
35
36
37 Palas Butea monosporne
38 Sunflower Tridax procambers
39
40 Khajawe
41 Sweet potato Ipomebatata
42 Kandhar
43 Lakh Acacia
44 Sag Tectona Gran
45 Chirisidium caprsium
SEED:
1 Mug Phaseomus aureus
2 Mahsoor Lens esculenta
3 Macca Zea maize
4 Chawali Vigna sinensis
5 Udit Phaseolus mango
6 Tur Cajanus cajan
7 watana Pisum Sativum
8 Hulga
9 Jawari Sorghum valgare
10 Tag
11 Methi Trigonella toenum
12 Ubal bee
13 Math
14 Soyabeen Glycine max
15 Karale
16 Kabuli Harbala Cicer grientum
17 Chinchuka Tamarindus indica
18 Airnda Ricimus communis
19 Tandul Orizha Sathiva
20 Bhagar
21 Dudhi Bhopla
22 Kharbuz Cacumis melo
23 Gokarn Clitonia kleio
24 Rale
25 Wange Solalum righhi
26 Khas khas
27 Shengadane Arachis hypogea
28 Coffee Caffia Arabica
29 Danger
30 Mohari Brassica kmphesis
31 Rajgira Amaranthus
32 Mire Piper nigrum
33 Rajma
34 Elayachi Elettoria cordomomum
35 Tulas Ocium santum
36 Hawari
37 Jira Caminum cyminum
38 Dhane Corriander sativa
39 Mirchi Capsium annum
40 Apte Bahunia racemose
41 Badishop Foenicumum valgari
42 Jawas Alhagi pseudalhagi
43 Bajari Pennisenem typhoids
44 Ghosale Laffer cylindrical
45 Sabja Ocimum bascilium
46 God babhul Accacia
47 Gahu Triticum aestivam
METHOD
Extraction of amylase inhibitors:-
extraction if amylase inhibitors leaves, flower, seed are done by same procedure.
1. Decorticated seeds of all sample were dried and ground in blender to obtain fine flour.
2. Leaves are crused making the fine as possible as in small amount of acetone.
3. Same procedure was used for the flower sample.
4. After removing pigments all the sample are kept in hexane for overnight to remove the fatty material.
5. This procedure was repeated 2 to 3 times.
6. Then samples are carefully dried and packed.
7. Proper method for numbering and identification was used
8. The defatted seed powder was stirred with 1% PVP in 1:5 ratio for 48 hour.
9. Inclusion of PVP helpful in removal of phenolics from extract.
10. The suspension was centrifuged on cooling centrifuged at 10000 rpm for 20 min at 4oC
11. addition of PVP and centrifugation was repeated twice for same sample.
12. The clear supernatant contained amylase inhibitors was stored in freeze and used for analysis.
(Here after mentioned as partially purified extract.)
DOT BLOT INHIBITOR ASSAY:-
1. Extracted sample was tested on agar-starch plat in different concentration for screnning.
2. Using the enzyme (Amylase) Buffer (6.8PH) inhibitor at different proportion dot-blot assay was done.
3. For that 2% agar and 1% starch was used.
4. These are screening method due to this reaction sample which show inhibition are repeated once again and confirm that they show inhibition for amylase.
5. Among the 146 sample 58 sample shown inhibitions.
6. In that these are further categorized in strong inhibitor, medium inhibitor, weak inhibitor.
7. In 146 samples 27 samples are strongly inhibitor, 22 samples are inhibitor, and 9 samples are weak inhibitor.
8. After the screening of 146 samples the extracts which show strong inhibition and medium inhibition was taken for their amylase inhibitor assay by DNSA.
AMYLASE INHIBITOR ASSY :
Amylase activity was assayed by measuring liberated maltose. amylase inhibitory ativity was assyed by measuring reduction in maltose liberated by salivary amylase using dinitrosalisylic acid reagent.
One amylase activity unit is defined as activity resulting in to liberation of 1 mg of maltose from starch at ph 6.9 at 37o C in 3 min.
one amylase inhibitor unit is one amylase unit inhibited under the given assay condition.
In this assay same extract showing the exact inhibition but here again same extract does not show inhibition.
The samples which show inhibition are again tested for the endogenous amylase and amylase activator.
ENDOGENOUS AMYLASE:-
1. Presence of amylases in biological sample particularly from plant case a major problem in detection of amylase inhibitor. The detection of endogenous amylase is possible with DNSA method.
2. In that tow test are taken in one reaction was arrested by addition of DNSA and in anther the reaction are carried out complete and then DNSA was added.
AMYLASE ACTIVATOR:-
Amylase activator also present within the extract there can be detected by DNSA.
In one test tube enzyme, buffer and substrate are added and in another activator was added with enzyme, buffer substrate. Incubate the both test tube and add DNSA activeter was add in first test tube after incubation the difference in OD indicated the amylase activator.
ELECTROPHORETIC SEPRATION OF AMYLASE INHIBITOR :-
Amylase inhibitors in the partially purified extract were analyzed on a vertical slab gel electrophoresis system in 7% polyacrylamide gels containing 0.5% soluble starch without stacking gels containing 0.5% soluble starch without stacking gel. For that tris-glycine (PH 8.9) both in gel and electrode tank was used.
VISUALIZATION OF AMYLASE INHIBITORS:-
After electrophoresis gels are placed in 20mm phosphate buffer (PH 6.9) containing 6.7mm NaCl for 5-10 mm for equilibration and incubated in salivary amylase in phosphate buffer (PH 6.9) for 30 min at 37 C. after incubation the gels are wash with D/W and placed in iodine solution. (10 MM iodine in 14mm KI) for 4 to 5 min.
Exact band of amylase inhibiter way not found.
RESULT AND DISCUSSION:-
Amylase inhibitors are detected by using three different concentrations the strong inhibitor show the inhibitor at all three concentration. Where the weak inhibitor show inhibition only at low concentration of amylase by using this technique determination of amylase activator is also possible. Same extract were help to the activation. amylase hydrolyzes the starch present in the agar. After staining with iodine. It show white area indicate that it is not inhibitor. When these area are broad than the control, then it is activator
DOT-BLOT ASSAY :-
TEST ENZYME BUFFER INHIBITOR
CONTROL 40 40 -
STRONG 40 28 12
MEDIUM 28 24 28
WEAK 12 28 40
SAMPLE NO TYPE OF INHIBITION
Seed- 1,4,7,8,11,9,23,24,26,27,42,43,15,19 * * *
25,30,31,28,43,3,5, * *
6,12,13,15,16,19,20 *
Flower-
1,2,8,10,20,23,27,30 * * *
5,6,7,11,43 * *
9 *
Leaves-
25,29,32,35,49, * * *
4,18,31,33,36,41,46,47,48,49 * *
26 *
*** = Strong Inhibitor
** = Medium Inhibitor
* = Weak Inhibitor
The extracts which show inhibitions for amylase are tested by DNSA assay for each sample two test are done. In first test extract were added after incubation and another, extract added before incubation. amylase inhibitory activity was assayed by measuring reeducation in maltose liberated by salivary amylase using DNSA reagent
AMYLASE INHIBITOR (DNSA)
TEST ENZ EXTRACT BUFF SUB EXTRACT DNSA
(ML) DILU
(ML) O.D
Control - - 1.0 0.5 Incubation for twenty min - 1 Bwb for 10 min 2 -
Test-1 0.4 - 0.5 0.5 0.1 1 2 -
Test-2 0.4 0.1 0.5 0.5 - 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
1 S2 0.53 0.62 L31 0.77 0.87
2 S3 0.49 0.39 L32 0.79 0.85
3 S5 0.44 0.50 L33 0.78 0.78
4 S7 0.84 0.81 L35 0.80 0.77
5 S8 0.97 1.02 L36 0.76 0.83
6 S9 0.85 0.85 L41 0.89 0.90
7 S10 0.83 0.81 L46 0.93 0.86
8 S12 1.15 1.19 L47 0.97 1.01
9 S13 0.86 0.84 L49 0.88 0.85
10 S16 0.84 0.84 F1 1.38 1.42
11 S19 0.85 0.85 F2 1.40 1.30
12 S24 0.62 0.55 F6 1.38 1.35
13 S26 0.75 0.58 F8 1.41 1.40
14 S27 0.56 0.43 F9 1.31 1.38
15 S28 0.96 0.78 F10 1.40 1.38
16 S30 1.01 0.97 F23 1.11 0.98
17 S31 0.65 0.59 F26 1.32 1.38
18 S4 0.76 0.73 F29 1.07 1.14
19 S42 0.63 0.53 F30 1.19 0.93
20 S43 0.58 0.67 F39 1.28 1.36
21 L4 0.80 0.74 F42 0.98 1.03
22 L18 0.78 0.79 F43 1.17 0.97
23 L26 0.77 0.74
24 L29 0.78 0.75
Amylase Inhibitor:-
S3, S24, S26, S27, S28, S31, S4, S42, L4, L26, L46, L49, F2, F23, F30, F43.
Presence of amylases in biological sample particularly from plant is a major problem in detection of amylase inhibitors. The detection of amylase inhibitors in sample containing endogenous amylases is possible. Endogenous amylase in sample can be detected. In one test tube DNSA are added before incubation and reaction are stopped. In another reaction are carried out. Difference between the O.D. indicate the concentration of endogenous amylase.
ENDOGENOUS AMYLASE :
TEST EXTRACT
(ML) BUFF.
(ML) DNSA
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 0.5 1 2 -
T1 0.1 0.5 1 0.5 - 2 -
T2 0.1 0.5 0.5 1 2 -
SAMPLE T1 T2 SAMPLE T1 T2
S3 0.80 0.93 L35 0.63 0.58
S24 0.90 0.94 L49 0.51 0.52
S26 1.26 1.29 L33 0.60 0.63
S27 0.81 1.31 L10 1.06 0.98
S30 1.40 1.49 F2 0.78 0.81
S43 1.40 1.53 F8 0.93 0.95
S5 0.89 1.20 F10 1.10 1.17
S42 0.51 0.84 F23 1.03 1.07
L4 0.41 0.80 F30 1.40 1.51
L26 0.50 0.58 F43 0.84 0.97
Amylase activator is also present within the extract. This can also detect. In one test tube extract were added after addition of DNSA and in another test tube extract were added before incubation. Differences between those indicate the presence or absence of amylase activator.
AMYLASE ACTIVATOR
TEST ENZ
(AMY) BUFF.
(ML) ACT.
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML) ACT.
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 - 0.5 1 - 2 -
T1 0.4 0.5 - 0.5 - 0.05 2 -
T2 0.4 0.5 0.05 0.5 1 - 2 -
SAMPLE T1 T2
L31 0.42 0.50
L32 0.52 0.54
L19 0.56 0.70
L25 0.60 0.58
S43 0.57 0.72
S12 0.98 1.09
S8 1.01 1.10
F9 1.29 1.41
Salivary amylase inhibitors in extract also separated by using starch polyacrylamide gel. But in many attempt the amylase inhibitor bands were not found clearly. The gels are incubate in the amylase solution, amylase hydrolyze the starch as it enters the gel. However, starch in the vicinity of amylase inhibitor band is protected from hydrolysis due to inhibition band is protected from hydrolysis due to inhibition of amylase and appears as blue band after staining with iodine. The size and intensity of blue bands correspond to the extent of inhibition of amylase which depends upon the concentration and activity of amylase inhibitor protein in gel.
This method is sensitive entire procedure takes about one hour after electrophoresis run. In the particle purification of samples by ammonium sulfate fractionation. Increase the possibility of band formation by enriching amylase inhibitory activity. Most of the amylase inhibitor is temp sensitive. So storage at cooling condition is necessary.
Albumins in the seed extract also inhibit starch-iodine complex formation by sequestering iodine and may be confuse for amylase activity bands. In the gel same time starch having the tendency to form clumps and precipitate during polymerization of gels. Slow polymerization and overnight keeping of gels prior to run help in even distribution of starch in the gel.
However, our attempts on visualizing amylase inhibitor band in starch poly acryl amide gas were unsuccessful.
REFRANCES
1. Alam n. And S, Gourinath, 2001. Substratre-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase / trypsin inhibitor (RATT) and various N-terminal fragments. Biochem, apr. 10:40: 4229-33
2. Barbar D, Sanchez, R. Mong, I. Gomez and G. Salcedo 1989, FEBSKett, 248:119-122.
3. Brieteneder, H and C.A. Radauer, 2004, Classification of plant food allergens. J. Allergy and Clin. Immunol. May 113:821-830.
4. K. Murayama R, Dimango EP, Character of a wheat amylase inhibitor preparation and effects on fasting human pancreaticobliliary secretions and hormones. Gastroenterology 1996:111:1313-20
5. M.M Pichare and M.S. Kachole. J. Biochem. Biophys. Methods 28 (1994) 215-224
6. CM, Purification and properties of phaseolamin , an inhibitor of alpha-amylase, from the kidney bean, phaseolus vulgaris. J Biol Chem 1975:250:8030-7
7. Octavio, L. and D. Rigden, 2002. Plant amylase inhibitos and their interaction with amylases. Eur. J. Biochem. 269:397-412
8. Octiva L. And D. Rigden, 2000. Activity of wheat amylase inhibitors towards bruchid. Amylase and structural explanation of observed specificities. Eur. J. Biochem, 267: 2166-2173
9. Richardson, M. 1991, Methods in Biochemistry, volume 5, Academic press, pp:259-
project on woodely land
DECLARATION
I do herby declare that this project titled “Detection of Enzyme Amylase From Woolly Aphids”. Submitted by me to the department of Chemistry New Arts, Commerce and Science College, Ahmednagar is the product of my own efforts. To the best of my knowledge and belief that this report is based on reliable scientific measurements. This dissertation has not been submitted to any university for the award of the any degree.
Date:- Chopde R.M.
M.Sc.-II
(Bio Chemistry)
New Arts, Commerce
& Science College,
Ahmednagar.
Place:-Ahmednagar
ACKNOWLEDGMENT
It is indeed a great pleasure and moment of immense satisfaction for me to express my deep sense of gratitude towards project guide Dr.M.V.Padul for ken inspiring interest and Dr. A.D. Chaugale invaluable guidance at each stage, during the entire course the entire course of my project.
I am grateful to Prof. Humbe A.M.head of chemistry department, for providing the requisite laboratory facilities without which this project could not have taken the present shape.
I am grateful to Prof. Mr.Bladye & Prof. Mrs. Shaikh R.R. my classmates and laboratory assistants for their timely help and suggestions.
Last but not the least I thank my parents my friends for their everlasting support in all my endeavors.
Mr. Chopde Ramakant Maruti.
INDEX
SR.NO. TOPIC PAGE NO.
1 Abstract
2 Introduction
3 Life Stages of C. lanigera
4 Material and Method
a) Apparatus
b) Chemical
c) Method
1. Agar diffusion assay technique
2. Comparative study between woolly aphide. Fungal, salivary, amylase at different pH tem. By DNS method
3. Biogel chromatography
4. Pricipitation Polyacrylamide gel electophoresis & Agrose gel Electrophoresis.
5 Result and Discussion
6 References
ABSTRACT
The amylase activity of woolly acid aphids was determine by agar plate diffusion assay method. Amylase activity was also determined different pH by agarplate diffusion method. Comparative study between woolly aphids, salivary, fungal amylase was the different pH-temp. by chemical method that is by DNSA method.
Protein concentration determined by Lawry method. substrate concentration also determine. Biogel chromatography was also done precipitation was carry out higher activity shown by fraction was used to determine further study. Pattern of protein was determine by poly acrylamide gel electrophoreses and agarosse gel electrophoresis.
NOT AFFECTED LESS AFFECTED HIGHLY AFFECTED
INTRODUCTION
Sugar woolly afide has been recently reported inout break proportion from westerm and southern India. Through the pest was first reported from west bangol in 1958 and later from other part of India. Resent survey in part of past affected area of maharashtra and Karnataka.
Sugar is a universal sweetening agent and sugarcane (Saccharum affleinarum L.) is the primary age old source of it.It is mainly cultivated in tropics in the world, but in India it is grown even under subtropical areas.
Sugarcane occupies an area of 42.97 lakhs hectares with a production of 278.56 mt(Anonymous, 2003a) the area in Maharashtra was 5.9 lakhs hectares and annual production of 45.14 mt (Anonymous, 2003 a.) Thus Maharashtra account for 15.4 % of the total production in India.
It is a prerennial crop and occupies the land for 12-18 month. It is propagated by setts. Planting of sugarcane is done in 3 seasons, viz. Suru ( Dec-jan plating), adsali ( June-July planting) and preseason ( Oct.-Nov.planting) in The crop requires a long warm growing season with adequate amount of rainfall, Maharashtra. a fairly sunny and cool conditions, nut frost free condition during ripening and haresting freedom from stormy and severe winds.
Sugarcane is damaged by about 288 species of insects and non insets (David and Nandagopal 1986) and tissue borers, whte grubs,white flies, rodends, mealy bugs, pyrilla, scale insects etc. are of an economic
Important. Avasthy (1977) reported losses to the extent of 20 and 15 percent in cane yield and sugar recovery, repectively, due to the ravages of the pests. Recently,in Maharashtra.state during July-2002,an epidemic of sugarcane wooly aphid, Ceratovacuna Lanigera Zehnter was noticed in sangli, kolhapur and Satara districts and later on spread in parts of Solapur, pune and Ahmednagar districts. By September, 2002 it spread to neighboring Karnataka state. (Anonymous,2003b0 upto February 203, 1.32 lakh hectares of sugarcane crop was found affected by this pest in maharashtra.
In other countries, ceretovacuna lanigera (Takara and Auma, 1968, hill, 19930 C japonica (Anonymous, 202 and C. graminum (David and nandagopal, 1986) are the common species of white woolly sugarcane aphids damaging the sugarcane. In Indonesia,Farina (19940 observed the loss due to heavy incidence of C. lanigera by 26% in sugarcane yield and 24% in sugar content.
NATURE OF DAMAGE:-
Initial aphid infestation was seen on the under surface of leaves along the midrib and then over the entire under surface, covering it with flocculent, waxy secretion. Co-pious honeydew excretion often covers the entire upper surface of the leaves, leading to growth of sooty molded. (lopez A.W.) and continuous infestation leads to reduction in the length. Circumference, weight and sugar content of the stalk, and in susceptible varieties in vienam,( Vietnam 1963, P 266) loss in tonnage as well as sugar recovery (Tripathi G.M.) Gupta and Goswami assessed the effect of 25 and 100% alphid infested leaves on some yield and quality parameters of sugarcane and found that 100% infested had detrimental effects on the length (11% reduction), girth (3.5% reduction) weight (16.6 % Reduction) length of internodes (18.4% reduction) and width of leaf (4.9% reduction) juice quality parameters also exhibited considerable reduction. The percent reduction in sucrose, brix, glucose, purity and commercial cane sugar (CSS) was 53.3. 32.3, 25.3, 31.7, and 64.0 respectively. Preliminary studies on loss estimation being conducted at University of agricultural Sciences, Dharwad indicate adverse effects of aphid infestation on yield and quality parameters(Patil R.K. Lingappa S.)
LIFE STAGES OF C. LANIGERA
Systematic position:
Scientific name of species : Ceratovacuna Lanigera zehnt.
Family : Pomphigidae.
Superfamily : Aphidoidea
Order : Homoptera
English (Common) name : White Woolly
Marathi (Local) Name : Pandhara Lokari Oosmava
Obeservation on duration of different life stages of C.I. lanigera are follows
First instar nymph
The first instar nymphs are yellowish or greenish yellow in colour. It is very active and moves fast on the surface of leaf when exposed to sunlight. A pair of hollow tube is present at the posterior end of the abdomen oat the firth segment and is mainly used to secrete the defensie fuluid. The average length of the 1st instar was 0.87 mm and the width was 0.36 mm. ghosh (1974) reported that early instar nymph length ranges from 0.82-096 mm and width ranges from 0.3-0.45 mm.
Table duration of nymphal instars of C lanigera
Nymphal instars (Days)
Total
Duration
(Days)
I
II
III
IV
6 7 7 9 29
Patil (*20020 reported that first instar nymph length averaged 0.77 mm and width between 0.27-0.38 mm. the average duration of the first instar nymph was found to be 6.33 days.
Second instar nymph
The second instar nymphs are found to be less active and brownish in colour with black marking on the body. The white wooly filaments are absent. The average length of the 2nd instar was 1.17 mm and was 0.57 mm.
Patil (2002) reported that second instar nymph length as 1.23 mm and width ranged between 0.3-0.46 mm.
The duration of the second instar was found to be 6.66 days.
Third instar nymph
The third instar nymph is partially convered by white woolly filaments. These filaments are dense at the posterior end of the abdomen and also covered thorax portion but not head. The white wooly filaments are very dry, soft, nonpolar and dydrophobic. The average length length of the third instar nymph was 1.81 m and width was 1.05mm.
Patil(2002) reported the average length of this instar nymph as 1.83 mm and width as 0.46-1.07 mm.
The duration of this instar was found to be 7.99 days.
Fourth instar nymph
The fourth instar is covered with dense wooly filaments. They are sedentary on the leaf. A four segmented antenna is seen which was very short and measured 0.15 mm length. The length of fourth instar nymph was 1.98 mm and width was 1.02 mm.
Patil (2002) reported that length of fourth instar nymph as 2.09 mm and width as 0.47-0.78 mm.
The duration of this instar nymph was found to be 8.22 days.
Adult
The alate form adult emerged after fourth moult is blackish in colour, two pairs of transparent wings with clear wing venation are observed. Wings expanse of female is 2.7 mm in length and 1.2 mm in width. Adult possesses a pair of compound eyes. The winged adult was 2.00 in length and 1.0 mm in width.
Apterous forms lived for 30 days and alate forms lived for 10 days. The alate forms were responsible for the spreads for colony to new areas.
Pan and yang (1989) reported the longevity of adult from 32 to 57 days. Cheng et al. (2000) reported the longevity of adults as 20.5-24.1 days.
Life span
The nymphs undergo 4 instars and average of 29 days was required to complete the four instars on potted sugarcane plants. Total adult longevity was 30 days in case of apeterous aphids and 10 days in case of alate forms.
However, change et. Al. (20000 reported that a period of 15.8 to 16.5 days was required to complete nymphal stages.
Patil (20000 reported that life cycle is completed in one month period.
Sex Ratio
A number of aphids were critically observed and it was found that no sexual were recorded in the colony. The colony consists of females only and this was responsible for rapid increase in number.
Ghosh (1974) reported that no sexual were reported and the results were found to be in conformity with it.
In view of the above, it became indispensable to have a through knowledge of the pest so as to frame effective control measures against it. The available literature showed that so far into much work has been reported on this pest in India. It was therefore felt necessary to enzymatic study of woolly aphid
MATERIALS AND METHODOLOGY
Materials:-
A) Apparatus:-
1. mortar & pestle
2. Knife or Sissor
3. Petri Plates
4. Measuring Cylinder
5. Conical Flask
6. Beakers
7. Test Tube
8. Colorimeter
9. Spectrophotometer
10. Cold Centrifuge
11. Refrigerator
12. Ependrop tube
13. pH meter
14. Micropipett
B) Chemical:-
1. Soluble starch powder
2. agar Powder
3. Dintiro Salicylic Acid
4. Naoh
5. Glycine
6. Sodium Acetate
7. acetic Acid
8. Monobasic Sodium Phosphate
9. Dibasic Sodium Phosphate
10. Biogelp100
11. Disterase
METHODS:-
Collection of woolly aphids
Woolly aphids were collected from the rahuri sugar cane fields which were affected by woolly aphids. After collection of the woolly aphids were separated from the infected leaf and taken into separate dish. Then woolly aphids are preserved deepfreeze.
PREPARATION OF DIFFERENT PH BUFFERS:-
1. acetate buffer ( pH 4,5)
2. Phosphate buffer (pH 6,7,8)
3. Glycine NaOH buffer (pH 9,10)
EXTRACTION OF ENZYME FROM WOOLLY APHIDS:-
For this experient the 2 gm of woolly aphids is taken in seprate dish and 10 to 15 ml of buffer solution is added then the content is crushed in mortar pastle and the pest is prepared. Then after complete homogenation the sample is cold centrifuged the supernantant obtained is used for the further study of enzyme analysis. If some turbidity was observed then supernatant again centrifuge at high speed about 15000 rpm and time 30 min. the super natant filter by whatman filter paper.
CHECKING AMYLASE ACTIVITY BY DIFFUSION ASSAY TECHNIQUE:-
Starch and agar (3gm + 1.5 gm ) was taken in 100 ml distil water. Heat the solution and pour in to the petriplates. When the medium was solidified. After solidification the wells were prepared with the help of borer. After same time sample was added different wells with different pH. Enzyme extract then incubate 1 hour. The iodine solution was added in the petridish for staining after 4-5 min. the petridish is washed with D/W 3-4 times. Then incubating the petridish for 1 hour. The activity of aylase was seen at alkaline pH.
EXTIMATION OF AMYLASE ACTIVITY BY DINTRO SALICYLIC ACID METHOD REAGENT:-
a) Dinitro salicylic acid reagent (DNSA)
Dissolved 1 gram DNSA + 200 mg crystal phenol & 50 mg Sodium sulphate in 100 ml 1 % NaOH stored at 40 C.
Method:-
For estimation of amylase activity by DNSA method the starch is taken as substrate.
For the enzyme amylase, the activity of enzyme is estimated by checking optical density on the colorimeter.
REAGENTS TEST CONTROL
Enzyme extract 0.5 ml 0.00ml
Substrate (Sttarch) 1.00 ml 1.00 ml
Distilled water 0.00 ml 05 ml
Incubate at 37 c for 30 minutes
DNSA 1.00 ml 1.00 ml
Keep in boiling water bath for 10 minutes
After all addition ckecked the optical density on the colorimeter at 530 mm.
BIOGEL CHROMATOGRAPHY:-
Take 5 gm of biogel in 100 ml distil water and kept for one hour for the purpose of swelling. The coloum was properly cleaned with distil water the glass wool was wetted. And pour into the coloum with the help of long glass rod. The biogel was added to the coloum. The distil water was slowely added setting of gel at the bottom of coloum. The coloum was equillibriated with equilibration buffer pH-8 (phosphate buffer)
The one ml of crude sample is loaded on coloum with the help of pipette. The flow rate was adjested to 2 ml per 5 min. phosphate buffer was added help of pipette when fraction was collected. Each ependrop tube 2 ml fraction collected. Activity of each fraction was measured by DNSA method and protein concentration also measured.
Some number of fraction get highest activity they get separated from other and acetone preptation was carried out.
ACCETONE PRICIPITATION:-
The equal volume of sample and chilled acetone was mixed by drop by drop. After mixing the sample were placed in freeze for over night. The next day the sample were centrifugal at 12000 rpm for 15 min at 100 C. the supernatant was discarded and pellet was collected by dissolving 1 ml buffer and sample used as electophorotic study.
Reagents:
1. Stock buffer for resolving gel (1.5 M Tris HCL pH 8.8) :-
Tris-hydrozymethyl aminomethane (23.64g) was dissolved in 50 ml distilled water and the pH adjusted to 8.8 by adding conc. HCL drop wise the final volume was made up to 100 ml and the solution was stored in refrigerator.
2. Stock buffer for stacking gel (0.5 M Tris-HCL, pH 6.8)
Tris (7.88g0 was dissolved in 50 mlo of distilled water and the pH was adjusted to 6.8 by adding conc. HCL drop. Final volume was made to 100 ml using distilled water.
3. Electrode (tank) buffers (pH8.3):-
9.0g Tris, 42.3 g glycine were dissolved in distilled water to make 3 liters (electrode buffer was used for 2-3 subsequent runs)
4. Staining Solution:-
10 ml of 1 per cent coomassie blue prepared in methanol solution starining solution was filtered two to three times before use.
5. 30% acryl amide for stacking gel:-
75g acryl amideand 2 g bis-acryl amides were dissolved in distilled water. Volume was made to 250 mlo by addition of istilled water.
Apparatus & equipments :-
1. The vertical slab gel electrophoresis apparatus of atto with 16*16 cm glass plates.
2. Gasket.
3. Well comb & power supply.
Chemicals:-
The following chemicals (analytical grade) ware used for preparation of different solution
1. Acryl amide ”Electrophoresis grade “
2. Bisacreylamide”Electrophoresis grade “(N’N’ methylene bisacrlymide)
3. Tris ( hydroxymethyl aminomethane)
4. Glycine
5. HCL
6. Glycerol
7. Ammonium persulphate
8. TEMED (N, N, N’N’ tetranmethylmethylene diamine)
9. glycial acetic acid
10. Coomassie brilliant blue
11. bromophenol blue
Preparation of Gel
The gel plates ware cleaned and dried and cassettes ware assembled as per the instruction.
1. Separating Gel
The following solution ware mixed (10%)
Buffer pH 8.8 :7.5ml
Water : 12.3ml
30% running gel acrylamide : 9.9ml
5% ammonium persulphate : 0.15ml
TEMED : 0.02
(TEMED was added just before pouring gel)
All the reagents were mixed well and poured between the plates of the cassette.care was taken, so that no air bubble was trapped in the gel solution. The cassettes was filled ¾ with 3-4 cm left from the top the polymerized gel solution was overlayed with butanol and the gel was allowed to polymerize (it took about 30-60 min)
2. Stacking gel (4%)
After the polymerization of the separating gel the butanol layer was removed using filter paper and the cassette was blotted. The following stacking gel mixture was carefully poured into it.
Tris buffer 6.8 : 1.25 ml
Water : 3.075 ml
30% stacking gel acryl amide : 0.67 ml
10% ammonium per sulphate : 0.025 ml
TEMED : 0.005 ml
(TEMED was added just before pouring the gel)
After puring the stacking gel solution an acrylic comb having 11 teeth or wells was set without trapping any bubble. The gel was allowed to polymerize (it look about 10-15 min,)
After polymerization of gel the comb was carefully removed from the gel without disturbing the shape of wells. The sample wells were washed with tank buffer to remove the unpolymerised material. The tank filled with an appropriate volume of tank buffer solution.
ELECTROPHORESIS:-
The gel casstte was fixed into the electrophoresis unit. An aliquot soluble protein (enuivalent of 100 mg) was loaded to each well w8ith help of micro syringes. The gel plates were fixed over cathode chamber cum heat exchange with clamps and screws. The cathode chamber was placed into the4 tank. The upper tank was filled in with power supply with the anode connected to the lower reservoir and cathode to upper reservoir. The electrophoresis was conducted at 35 mA till the samples migrate into the running gel and subsequently at 60 mA.
The unit was disconnected from the power supply and cassette was removed from the electrophoresis tank as soon as the tracking dye reached the bottom of gel.
Fixing and staining the gel:-
The cassette was removed from the unit and the gel was taken out gently. Gel was placed in a staining tray and sufficient staining solution was poured of cover the gel uniformly. It was incubated for 6 hrs. And the stain was decanted, rinsed with water. To clear the gel back ground detaining in water was followed for a day or two. The pattern of staining drawn as well as photographed.
Agarose gel electropheresi:
Principle:- Agarose gel electophoresis refers to movement of charged particle/ molecules under the influence of electric field.
Agrose gel is more porous and used to separation of large particles. In this method charge molecules moves to the charged when electric field supplied. This movment is depend on the size of molecules and molecular weight smaller size protein molecules move faster than larger size molecules.
Procedure:-
1. Phosphate buffer (0.2 m) pH-8 is prepared about 200 ml with distil water.
2. 0.5 gm of agarose was dissolved in 50 ml distill water.1% agrose gel from it dissolved by heating to form clear solution.
3. Above containt cooled at 600 C and pored in to tray and comb was inserted in to it. Precautions were taken that no air bubble should enter.
4. Gel thickness were made up to 4 to 5 mm. and agarose solution were poure and allowed to cool comb was removed gentle when agar solidified.
5. phosphate buffer poured into tank to immerse the gel by about 5 ml
6. Power set was connected.
7. sample is loaded in well in derived order
8. Voltage was set to 50 V and switch was start of the power supply.
9. Electrophoresis was run until dye migrate to the other end of the gel ( approx.1 hr)
10. Power supply was turn off. The agrose gel was removed from the electro phorsis tank.
11. Kept in to the staining solution for 1 to 2 hr.
12. It was distained by water or solution.
13. the band pattern was observed
RESULT AND DISCUSSION:-
1. Amylase activity
pH7 pH8 pH9
The enzyme amylase present in woolly aphides activity is checked at pH 4, 5,6,7,8,9,10 woolly aphids amylase shown activity in alkaline pH 7,8,9 but pH-8 show maximum activity. pH-8 get maximum zone of activity.
2. Protein Concentration
NO ENZYME(ml) WATER(ml) LAWARYREAG. F.P. O.D.
Reg. constant one 0.0 1 3 0.5 0.0
Enzyme const. 2 0.1 4.4 0.0 0.0 0.2
Test 3 0.1 0.9 3 0.5 0.34
Test 4 0.1 0.9 3 0.5 0.34
O.D. = Test – Enzyme control
= 34-2
O.D. = 32
O.D. plotted on the standard graph
1 ml sample = 124mg/ml
But sample is diluted with distil water up to 10 ml.
1 ml = 124 µg X 10
I.e. 1 ml = 1240 µg/ml
I.e. 1 ml = 1.245 mg/ml
1 ml = 0.00124 gm/ml
i.e. 1 gm woolly aphide contain = 0.00124 X 10
i.e. 1 gm contain = 0.0124 gm/ml
3. AMYLASE ACTIVITY BY DNSA METHOD:-
Standard Graph for Activity by using DNS method.
O.D.
Constration of Maltose (µgm)
A) Substrate Concentration:-
O.D.
Substrate Cons.
Substrate conc in % O.D Activity(µgm/ml/min)
2 0.73 96
1.8 0.73 96
1.6 0.73 96
1.4 0.73 96
1.2 0.66 176
1 0.54 144
0.8 0.48 128
0.6 0.34 89.33
0.4 0.27 70.33
0.2 0.18 48
Maximum substrate concentration for woolly aphids amylase is 1.2%
Graph shown increase any activity with increase in substrate const. but further steady state observed.
B) Different pH with comparative study between salivary, fungal, woolly aphides amylase
pH Woolly Aphid Activity(µgm/ml/min) Fungel Activity(µgm/ml/min) Salivary Activity(µgm/ml/min)
4 0.18 48 0.64 171.66 0.65 173.33
5 0.41 109.33 0.79 212 0.7 186.60
6 0.58 154.66 0.8 213 0.8 213
7 0.64 171.66 0.61 162.66 0.65 173.33
8 0.71 189.33 0.43 154.66 0.6 160
9 0.42 105.33 0.16 41.33 0.55 146.66
10 0.36 96 0.15 40 0.51 122.66
In above graph shows woolly aphids amylase show maximum activity at pH-8 salivary, fungal amylase shown maximum activity at pH-6.
The graph shown the woolly aphides amylase is different from salivary and fungal amylase.
C) Different temperature with comparative study between woolly aphids salivary, fungal amylase.
Temp Woolly Aphid Activity(µgm/ml/min) Salivary Activity(µgm/ml/min) Fungel Activity(µgm/ml/min)
20 0.16 41.33 0.22 60 0.24 64
30 0.2 53.33 0.24 64 0.24 64
40 0.22 60 0.28 74.66 0.28 74.66
50 0.18 48 0.26 69.33 0.25 66.66
60 0.16 41.33 0.19 50.66 0.21 56
70 0.11 28 0.16 41.33 0.18 48
The woolly aphids, salivary, fungal amylase shown maximum activity at 40o C temperature but at higher temperature suddenly decrease the activity. It clear that enzyme is not thermos table..
4) BIOGEL CHROMATOGRAPHY:-
The above graph peak has shown maximum activity. These fractions is collected and carry out acetone precipitation.
5) ELECTROPHORESIS STUDY:-
Crude sample
Band Accetone Preciption Sample
band
a) Electrophoresis pattern of protein is woolly aphide shown only one band in chromatography fraction but crude sample get diffused pattern of band.
b) Agarosegel Electrophoresis:-
Crude Sample Band with
Diff Concentration
in agarosegel Electrophoresis load crude sample but not get clear band some diffused band observed
6) Molecular weight determination:-
REFERANCE
1. Ghosh A.K, Homoptera: Aphidoidea, 4, Subfamily phloemyzinae, Anoeciinae and Hormaphidinae. In the fauna of India and adjacent countries, Zoological survey of India, kolkata, 1988 p 429
2. Lingappa, S. Kulkarni. K.A. Patil R.K. Thippanavar, P.S. and shekhappa, status of woolly aphid ceratovacuna lanigera zehntner on sugarcane in Karnataka. Paper presented at brain storming session on sugarcane woolly aphid, 10 june 2003.
3. Gupta, M.K. and Goswami, P.K. incidence of sugarcane woollyu aphid and its effect on yield attributes and juice quality. Indian sugar, 1995, 44, 883-885. Lopez, A.W.Ann. Rep. Res. Bur. Philipp. Sugar assoc, Entomology department report, 1931, p 273
4. Anonymous, annual work progress report on crop improvement program of rice, sugarcane, vegetable and field crops, directorate of rural Affairs, Vietnam, 1963, p, 263
5. Tripathi, G.M. Record of parasite and predator complex of sugarcane woolly aphide, ceratovacuna lanigera Zehnter in Nagaland India sugar, 1995, 44, 883-885.
6. Raychaudhari. D. N. food plant catalogue of India aphidede, aphidological socity of India, kolkata, 1984, p 188.
7. Mote U.N. & Puri.S.N., present status of sugarcane woolly aphid, ceratovacuna lanigera, a new pest problem on sugarcane in maharashtra. Paper presented at brain stroming.
8. David & Nandagopal, study of pest problem on sugarcane in India 1986
9. Avasthy, Study on losses percent in cane yield and sugar recovery due to the ravage of the pests.
I do herby declare that this project titled “Detection of Enzyme Amylase From Woolly Aphids”. Submitted by me to the department of Chemistry New Arts, Commerce and Science College, Ahmednagar is the product of my own efforts. To the best of my knowledge and belief that this report is based on reliable scientific measurements. This dissertation has not been submitted to any university for the award of the any degree.
Date:- Chopde R.M.
M.Sc.-II
(Bio Chemistry)
New Arts, Commerce
& Science College,
Ahmednagar.
Place:-Ahmednagar
ACKNOWLEDGMENT
It is indeed a great pleasure and moment of immense satisfaction for me to express my deep sense of gratitude towards project guide Dr.M.V.Padul for ken inspiring interest and Dr. A.D. Chaugale invaluable guidance at each stage, during the entire course the entire course of my project.
I am grateful to Prof. Humbe A.M.head of chemistry department, for providing the requisite laboratory facilities without which this project could not have taken the present shape.
I am grateful to Prof. Mr.Bladye & Prof. Mrs. Shaikh R.R. my classmates and laboratory assistants for their timely help and suggestions.
Last but not the least I thank my parents my friends for their everlasting support in all my endeavors.
Mr. Chopde Ramakant Maruti.
INDEX
SR.NO. TOPIC PAGE NO.
1 Abstract
2 Introduction
3 Life Stages of C. lanigera
4 Material and Method
a) Apparatus
b) Chemical
c) Method
1. Agar diffusion assay technique
2. Comparative study between woolly aphide. Fungal, salivary, amylase at different pH tem. By DNS method
3. Biogel chromatography
4. Pricipitation Polyacrylamide gel electophoresis & Agrose gel Electrophoresis.
5 Result and Discussion
6 References
ABSTRACT
The amylase activity of woolly acid aphids was determine by agar plate diffusion assay method. Amylase activity was also determined different pH by agarplate diffusion method. Comparative study between woolly aphids, salivary, fungal amylase was the different pH-temp. by chemical method that is by DNSA method.
Protein concentration determined by Lawry method. substrate concentration also determine. Biogel chromatography was also done precipitation was carry out higher activity shown by fraction was used to determine further study. Pattern of protein was determine by poly acrylamide gel electrophoreses and agarosse gel electrophoresis.
NOT AFFECTED LESS AFFECTED HIGHLY AFFECTED
INTRODUCTION
Sugar woolly afide has been recently reported inout break proportion from westerm and southern India. Through the pest was first reported from west bangol in 1958 and later from other part of India. Resent survey in part of past affected area of maharashtra and Karnataka.
Sugar is a universal sweetening agent and sugarcane (Saccharum affleinarum L.) is the primary age old source of it.It is mainly cultivated in tropics in the world, but in India it is grown even under subtropical areas.
Sugarcane occupies an area of 42.97 lakhs hectares with a production of 278.56 mt(Anonymous, 2003a) the area in Maharashtra was 5.9 lakhs hectares and annual production of 45.14 mt (Anonymous, 2003 a.) Thus Maharashtra account for 15.4 % of the total production in India.
It is a prerennial crop and occupies the land for 12-18 month. It is propagated by setts. Planting of sugarcane is done in 3 seasons, viz. Suru ( Dec-jan plating), adsali ( June-July planting) and preseason ( Oct.-Nov.planting) in The crop requires a long warm growing season with adequate amount of rainfall, Maharashtra. a fairly sunny and cool conditions, nut frost free condition during ripening and haresting freedom from stormy and severe winds.
Sugarcane is damaged by about 288 species of insects and non insets (David and Nandagopal 1986) and tissue borers, whte grubs,white flies, rodends, mealy bugs, pyrilla, scale insects etc. are of an economic
Important. Avasthy (1977) reported losses to the extent of 20 and 15 percent in cane yield and sugar recovery, repectively, due to the ravages of the pests. Recently,in Maharashtra.state during July-2002,an epidemic of sugarcane wooly aphid, Ceratovacuna Lanigera Zehnter was noticed in sangli, kolhapur and Satara districts and later on spread in parts of Solapur, pune and Ahmednagar districts. By September, 2002 it spread to neighboring Karnataka state. (Anonymous,2003b0 upto February 203, 1.32 lakh hectares of sugarcane crop was found affected by this pest in maharashtra.
In other countries, ceretovacuna lanigera (Takara and Auma, 1968, hill, 19930 C japonica (Anonymous, 202 and C. graminum (David and nandagopal, 1986) are the common species of white woolly sugarcane aphids damaging the sugarcane. In Indonesia,Farina (19940 observed the loss due to heavy incidence of C. lanigera by 26% in sugarcane yield and 24% in sugar content.
NATURE OF DAMAGE:-
Initial aphid infestation was seen on the under surface of leaves along the midrib and then over the entire under surface, covering it with flocculent, waxy secretion. Co-pious honeydew excretion often covers the entire upper surface of the leaves, leading to growth of sooty molded. (lopez A.W.) and continuous infestation leads to reduction in the length. Circumference, weight and sugar content of the stalk, and in susceptible varieties in vienam,( Vietnam 1963, P 266) loss in tonnage as well as sugar recovery (Tripathi G.M.) Gupta and Goswami assessed the effect of 25 and 100% alphid infested leaves on some yield and quality parameters of sugarcane and found that 100% infested had detrimental effects on the length (11% reduction), girth (3.5% reduction) weight (16.6 % Reduction) length of internodes (18.4% reduction) and width of leaf (4.9% reduction) juice quality parameters also exhibited considerable reduction. The percent reduction in sucrose, brix, glucose, purity and commercial cane sugar (CSS) was 53.3. 32.3, 25.3, 31.7, and 64.0 respectively. Preliminary studies on loss estimation being conducted at University of agricultural Sciences, Dharwad indicate adverse effects of aphid infestation on yield and quality parameters(Patil R.K. Lingappa S.)
LIFE STAGES OF C. LANIGERA
Systematic position:
Scientific name of species : Ceratovacuna Lanigera zehnt.
Family : Pomphigidae.
Superfamily : Aphidoidea
Order : Homoptera
English (Common) name : White Woolly
Marathi (Local) Name : Pandhara Lokari Oosmava
Obeservation on duration of different life stages of C.I. lanigera are follows
First instar nymph
The first instar nymphs are yellowish or greenish yellow in colour. It is very active and moves fast on the surface of leaf when exposed to sunlight. A pair of hollow tube is present at the posterior end of the abdomen oat the firth segment and is mainly used to secrete the defensie fuluid. The average length of the 1st instar was 0.87 mm and the width was 0.36 mm. ghosh (1974) reported that early instar nymph length ranges from 0.82-096 mm and width ranges from 0.3-0.45 mm.
Table duration of nymphal instars of C lanigera
Nymphal instars (Days)
Total
Duration
(Days)
I
II
III
IV
6 7 7 9 29
Patil (*20020 reported that first instar nymph length averaged 0.77 mm and width between 0.27-0.38 mm. the average duration of the first instar nymph was found to be 6.33 days.
Second instar nymph
The second instar nymphs are found to be less active and brownish in colour with black marking on the body. The white wooly filaments are absent. The average length of the 2nd instar was 1.17 mm and was 0.57 mm.
Patil (2002) reported that second instar nymph length as 1.23 mm and width ranged between 0.3-0.46 mm.
The duration of the second instar was found to be 6.66 days.
Third instar nymph
The third instar nymph is partially convered by white woolly filaments. These filaments are dense at the posterior end of the abdomen and also covered thorax portion but not head. The white wooly filaments are very dry, soft, nonpolar and dydrophobic. The average length length of the third instar nymph was 1.81 m and width was 1.05mm.
Patil(2002) reported the average length of this instar nymph as 1.83 mm and width as 0.46-1.07 mm.
The duration of this instar was found to be 7.99 days.
Fourth instar nymph
The fourth instar is covered with dense wooly filaments. They are sedentary on the leaf. A four segmented antenna is seen which was very short and measured 0.15 mm length. The length of fourth instar nymph was 1.98 mm and width was 1.02 mm.
Patil (2002) reported that length of fourth instar nymph as 2.09 mm and width as 0.47-0.78 mm.
The duration of this instar nymph was found to be 8.22 days.
Adult
The alate form adult emerged after fourth moult is blackish in colour, two pairs of transparent wings with clear wing venation are observed. Wings expanse of female is 2.7 mm in length and 1.2 mm in width. Adult possesses a pair of compound eyes. The winged adult was 2.00 in length and 1.0 mm in width.
Apterous forms lived for 30 days and alate forms lived for 10 days. The alate forms were responsible for the spreads for colony to new areas.
Pan and yang (1989) reported the longevity of adult from 32 to 57 days. Cheng et al. (2000) reported the longevity of adults as 20.5-24.1 days.
Life span
The nymphs undergo 4 instars and average of 29 days was required to complete the four instars on potted sugarcane plants. Total adult longevity was 30 days in case of apeterous aphids and 10 days in case of alate forms.
However, change et. Al. (20000 reported that a period of 15.8 to 16.5 days was required to complete nymphal stages.
Patil (20000 reported that life cycle is completed in one month period.
Sex Ratio
A number of aphids were critically observed and it was found that no sexual were recorded in the colony. The colony consists of females only and this was responsible for rapid increase in number.
Ghosh (1974) reported that no sexual were reported and the results were found to be in conformity with it.
In view of the above, it became indispensable to have a through knowledge of the pest so as to frame effective control measures against it. The available literature showed that so far into much work has been reported on this pest in India. It was therefore felt necessary to enzymatic study of woolly aphid
MATERIALS AND METHODOLOGY
Materials:-
A) Apparatus:-
1. mortar & pestle
2. Knife or Sissor
3. Petri Plates
4. Measuring Cylinder
5. Conical Flask
6. Beakers
7. Test Tube
8. Colorimeter
9. Spectrophotometer
10. Cold Centrifuge
11. Refrigerator
12. Ependrop tube
13. pH meter
14. Micropipett
B) Chemical:-
1. Soluble starch powder
2. agar Powder
3. Dintiro Salicylic Acid
4. Naoh
5. Glycine
6. Sodium Acetate
7. acetic Acid
8. Monobasic Sodium Phosphate
9. Dibasic Sodium Phosphate
10. Biogelp100
11. Disterase
METHODS:-
Collection of woolly aphids
Woolly aphids were collected from the rahuri sugar cane fields which were affected by woolly aphids. After collection of the woolly aphids were separated from the infected leaf and taken into separate dish. Then woolly aphids are preserved deepfreeze.
PREPARATION OF DIFFERENT PH BUFFERS:-
1. acetate buffer ( pH 4,5)
2. Phosphate buffer (pH 6,7,8)
3. Glycine NaOH buffer (pH 9,10)
EXTRACTION OF ENZYME FROM WOOLLY APHIDS:-
For this experient the 2 gm of woolly aphids is taken in seprate dish and 10 to 15 ml of buffer solution is added then the content is crushed in mortar pastle and the pest is prepared. Then after complete homogenation the sample is cold centrifuged the supernantant obtained is used for the further study of enzyme analysis. If some turbidity was observed then supernatant again centrifuge at high speed about 15000 rpm and time 30 min. the super natant filter by whatman filter paper.
CHECKING AMYLASE ACTIVITY BY DIFFUSION ASSAY TECHNIQUE:-
Starch and agar (3gm + 1.5 gm ) was taken in 100 ml distil water. Heat the solution and pour in to the petriplates. When the medium was solidified. After solidification the wells were prepared with the help of borer. After same time sample was added different wells with different pH. Enzyme extract then incubate 1 hour. The iodine solution was added in the petridish for staining after 4-5 min. the petridish is washed with D/W 3-4 times. Then incubating the petridish for 1 hour. The activity of aylase was seen at alkaline pH.
EXTIMATION OF AMYLASE ACTIVITY BY DINTRO SALICYLIC ACID METHOD REAGENT:-
a) Dinitro salicylic acid reagent (DNSA)
Dissolved 1 gram DNSA + 200 mg crystal phenol & 50 mg Sodium sulphate in 100 ml 1 % NaOH stored at 40 C.
Method:-
For estimation of amylase activity by DNSA method the starch is taken as substrate.
For the enzyme amylase, the activity of enzyme is estimated by checking optical density on the colorimeter.
REAGENTS TEST CONTROL
Enzyme extract 0.5 ml 0.00ml
Substrate (Sttarch) 1.00 ml 1.00 ml
Distilled water 0.00 ml 05 ml
Incubate at 37 c for 30 minutes
DNSA 1.00 ml 1.00 ml
Keep in boiling water bath for 10 minutes
After all addition ckecked the optical density on the colorimeter at 530 mm.
BIOGEL CHROMATOGRAPHY:-
Take 5 gm of biogel in 100 ml distil water and kept for one hour for the purpose of swelling. The coloum was properly cleaned with distil water the glass wool was wetted. And pour into the coloum with the help of long glass rod. The biogel was added to the coloum. The distil water was slowely added setting of gel at the bottom of coloum. The coloum was equillibriated with equilibration buffer pH-8 (phosphate buffer)
The one ml of crude sample is loaded on coloum with the help of pipette. The flow rate was adjested to 2 ml per 5 min. phosphate buffer was added help of pipette when fraction was collected. Each ependrop tube 2 ml fraction collected. Activity of each fraction was measured by DNSA method and protein concentration also measured.
Some number of fraction get highest activity they get separated from other and acetone preptation was carried out.
ACCETONE PRICIPITATION:-
The equal volume of sample and chilled acetone was mixed by drop by drop. After mixing the sample were placed in freeze for over night. The next day the sample were centrifugal at 12000 rpm for 15 min at 100 C. the supernatant was discarded and pellet was collected by dissolving 1 ml buffer and sample used as electophorotic study.
Reagents:
1. Stock buffer for resolving gel (1.5 M Tris HCL pH 8.8) :-
Tris-hydrozymethyl aminomethane (23.64g) was dissolved in 50 ml distilled water and the pH adjusted to 8.8 by adding conc. HCL drop wise the final volume was made up to 100 ml and the solution was stored in refrigerator.
2. Stock buffer for stacking gel (0.5 M Tris-HCL, pH 6.8)
Tris (7.88g0 was dissolved in 50 mlo of distilled water and the pH was adjusted to 6.8 by adding conc. HCL drop. Final volume was made to 100 ml using distilled water.
3. Electrode (tank) buffers (pH8.3):-
9.0g Tris, 42.3 g glycine were dissolved in distilled water to make 3 liters (electrode buffer was used for 2-3 subsequent runs)
4. Staining Solution:-
10 ml of 1 per cent coomassie blue prepared in methanol solution starining solution was filtered two to three times before use.
5. 30% acryl amide for stacking gel:-
75g acryl amideand 2 g bis-acryl amides were dissolved in distilled water. Volume was made to 250 mlo by addition of istilled water.
Apparatus & equipments :-
1. The vertical slab gel electrophoresis apparatus of atto with 16*16 cm glass plates.
2. Gasket.
3. Well comb & power supply.
Chemicals:-
The following chemicals (analytical grade) ware used for preparation of different solution
1. Acryl amide ”Electrophoresis grade “
2. Bisacreylamide”Electrophoresis grade “(N’N’ methylene bisacrlymide)
3. Tris ( hydroxymethyl aminomethane)
4. Glycine
5. HCL
6. Glycerol
7. Ammonium persulphate
8. TEMED (N, N, N’N’ tetranmethylmethylene diamine)
9. glycial acetic acid
10. Coomassie brilliant blue
11. bromophenol blue
Preparation of Gel
The gel plates ware cleaned and dried and cassettes ware assembled as per the instruction.
1. Separating Gel
The following solution ware mixed (10%)
Buffer pH 8.8 :7.5ml
Water : 12.3ml
30% running gel acrylamide : 9.9ml
5% ammonium persulphate : 0.15ml
TEMED : 0.02
(TEMED was added just before pouring gel)
All the reagents were mixed well and poured between the plates of the cassette.care was taken, so that no air bubble was trapped in the gel solution. The cassettes was filled ¾ with 3-4 cm left from the top the polymerized gel solution was overlayed with butanol and the gel was allowed to polymerize (it took about 30-60 min)
2. Stacking gel (4%)
After the polymerization of the separating gel the butanol layer was removed using filter paper and the cassette was blotted. The following stacking gel mixture was carefully poured into it.
Tris buffer 6.8 : 1.25 ml
Water : 3.075 ml
30% stacking gel acryl amide : 0.67 ml
10% ammonium per sulphate : 0.025 ml
TEMED : 0.005 ml
(TEMED was added just before pouring the gel)
After puring the stacking gel solution an acrylic comb having 11 teeth or wells was set without trapping any bubble. The gel was allowed to polymerize (it look about 10-15 min,)
After polymerization of gel the comb was carefully removed from the gel without disturbing the shape of wells. The sample wells were washed with tank buffer to remove the unpolymerised material. The tank filled with an appropriate volume of tank buffer solution.
ELECTROPHORESIS:-
The gel casstte was fixed into the electrophoresis unit. An aliquot soluble protein (enuivalent of 100 mg) was loaded to each well w8ith help of micro syringes. The gel plates were fixed over cathode chamber cum heat exchange with clamps and screws. The cathode chamber was placed into the4 tank. The upper tank was filled in with power supply with the anode connected to the lower reservoir and cathode to upper reservoir. The electrophoresis was conducted at 35 mA till the samples migrate into the running gel and subsequently at 60 mA.
The unit was disconnected from the power supply and cassette was removed from the electrophoresis tank as soon as the tracking dye reached the bottom of gel.
Fixing and staining the gel:-
The cassette was removed from the unit and the gel was taken out gently. Gel was placed in a staining tray and sufficient staining solution was poured of cover the gel uniformly. It was incubated for 6 hrs. And the stain was decanted, rinsed with water. To clear the gel back ground detaining in water was followed for a day or two. The pattern of staining drawn as well as photographed.
Agarose gel electropheresi:
Principle:- Agarose gel electophoresis refers to movement of charged particle/ molecules under the influence of electric field.
Agrose gel is more porous and used to separation of large particles. In this method charge molecules moves to the charged when electric field supplied. This movment is depend on the size of molecules and molecular weight smaller size protein molecules move faster than larger size molecules.
Procedure:-
1. Phosphate buffer (0.2 m) pH-8 is prepared about 200 ml with distil water.
2. 0.5 gm of agarose was dissolved in 50 ml distill water.1% agrose gel from it dissolved by heating to form clear solution.
3. Above containt cooled at 600 C and pored in to tray and comb was inserted in to it. Precautions were taken that no air bubble should enter.
4. Gel thickness were made up to 4 to 5 mm. and agarose solution were poure and allowed to cool comb was removed gentle when agar solidified.
5. phosphate buffer poured into tank to immerse the gel by about 5 ml
6. Power set was connected.
7. sample is loaded in well in derived order
8. Voltage was set to 50 V and switch was start of the power supply.
9. Electrophoresis was run until dye migrate to the other end of the gel ( approx.1 hr)
10. Power supply was turn off. The agrose gel was removed from the electro phorsis tank.
11. Kept in to the staining solution for 1 to 2 hr.
12. It was distained by water or solution.
13. the band pattern was observed
RESULT AND DISCUSSION:-
1. Amylase activity
pH7 pH8 pH9
The enzyme amylase present in woolly aphides activity is checked at pH 4, 5,6,7,8,9,10 woolly aphids amylase shown activity in alkaline pH 7,8,9 but pH-8 show maximum activity. pH-8 get maximum zone of activity.
2. Protein Concentration
NO ENZYME(ml) WATER(ml) LAWARYREAG. F.P. O.D.
Reg. constant one 0.0 1 3 0.5 0.0
Enzyme const. 2 0.1 4.4 0.0 0.0 0.2
Test 3 0.1 0.9 3 0.5 0.34
Test 4 0.1 0.9 3 0.5 0.34
O.D. = Test – Enzyme control
= 34-2
O.D. = 32
O.D. plotted on the standard graph
1 ml sample = 124mg/ml
But sample is diluted with distil water up to 10 ml.
1 ml = 124 µg X 10
I.e. 1 ml = 1240 µg/ml
I.e. 1 ml = 1.245 mg/ml
1 ml = 0.00124 gm/ml
i.e. 1 gm woolly aphide contain = 0.00124 X 10
i.e. 1 gm contain = 0.0124 gm/ml
3. AMYLASE ACTIVITY BY DNSA METHOD:-
Standard Graph for Activity by using DNS method.
O.D.
Constration of Maltose (µgm)
A) Substrate Concentration:-
O.D.
Substrate Cons.
Substrate conc in % O.D Activity(µgm/ml/min)
2 0.73 96
1.8 0.73 96
1.6 0.73 96
1.4 0.73 96
1.2 0.66 176
1 0.54 144
0.8 0.48 128
0.6 0.34 89.33
0.4 0.27 70.33
0.2 0.18 48
Maximum substrate concentration for woolly aphids amylase is 1.2%
Graph shown increase any activity with increase in substrate const. but further steady state observed.
B) Different pH with comparative study between salivary, fungal, woolly aphides amylase
pH Woolly Aphid Activity(µgm/ml/min) Fungel Activity(µgm/ml/min) Salivary Activity(µgm/ml/min)
4 0.18 48 0.64 171.66 0.65 173.33
5 0.41 109.33 0.79 212 0.7 186.60
6 0.58 154.66 0.8 213 0.8 213
7 0.64 171.66 0.61 162.66 0.65 173.33
8 0.71 189.33 0.43 154.66 0.6 160
9 0.42 105.33 0.16 41.33 0.55 146.66
10 0.36 96 0.15 40 0.51 122.66
In above graph shows woolly aphids amylase show maximum activity at pH-8 salivary, fungal amylase shown maximum activity at pH-6.
The graph shown the woolly aphides amylase is different from salivary and fungal amylase.
C) Different temperature with comparative study between woolly aphids salivary, fungal amylase.
Temp Woolly Aphid Activity(µgm/ml/min) Salivary Activity(µgm/ml/min) Fungel Activity(µgm/ml/min)
20 0.16 41.33 0.22 60 0.24 64
30 0.2 53.33 0.24 64 0.24 64
40 0.22 60 0.28 74.66 0.28 74.66
50 0.18 48 0.26 69.33 0.25 66.66
60 0.16 41.33 0.19 50.66 0.21 56
70 0.11 28 0.16 41.33 0.18 48
The woolly aphids, salivary, fungal amylase shown maximum activity at 40o C temperature but at higher temperature suddenly decrease the activity. It clear that enzyme is not thermos table..
4) BIOGEL CHROMATOGRAPHY:-
The above graph peak has shown maximum activity. These fractions is collected and carry out acetone precipitation.
5) ELECTROPHORESIS STUDY:-
Crude sample
Band Accetone Preciption Sample
band
a) Electrophoresis pattern of protein is woolly aphide shown only one band in chromatography fraction but crude sample get diffused pattern of band.
b) Agarosegel Electrophoresis:-
Crude Sample Band with
Diff Concentration
in agarosegel Electrophoresis load crude sample but not get clear band some diffused band observed
6) Molecular weight determination:-
REFERANCE
1. Ghosh A.K, Homoptera: Aphidoidea, 4, Subfamily phloemyzinae, Anoeciinae and Hormaphidinae. In the fauna of India and adjacent countries, Zoological survey of India, kolkata, 1988 p 429
2. Lingappa, S. Kulkarni. K.A. Patil R.K. Thippanavar, P.S. and shekhappa, status of woolly aphid ceratovacuna lanigera zehntner on sugarcane in Karnataka. Paper presented at brain storming session on sugarcane woolly aphid, 10 june 2003.
3. Gupta, M.K. and Goswami, P.K. incidence of sugarcane woollyu aphid and its effect on yield attributes and juice quality. Indian sugar, 1995, 44, 883-885. Lopez, A.W.Ann. Rep. Res. Bur. Philipp. Sugar assoc, Entomology department report, 1931, p 273
4. Anonymous, annual work progress report on crop improvement program of rice, sugarcane, vegetable and field crops, directorate of rural Affairs, Vietnam, 1963, p, 263
5. Tripathi, G.M. Record of parasite and predator complex of sugarcane woolly aphide, ceratovacuna lanigera Zehnter in Nagaland India sugar, 1995, 44, 883-885.
6. Raychaudhari. D. N. food plant catalogue of India aphidede, aphidological socity of India, kolkata, 1984, p 188.
7. Mote U.N. & Puri.S.N., present status of sugarcane woolly aphid, ceratovacuna lanigera, a new pest problem on sugarcane in maharashtra. Paper presented at brain stroming.
8. David & Nandagopal, study of pest problem on sugarcane in India 1986
9. Avasthy, Study on losses percent in cane yield and sugar recovery due to the ravage of the pests.
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