MOLECULAR CHARACTERIZATION OF CERTAIN GYMNEMA SYLVESTER ECOTYPES THROUGH RAPD ANAYLYSIS

ABOUR AUTHORS:
G.M.Reddy, *D.Gopi Krishna
Sathyabama University
Chennai, TN, India
*gopikrishnamsc@yahoo.com

ABSTRACT
Gymnema sylvestre is a large, stout, woody , vine-like plant which climbs on bushes and trees. It is known in sankrit- meshashiringi, madhu nashinin (madhu=sugar, nashini= destroy), in telugu- podapatri, in hindi- gur-mar. the latin name of gymnema sylvestre means sugar destroyer and is considered a herbal remedy for high blood sugar. traditionally it was recommended for stomach problems, constipation, water retention and liver disease but the recent studies conducted in india have further shown that extract of gymnema sylvestre is useful in controlling bloodsugar to treat type-ii diabetes. gymnema has been clinically proven to reduce excessively high blood sugar levels, perhaps as a result of boosting the production of insulin required to process sugar.

The medicinally active parts of the plant are the leaves and the roots. It is known as destroyer of sugar because, in ancient times, Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestre suppressed the taste of sugar i.e, sweet foods no longer tasted sweet, but rather became almost completely tasteless. In later generations, clinical tests showed that regular use of over a period of three to four months helped to reduce glycosuria, or the appearance of carbohydrates in urine.

The present investigation entitled “Molecular Characterization of five ecotypes of Gymnema sylvester an Anti-diabetic plant”. The main objective of the project is to identify the variation between different ecotypes of Gymnema sylvestre.

Molecular characterization/genetic diversity of the five different ecotypes of Gymnema sylvester was studied using Random Amplified Polymorphic DNA (RAPD) analysis. A preliminary study was carried out using only four primers namely, OPL 01, OPL 02, OPL 06, and OPL 07. Except OPL-01 Primer all the primers gave satisfactory amplification of 63 bands. OPL-01 Primer gave the monomorphic banding pattern. Out ofthe total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype.

The preliminary study suggested that existence of genetical differences among five ecotypes of the Gymnema sylvestre and RAPD analysis exhibits the differences in ecotypes.

REFERENCE ID: PHARMATUTOR-ART-1841

INTRODUCTION
Gymnema sylvestre is a large, stout, woody , vine-like plant which climbs on bushes and trees. It is known in sankrit- Meshashiringi, Madhu nashinin ( Madhu=sugar, nashini= destroy), in Telugu- podapatri, in Hindi- Gur-mar. The Latin name of Gymnema Sylvestre means “ sugar destroyer” and is considered a herbal remedy for high blood sugar. Traditionally it was recommended for stomach problems, constipation, water retention and liver disease but the rescent studies conducted in India have further shown that extract of Gymnema sylvestre is usefull in controlling bloodsugar to treat type-II Diabetes. Gymnema has been clinically proven to reduce excessively high blood sugar levels, perhaps as a result of boosting the production of insulin required to process sugar.

The medicinally active parts of the plant are the leaves and the roots. It is known as “destroyer of sugar” because, in ancient times, Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestre suppressed the taste of sugar i.e, sweet foods no longer tasted sweet, but rather became almost completely tasteless. In later generations, clinical tests showed that regular use of over a period of three to four months helped to reduce glycosuria, or the appearance of carbohydrates in urine. Gymnema sylvester acts on the taste buds of the oral cavity and on the absorptive surface of the intestine. The recent studies conducted in India as early as 1930 showed that G. Sylvestre cause hypoglycemia, probably by indirectly stimulating the insulin secretion of the pancreas. In the present study the Gymnema Sylvester is used as model plant in the control of Diabetes specially in carrying out the molecular characterization.

Classification:
Kingdom: Plantae.
Division: Angiospermae.
Class: Dicotyledoneae.
Order: Contortae.
Family: Asclepiadaceaeous. (Milkweed)
Genus: Gymnema.
Species: slyvestre

DISTRIBUTION
Gymnema sylvestre is distributed in Deccan peninsula, extending to parts of northern and western India , wild in the forests of India. These plants grown in central and southern India and also widely distributed thought out equatorial Africa.

Gymnema sylvestre belongs to “ASCLEPIADACAE” family,” LATICIFEROUS “genus. About 10 species in India – G. hirsutasm, G. montanum, G. tingens, G. acuminatum. Gymnema sylvestre is a tropical and native to central and western India and can also be found growing in tropical Africa and Australia.

It is a large or more pubescent shrub with young stems and branches, terete, pubscent. Leaves sub-coriaceous, 2.5-6 cm long, ovate or elliptical, acute or shortly acuminate, often glabrous above. Petiolcus 0.6 to 1.2 cm long.

ACTIVE COMPOUNDS:
The medicinally active parts of the plant are the leaves and roots. The important active ingredient of Gymnema sylvestre is an organic acid called gymnemic acid. Gymnemic acid is made up of molecules whose seatom arrangement is similar to that of glucose molecules.

Those molecules fill the receptor locations on the taste buds for a period of one to two hours, there by preventing the taste buds from being activated by any sugar molecules in the food. Similarly, the glucose- like molecules in the gymnemic acid fill the receptor locations in the absorptive external layers of the intestine, there by preventing the intestine from absorbing the sugar molecules.

The active principle of this plant suppresses the sweet taste not only of sucrose, but also the sweetness effects of sodium saccharin, cyclamate, glycine, D- alanine , D- tryptophan, D- leucine,beryllium chloride and lead acetate, but not that of chloroform.

As for the mode of action of anti-sweet active principles obtained from Gymnema sylvestre , Warren et al., (1969) and Yackzan (1969) have independently suggested that the compounds might act at the taste receptor surface, by chemically and physically altering it so that originally stimulating sweet molecules provoke lesser degrees of stimulation.

MEDICINAL VALUES:
Commonly referred to as “sugar – killer “gymnema leaves comprised of gymnemic acids which suppress the absorption of glucose molecules, and it reduces the sensation of sweet ness of foods. Gymnema sylvestre reduces the blood sugar levels, lower blood cholesterol levels and balance insulin levels .

It is used as astringent, stomachinic, tonic, refrigerant, antidiabetic. Leaves have a peculiar property neutralizing temporarily the sensation for sugar and is used in diabetes., Liver disorders, cardiac Amenorrhoea, Sec . Amenorrhoea, Cough and Asthma.

What is Diabetes mellitus?
Greek: diabetes- a siphon or running through; mellitus-sweet

Diabetics is one of the oldest wide spread disease of the world which results from defects in Insulin Secretion, or action, or both. Diabetes mellitus, commonly referred to as a diabetes, means “Sweet Urine” There are two major types of diabetes mellitus, called type 1 and type 2 .Type 1 diabetes mellitus was also called insulin dependent diabetes mellitus (IDDM) or juvenile onset diabetes mellitus. Whereas Type 2 diabetes was known as insulin independent diabetes.

The control of blood glucose level is necessary to avoid the damage of blood vessels. In several cases this may affect retina, renal glimerus and nerve sheath. Over time, diabetes mellitus can lead to blindness, kidney failure and nerve damage. Diabetes mellitus is also an important factor in accelerating the hardening and narrowing of the arteries (atherosclerosis), leading to strokes, coronary hearth diseases, and other blood vessel disease. Diabetes mellitus is a chronic medical condition, meaning it can last a lifetime.

Diabetes mellitus is gene controlled disorder besides the expression is regulated by another factor,

Heredity (Heredity is considered an important cause. Children of parents with no diabetes are less likely to develop diabetes than the children of parent with diabetes.)

Obesity and sedentary lifestyle

Alcoholic beverages (when taken in Excess, damage the pancreas and cause diabetes.

Higher consumption of bitter tapioca, maize, sugar, salt and low protein diet during childhood increase the risk of developing diabetes.

Those with low (1.5 Kg or lower), high (3.8Kg higher) birth weight,stress,high blood sugar (during an infection, hearth attack and mental stress),diabetes during pregnancy and men and women with hypertension run the risk of developing diabetes

Viral gastroenteritis, mumps and chicken pox may precede development of type 1 diabetes.

Diabetics mellitus (DM) is the most common form caused by deficiency or diminished effectiveness of insulin and is controlled by exogenous supply of Insulin. Only approximately 10% of the diabetes mellitus have type 1 and the remaining 90% have type 2 diabetes mellitus.

Types of diabetes mellitus:
Recently the WHO has recognized four types of Diabetics:
1. Type 1 diabetes mellitus:

In this type of Diabetes, the pancreas produces little or no insulin.
This type of Diabetes generally develops at young age.
Their blood glucose is very high.
Patients need insulin treatment lifelong.

2. Type 2 diabetes mellitus(Adult onset diabetes mellitus (AODM)

In this type of Diabetes, the pancreas produces less insulin than required to maintain normal blood glucose.
It generally develops after 40-45 years of age and progresses slowly
Several years may pass before diagnosis is actually made.
All patients need diet control, tablets or insulin to keep their diabetes under control

3. Secondary Diabetes Mellitus:

This includes diabetes due to genetic defects in insulin secretion and action, drugs (steroids, contraceptive pills, Adrenocorticotropic hormone ACTH etc..,) and diabetes in association with endocrine disorders. Most of them require insulin to control diabetes.
Most of them require insulin to control diabetes.

4. Gestational Diabetes Mellitus (GD)

Usually occurs in pregnant women.

REVIEW OF LITERATURE.
In 1967, Stocklin et al., reported that gymnemic acid was the D- glucuronide ofa new hexahydroxy Oleanene triterpene named gymnemagenin, which is esterified with various combinations of formic, acetic, n- butyric , isovaleric and tiglic acids.

Koch et al., (1973) found that gymnemic acids are inhibitory to the ATPase system from housefly brain and labellum, in addition to fish brain. There fore, they suggested that a portion of the ATPase system might be important biochemical in the sweetness recognition process.

Cagan (1974) experimented with gymnemic acid and discovered that it does not inhibit the binding of ¹⁴C – sucrose to bovine taste papillae, so he proposed that gymnemic acid might act at a step subsequent to the initial binding of sweet molecules.

As for the mode of action of anti-sweet active principles obtained from Gymnema Sylvester, Warren et al. (1969) and Yackzan ( 1969) have independently suggested that the compounds might act at the taste receptor surface, and chemically and physically alter it so that originally stimulating sweet molecules provoke lesser degrees of stimulation.

Hooper was the first investigator who attempted to isolate the active compounds from the leaves of Gymnema sylvester. He noted that the anti sweet principle could be precipitated from a crude alcoholic extract of the leaves as a brownish- black aggregate of resinous substances “ gymnemic acid “ and reported that it occurred in the potassium salt form in the plant.

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE

Gymnema sylvestre useful for reducing the blood sugar is known as “ sugar killer”. Several companies provided as Herbal tea and Capsules.

1. DIA-TEA.
Gymnema sylvestre DIA-TEA is made from Gymnema and Sinensis. An excellent health beverage, DIA-TEA prepared from the Gymnema herb is considered to be the best from as it has an effect in the mouth to reduce sugar craving as well in the intestine to reduce absorption of sugar. DIA- TEA is completely coffin free. Having regularly can reduce the appetite for sweet tasting food, which can result in substantial weight loss, promoting balanced blood sugar levels and healthy pancreatic activity.

Ingradients .
Gymnema sylvester----- 60%
Camellia Sinensis ------40%.
Coffen

2. Gymnema Herbal Tea :
Best Nutrition has introduced the beneficent powers of Gymnema sylvester in two forms. Herbal tea and capsules, both are equally effective.

An extract of ayurvedic herb, effects the:

Taste buds in the oral cavity, the acid prevents the taste buds from being activated by any sugar molecules in the food.
Absorptive surface of the intestines. The acid prevents the intestine from absorbing sugar molecules.

Gymnema sylvestere ( Ayurvedic Herbal extract)
Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestere suppressed the taste of sugar .

DIA-BOTICA Capsules :
Dia- Botica Capsules contain 4 most powerful anti sugar absorption herbal standardized extracts which helps for not absorbing sugar into our blood stream during digestion. G. sylvestere is one of the main herbal standardized extract.

4. Gymnema sylvestre Extract: (Standardized 25% Gumnemic acid)
Research indicates this amazing herb has positive benefits on blood sugar control, helps with sugar cravings and to regenerate the pancreas. In tests on diabetic rats, the pancreas doubled in size and grew new insulin- producing cells.

MATERIALS AND METHODS
Over the last three decades DNA markers are widely being used for genetic mapping, molecular taxonomical studies and for the detection of genetic changes in organisms caused due to mutations or genetic engineering. Arbitrary primed PCR has been used to detect dominant polymorphic markers in genetic mapping experiments (Williams et al., 1990). DNA markers have been widely used in genome mapping in a wide range of plant species and are now being increasingly employed for studies of genetic relatedness among species.

New source of high quality genetic markers, based on the identification of polymorphisms in DNA and protein was developed in the last three decades. They have been termed as Molecular markers. Selection with markers depends mainly on the quality of the polymorphism used. Williams et al., (1990) proposed the use of single short random primers (usually 10-mers) in polymerase chain reaction (PCR) as a method of generating polymorphic markers (RAPDs). DNA polymorphism result either from differences in the DNA sequence at primer binding sites or by chromosomal changes affecting the amplified regions. After Electrophoresis the amplified fragments can be visualized by florescence and the resulting patterns of bands can interpreted genetically

The present investigation entitled “Molecular Characterization of five ecotypes of Gymnema sylvester an Anti-diabetic plant” was conducted in the Molecular biology laboratory of Prof. G.M. Reddy Research Foundation, Hyderabad.

Experimental materials:
Different ecotypes of Gymnema plants present in and around the lab were used as source of experimental material for collection of explants.

Glass and plastic ware:
All the glassware used were of Borosil make for various studies, test tubes, Flasks, Bottles, beakers, MicroPipettes etc were used for preparations of various chemicals. All these glass and plastic ware were autoclaved at 121^o C for 15 min, at 15 lb pressure. The pipette tips and the micro pipettes used were of Qualigen make.

Chemicals:

Chemicals required for DNA isolation.
1) DNA extraction buffer

2) Chloroform : Isoamyl alcohol (24:1)

3) Chilled ethanol / Propanol.

4) 70% Ethanol.

5) RNase

6) TE buffer (T10 E1)

7) Phenol :Chloroform : Isoamyl alcohol. (25:24:1)

Chemicals required for Agarose gel electrophoresis.
1) TAE (50X) buffer

2) Agarose

3) Ethidium bromide

4) Running buffer (1X)

5) Loading dye.

Chemicals required for PCR (Polymerase Chain Reaction).
1) Thermostable DNA polymerase
2) A pair of synthetic Oligonucleotides to prime DNA synthesis
3) Deoxynucleoside triphosphates (dNTPs)
4) Mg +2 Buffer
5) 10X Buffer
6) Template DNA.

Procedure/protocols.
Isolation of DNA (Deoxy ribonucleic acid):
Genomic DNA is the blue print of life, which is inherited and passed on from generation to generation directing the development of cells, organelles and organisms. Isolation of DNA from RNA and proteins is essential for all molecular biology investigations.

For the isolation of DNA, the cell wall must be broken or digested in order to release cellular constituents, which is done by grinding tissue in dry ice or liquid nitrogen. Cell membrane is disrupted by using detergents such as CTAB (Cetyl Trimethyl Ammonium Bromide) or SDS (Sodium Dodecyl Sulphate). For the protection of DNA from endogenous nucleases, EDTA (Ethylene Diamine Tetra Acetic Acid.) is used. EDTA chellates Mg⁺⁺ ions, which is essential co-factor for most of the nucleases. The tissue mixture is emulsified with chloroform and phenol to denature proteins from DNA.

The DNA isolation from plant tissues is very difficult due the presence of various secondary plant products. A protocol works with one plant group and may fail with the others. Hence a number of DNA isolation methods have been developed for different target plant groups. But two methods originally developed by Dellaporta et al., (1983) and Murray and Thompson (1980) respectively are being commonly used with certain modifications for isolating high molecular weight DNA from small amounts of tissue.

Preparations of chemicals required for DNA isolation.

I) DNA extraction buffer (DEB) stock preparation.

Make upto the volume of 100 ml with distilled water and autoclaved.

Note:
The above stocks i.e 4M NaCl, 0.5M EDTA (pH 8.0), 1M Tris Cl (pH 8.0)10% CTAB were prepared for 100ml.

II) 24:1 Chloroform Isoamyl alcohol.
24 ml of chloroform was taken in a flask and 1 ml of isoamyl alcohol is added together.

III) Phenol :Chloroform : Isoamyl alcohol. (25:24:1)
25 ml of Phenol was taken in a clean conical flask 24 ml of chloroform and I ml of isoamyl alcohol were added to prepare the mixture of phenol, chloroform and isoamyl alcohol.

IV) TE Buffer
1ml of 1M Tris (pH – 8.0) was taken in a clean conical flask along with that 2 ml of EDTA (pH- 8.0) was added the volume was made up to 100 ml with distilled water. Then it was conical autoclaved.

V) 70% Ethanol.
70 ml of alcohol was taken in a clean conical flask and the final volume was made up to 100ml with distilled water.

Steps involved in extraction of DNA .
1) One gram of Gymnema leaf was taken from each different four ecotypes, washed under running tap water and then dried on filter paper.
2) These leaves were taken in different Motor and pistles and were made to fine powder with the help of liquid N_2.
3)The powder was then transferred to different centrifuge tubes (as labeled) and 5 ml of DEB was added to each tube.
4) The tubes were shaken gently for about 10 minutes and then put in a water bath for about one hour at 60°C.
5) After one hour the tubes were removed and equal volume of (24:1) Chloroform :Isoamyl alcohol was added and gently shaked for about 5 – 10 minutes.
6) The tubes were then centrifuged at 15,000 rpm for about 10 minutes at room temperature.
7) These tubes were carefully removed and the supernatant was collected using a micropipette.
8) To the above supernatant, equal volume of chilled ethanol was added and gently shaked.
9)A pellet of white fibrous structure, DNA was observed in the tube.
10) This DNA was then separated from the tube and washed twice or thrice with 70% Ethanol to remove the impurities.
11) finally, the DNA is taken in a small MCT(Micro Centrifuge Tube) tube and the mouth wrapped with a paraffin wax with a hole in the centre is dry overnight.
12) Next day, the dried DNA was dissolved with 150 – 300 microlitres of T.E buffer.
13) To the dissolved DNA, RNase was added and is stored in cool conditions for future use.

PROTOCOL FOR ISOLATION OF DNA FROM GYMNEMA sYLVESTRE LEAVES USING CTAB METHOD

Precautions
1)Time between thawing of frozen pulverized tissue and exposure to extraction buffer should be minimized to avoid nucleotide degradation of DNA.
2) DEB stock is light sensitive, hence it must be always covered with a black plastic cover/with aluminum foil.
3) The DEB should be preheated before using it.
4) The Ethanol used for precipitating DNA should be very chilled.

Electrophoresis
The idea of using Electrophorasis to analyze DNA came from Vin Thorne – 1960. The first modern electrophoresis was developed by Walter schffner. Electrophoresis is used to identify, separate and purify DNA fragements.The technique is simple, rapid to perform and can separate DNA fragments that can not be separated adequately by other procedures such as gradient centrifugation.

Generally two types of gels are being used. The choice depends mainly on the sizes of the fragments being separated.
1) Agarose gels.
2) Poly acrylamide gels.

Polyacrylamide gels are most effective for separating small fragments of DNA (5 – 500bp) and have high resolving power. But the polyacrylamide gels are difficult to handle and prepare.

Agarose gels are used for separating larger double stranded molecules and have low resolving power and have grater range of separation. DNAs from 50bp to several megabases in length can be separated by agarose gels.

Theory/Principle of DNA migration.
Nucleic acids being negatively charged will migrate towards the +ve pole i.e anode. The electrophoratic mobility of a macromolecule is determined by the volume fraction of pores within the gel that the macromolecule can enter. Since small DNA fragments can fit into more pores than large DNA fragments, small DNA fragments will migrate through the matrix faster than large DNA fragments. Larger molecules migrate more slowly because of greater frictional drag and because they worn their way though the pores of the gel less efficiently than the smaller molecules.

The velocity of the DNA fragments decreases as their length increases and is proportional to electric field strength. The greater the pore size of the gel, the larger the DNA that can be sieved. Agarose gels with low concentration of agarose (0.1 – 0.2% w/v) are capable of resolving larger DNA molecules, but are very fragile and must be run for several days and cannot resolve linear DNA molecules larger then 750kb in length.

Preparation of chemicals required for Agarose Gel Electrophoresis.

1) Buffers:
Two types of running buffers are commonly used for DNA gel electrophoresis.

A) 50X TAE buffer (Tris Acetate EDTA) stock solution.
242 g of Tris and 37.2 g. of Na₂ EDTA (2H₂O) were dissolved in 900 ml of water. To the above solution 57.1 ml of glacial acetic acid was added and the final volume is made up to 1 lit. Store at 4^o C.

B) TBE buffer (Tris Borate EDTA).
108g. of tris base and 55g. of Boric acid were dissolved in 900ml of water. To the above solution 40ml of 0.5 M EDTA (pH 8.0) was added and the final volume was made to 1 liter with the help of distilled water.

2) Ethidium bromide – Available readymade.

3) Agarose – Available readymade as powder.

4) Preparation of Tank buffer.
2ml of 50X TE was taken in a clean conical flask and was made upto 100ml with distilled water.

Steps involved in Electrophoresis of crude DNA.
Casting the gel.
1)2ml of the 50X TAE was taken and made up to 100ml with distilled water.
2) 1g of Agarose powder was added to the buffer solution.
3) The solution was then heated till all the agarose was completely dissolved.
4) The solution was then cooled on the bench and Ethidium bromide was added and is mixed gently with out formation of any air bubbles.
5) Meanwhile the casting tray along with the comb was set on the bench and the above solution was poured carefully in the tray with out formation of air bubbles.
6) The tray was left as such for about half an hour with out disturbing it to solidify.
7) Meanwhile tank buffer was prepared and poured it in the electrophoresis tank.
8) After the gel solidified, the gel was mounted in the electrophoretic tank carefully.
9) The DNA samples were taken out from the fridge and were gently shaked for uniformity.
10) Eight Micro lit. of the DNA sample was taken and transferred on to a paraffin paper and mixed with 2 micro lit loading dye
11) Then the solution was taken into a pipette carefully and loaded in the wells.
12)Gel was runned at 40- 80 volts for 3-4 hours.
14) After the electrophoresis is completed the gel was carefully removed from the electrophoretic tank and is kept in the gel documentation unit for viewing the DNA bands under U.V. light.

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE

PCR (Polymerase chain reaction):
The method of PCR was first proposed by H. Gobind Khorana in early 1970.The technique was put into practice by Kary Millis at Cetus Corporation in 1985 which described invitro amplification of single copy mammalian genes using the Klenow fragment of E. Coli – DNA Polymerase – I. The first description of PCR precise, laconic, impersonal was published by Kleppe et al in 1971.

Polymerase chain reaction discovered in 1985 is an ingenious new tool enabling amplification

Principle:
PCR involves synthesizing multiple copies of a region of a DNA which bind to opposite strands flanking the target sequences.

Essential components/Reagents of PCR are-
1) Thermostable DNA polymerase (Taq polymerase).
2) A pair of synthetic oligonucleotides to prime DNA synthesis (Primers).
3)Deoxynucleoside tri phosphates.(dNTPs)
4) Template DNA.
5) 10X buffer
6) Mg+2 buffer.

Temperature cycles involved in PCR are :
1) Pre denaturation – 94°C for 4 min.

2) Denaturation – 94^o C for 1 min.
In Both 1and 2, i.e Predenaturation and denaturation the two strands of target DNA are separated.

3) Annealing – It depends upon the primers used. Upon the primer bottles the nucleotide sequence will be given fom 3’ to 5’ end. Depending on the nucleotide sequence the annealing temperature is calculated using the two formulas:
4(G+C)

2(A+T)
A = Adenine
T = Thymene
G = Guanine
C = Cytosine.

This annealing temperature anneals two complementary primers to ends of separated single stranded DNA strands of targeted DNA.

4) Primer extension – 72°C for 2 min. This temperature allows thermostable Taq DNA polymerase to use single standard DNA strands of target and primers to synthesize new strands.

5) Final extension – 72°C for 5 min.

Protocol
Preparation of 20mlit. PCR reaction mixture.
1) 20 different sterile thin walled autoclaved PCR tubes were taken and numbered serially from 1-20. Four samples were tested with eight primers for amplification.

2) In these tubes 1mlit of DNA sample and 1mlit. of the respective primers are taken according to the table given below and stored in Ice.

3) In a sterile, autoclaved tubes prepare the cocktail mixture by adding the below contents as follows.

4) Then maintain the tube of the cocktail reaction mixture at cool temperatures.

5) With the help of micropipette (pipette tips should be autoclaved) pipette out 18mlit. of the cocktail mixture from the tube an add it to the PCR tubes.

6) Then tap the tubes gently and wipe the outer surface of the PCR tube with tissue paper and then place them in the PCR machine.

7) Program the PCR machine to the following conditions.

Predenaturation - 94°C for 4 min.
Denaturation - 94°C for 1 min.
Annealing - Depending on the Primer sequence.
Primerl extension - 72°for 2 min.
Final extension - 72°C for 5 min
No. of cycles - 40 cycles.
Hold temperature - 4°C.

8) After the 40 cycles are over the PCR machine comes to a hold temperature of 4°C. Then remove the samples and store them at 4°C.

The Table showing the distribution of primers to the sample.

Primers	OPL-01	OPL-02	OPL-06	OPL-07
Ecotype – I	1	2	3	4
Ecotype – II	5	6	7	8
Ecotype III	9	10	11	12
Ecotype IV	13	14	15	16
Ecotype V	17	18	19	20

Electrophoresis of PCR samples.
The PCR DNA samples should be subjected to electrophoresis for observing the amplification patterns of the DNA.

Steps involved in Electrophoresis of PCR samples.

Casting the gel.
1)2ml of the 50X TAE was taken and was made up to 100ml with distilled water.
2) 1.5g of Agarose powder was added to the buffer solution.
3) The solution was then heated till all the agarose is completely dissolved.
4) The solution was then cooled on the bench and Ethidium bromide is added and is mixed gently with out formation of air bubbles.
5) Meanwhile the casting tray along with the comb was set on the bench and the above solution was poured carefully in the tray with out formation of air bubbles.
6) The tray is left as such for about half an hour with out disturbing it to solidify.
7) Meanwhile prepare the tank buffer and pour it in the electrophoresis tank up to the level.
8) After the gel gets solidified, mount the gel in the electrophoresis tank carefully.
9) The DNA samples were taken out from the fridge and were gently shaken for uniformity.
10) 8mlit. of the DNA sample was taken and transferred on to a paraffin paper and to it 2mlit. of Loading dye was added to the sample and mixed properly. To the first well a DNA Ladder was loaded (500bp).
11) Then the solution is taken into a pipette carefully and loaded in the wells.
12) The electrophoresis is switched on and the voltage is adjusted in-between 40 – 80 volts.
13) It takes atleast 3 – 4 hours for the DNA to run completely.
14) After the electrophoresis is completed the gel is carefully removed from the electrophoresis tank and is kept in the gel documentation unit for viewing the DNA bands under U.V. light.

RESULTS
Molecular characterization/genetic diversity of the five different ecotypes of Gymnema sylvester was studied using Random Amplified Polymorphic DNA (RAPD) analysis. A preliminary study was carried out using only four primers namely, OPL 01, OPL 02, OPL 06, and OPL 07except OPL-01. All the primers gave satisfactory amplification of 63 bands. OPL-01 Primer gave the monomorphic banding pattern. Out of the total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype.

The primer OPL 02 resulted with a total number of 21 amplicons.Out of these amplified products, ten bands were found to be polymorphic and 3 were specific to the ecotypes Ecotype I, Ecotype III & Ecotype V.

A total number of 19 bands were amplified with the primer OPL 06 with the fragment sizes ranging from approximately 500 bp to 1500 bp.out of these 19 bands, eight out of these 19 bands were polymorphic and two bands were specific to the ecotypes Ecotype II & Ecotype III respectively .

Eighteen amplicons were observed with the primer OPL 07. All the bands were monomorphic except one, which is specific for the ecotype Il.

The preliminary study suggested that existence of genetical differences among five ecotypes of the Gymnema sylvestre and RAPD analysis exhibits the differences in ecotypes.

DISCUSSION
The main objectives of the project are acquisition of different ecotypes of Gymnema sylvester and their molecular characterization.

Different ecotypes of Gymnema sylvestre, were collected from various places of Andhra Pradesh including Tropical Botanical Garden Research Institute. Pallad, Kerala and were maintained in the net houses at the Foundation. The material required for the research work is taken from the plants grown in the net houses.

TA preliminary study was carried out using only five primers namely, OPL 01, OPL 02, OPL 06, and OPL 07. Except OPL-01 Primer all the primers gave satisfactory amplification of 63 bands. Out of the total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype. The primer OPL-01 gave the monomorphic banding pattern.

The primer OPL 02 resulted with a total number of 21 amplicons. Ten bands were found to be polymorphic and 3 were specific to the ecotypes I, ecotype III and ecotype V.

A total number of 19 bands were amplified with the primer OPL 06 with the fragment sizes ranging from approximately 500 bp to 1500 bp. Eight out of these 12 bands were polymorphic and two, with fragment size approximately 1500 bp were specific to the ecotype II and ecotype III and ecotype respectively.

Eight amplicons were observed with the primer OPL 07. All the bands were monomorphic except one, which is specific for the ecotype II.

The preliminary study suggested that existence of genetical differences among six ecotypes of the Gymnema sylvester and RAPD analysis exhibits the differences in ecotypes.

BIBLIOGRAPHY
1. George P. Datei, jr., and Louis Long, Jr. (1973). Gymnemic acid,the antisaccharine of Gymnema sylvestre. Studies on the isolation and Heterogeneity of Gymnemic acid A1. J. Agr. Food Chem., Vol. 21, No.5, 1973.
2.Joseph E. Sinshemer, G. Subba Rao, and Hugh M. Mcilhenny. Constituents from Gymnema sylvestre V: Isolation and Prelimunary Characteriation of the Gymnemic acids.Jour. of pharmaceutical sciences, Vol. 59, No. 5, 1970.
3.Rutt Suttisri, Ik-Soo, A. Douglas Kinghorn. Plant derived triterpinoid sweetness inhibitors. Jour. of Ethnopharmacology 47 (1995) 9 – 26.
4.Jakinovich, Jr., W. (1989) Specificity and mechanisms of gymnemic acid antagonism (inhibition). Chemical senses 14, 859 – 864.
5.Chakravarthy D. and Debanth, N.B (1981) isolation of Gymnemagenin, the sapogenin from Gymnema sylvestre . R.Br.(Asclepiadaceae). Jour. of the Institute of Chemists (India) 53, 155 – 158.
6.Rao. G.S and Sinsheimer, J.E (1971) Constituents from Gymnema sylvestre leavesVIII: isolation, chemistryand derivatives of gymnemagenin and gymnestrogenin. Jour. of Pharmaceutical Sciences 60,, 190 – 193.
7.Stocklin,W. weisis. E. and Reichstein. T. (1967) Gymnemasaure, das antisaccharine prinzip von Gymnema sylvestre R.Br Isolierungen and identifizierungen helvelica chimica acta 50, 474 – 490
8.Reddy M.B. , Reddy K.R., and Reddy M.N., (1989). A survey of plant crude drugs of Ananthapur district, Andhra Pradesh India. International Journal of Crude drug research 27, 145 – 155.
9.Bueding, E., J. Biol. Chem. 202, 505 (1953).
10.Coll, H., Separ. Sci. 6, 207 (1971).
11.Falconer, Pharm. J. 7, 351 (1848).
12.Fraser, R.R., Can. J. Chem. 38, 549 (1960).
13.Rao, G.S., Sinsheimer, J. E., Chem Comman 1681 (1968).
14.Rao, G.S., Sinsheimer, J. E., J. Pharm Sci. 60, 190 (1971).
15.Wagner,J., Schlemmer, W., Hoffmann, H., Arzneim. Forsch 20, 205 (1970).
16.D.E. pegg, J. Appl. Physiol., 19, 123 (1964).
17.B.A. Bouroncle, Cryobiology, 3, 445 (1967).
18.C.E. Huggins, Transfusion, 3, 483 (1963).