Volume 2; Issue 12; December, 2014 Int.J.Curr.Biotechnol. 16 International Journal of Current Biotechnology Journal Homepage : http://ijcb.mainspringer.com Chiluveru Mohan, Aireddy Karnakar, Surapaneni Sateesh, Bhukya Rama Devi, Pamuraju Manjula, Dasari Sreekanth, Bittlingu Keerthi and Prathibha Devi, Phytochemical analysis and in vitro propagation of Gymnema sylvestre R.Br. a valuable medicinal plant, Int.J.Curr.Biotechnol., 2014, 2(12):16-21. Phytochemical analysis and in vitro propagation of Gymnema sylvestre R.Br. a valuable medicinal plant Chiluveru Mohan*, Aireddy Karnakar, Surapaneni Sateesh, Bhukya Rama Devi, Pamuraju Manjula, Dasari Sreekanth, Bittlingu Keerthi and Prathibha Devi Biotechnology and Molecular Genetics Laboratory, Department of Botany, Osmania University, Hyderabad - 500007, India. ARTICLE INFO ABSTRACT Article History: Received 20 November 2014 Received in revised form 30 November 2014 Accepted 05 December 2014 Available online 10 December 2014 Key words: Gymnema sylvestre, qualitative and quantitative analysis, in vitro propaga- tion, nodal explants. The present paper reports the phytochemical and micropropagation studies of a threatened plant, Gymnema sylvestre. Gymnema sylvestre which belongs to the family Asclepiadaceae is a perennial slow growing medicinal woody climber commonly called as “Gudmar”. There is a growing demand for leaves of G. sylvestre in the pharmaceutical trade due to its use as a remedy for diabetes and also as a tonic of the nerves and as a laxative. Propagation of this plant is often hard and expensive. In the present study the qualitative analysis confirmed the presence of various phytochemicals like alkaloids, flavonoids, phenols, tannins, terpenoids and glycosides. Quantitative estimation of flavonoids and phenols was also carried out. In vitro propagation is an alternative method of propagation of the threatened and endangered plant which can aid its conservation. The nodal explants were cultured on MS medium containing different concentration and combinations of growth regulators like 6- benzylaminopurine (BAP) and indoleacetic acid (IAA). Multiple shoot buds were regener- ated successfully from the nodal explants which were efficiently rooted on ½ strength MS medium supplemented with IBA. The regenerated plantlets were successfully transferred to the glasshouse, acclimatized and transferred to the field. *Corresponding author. Email address: [email protected]Introduction Gymnema sylvestre R.Br. which belongs to the family Asclepiadaceae, is a vulnerable species. It is a perennial slow growing woody climber of tropical and subtropical regions (Anonymous, 1997). It is a potent antidiabetic plant and used in folk, homeopathic and ayurvedic systems of medicine (Mitra et al., 1995). It is also used in treating of asthma, eye complaints, inflammations, family planning and snake bite (Anonymous, 1956; Selvanayagam et al., 1995). In addition, it possesses antimicrobial, antihypercholesterolemic (Bishayee and Chatterjee, 1994), hepatoprotective (Rana and Avadhoot, 1992) and sweet suppressing (Kurihara, 1992) activities. It also acts as feeding deterrents to caterpillar, Prodenia eridania (Granich et al., 1974), prevent dental caries caused by Streptococcus mutans (Hiji Yasutake, 1990) and in skin cosmetics (Maeda et al., 1996). There is a growing demand for leaves of G. sylvestre in the pharmaceutical trade due to its use as a remedy for diabetes and also as a tonic of the nerves and as a laxative (Shanmugasundaram et al., 1983), as an anti-sweetner (Kurihara, 1992) and as an antihypercholesterolemic (Bishayee and Chatterjee,1994). It also has stomatic, diuretic and cough suppressant property (Kapoor, 1990). Increasing awareness of the side effects of Western drugs ISSN: 2321 - 8371 have made general public turn towards the herbal medicine, thus the demands for medicinal plants have drastically increased. Due to over exploitation, this plant species has become threatened and is listed in International Union for Conservation of Nature (IUCN) red data book (Shailasree et al., 2012). Gymnema sylvestre is a slow growing, perennial woody climber (Shrivastava and Singh, 2011). Seeds lose viability in a short period of storage (Reddy et al ., l998). Conventional propagation methods are hampered due to its poor seed viability, low rate of germination and poor rooting ability of vegetative cuttings (Komavali and Rao, 2000). The propagation of plant through seed results in less survivability under natural conditions (Anonymous, 1950). Therefore, to fulfill the increasing demand of this potent medicinal plant and population, in vitro culture and micropropagation could be an alternative method to aid in its conservation. Therefore, the propagation of this plant species by alternative methods is needed. The present study of Gymnema sylvestre has been taken up to carry out qualitative phytochemical analysis for alkaloids, flavonoids, tannins, saponins, phenols, steroids, terpenoids and glycosides present in leaves and quantify the flavonoids and phenols and in vitro propagation through direct organogenesis using nodal segments as explants.
The present paper reports the phytochemical and micropropagation studies of a threatenedplant, Gymnema sylvestre. Gymnema sylvestre which belongs to the family Asclepiadaceaeis a perennial slow growing medicinal woody climber commonly called as “Gudmar”. There isa growing demand for leaves of G. sylvestre in the pharmaceutical trade due to its use as aremedy for diabetes and also as a tonic of the nerves and as a laxative. Propagation of thisplant is often hard and expensive. In the present study the qualitative analysis confirmed thepresence of various phytochemicals like alkaloids, flavonoids, phenols, tannins, terpenoidsand glycosides. Quantitative estimation of flavonoids and phenols was also carried out. Invitro propagation is an alternative method of propagation of the threatened and endangeredplant which can aid its conservation. The nodal explants were cultured on MS mediumcontaining different concentration and combinations of growth regulators like 6-benzylaminopurine (BAP) and indoleacetic acid (IAA). Multiple shoot buds were regener-ated successfully from the nodal explants which were efficiently rooted on ½ strength MSmedium supplemented with IBA. The regenerated plantlets were successfully transferred tothe glasshouse, acclimatized and transferred to the field.
IntroductionGymnema sylvestre R.Br. which belongs to the familyAsclepiadaceae, is a vulnerable species. It is a perennialslow growing woody climber of tropical and subtropicalregions (Anonymous, 1997). It is a potent antidiabeticplant and used in folk, homeopathic and ayurvedicsystems of medicine (Mitra et al., 1995). It is also used intreating of asthma, eye complaints, inflammations, familyplanning and snake bite (Anonymous, 1956;Selvanayagam et al., 1995). In addition, it possessesantimicrobial, antihypercholesterolemic (Bishayee andChatterjee, 1994), hepatoprotective (Rana and Avadhoot,1992) and sweet suppressing (Kurihara, 1992) activities.It also acts as feeding deterrents to caterpillar, Prodeniaeridania (Granich et al., 1974), prevent dental cariescaused by Streptococcus mutans (Hiji Yasutake, 1990)and in skin cosmetics (Maeda et al., 1996). There is agrowing demand for leaves of G. sylvestre in thepharmaceutical trade due to its use as a remedy fordiabetes and also as a tonic of the nerves and as a laxative(Shanmugasundaram et al., 1983), as an anti-sweetner(Kurihara, 1992) and as an antihypercholesterolemic(Bishayee and Chatterjee,1994). It also has stomatic,diuretic and cough suppressant property (Kapoor, 1990).Increasing awareness of the side effects of Western drugs
ISSN: 2321 - 8371
have made general public turn towards the herbalmedicine, thus the demands for medicinal plants havedrastically increased. Due to over exploitation, this plantspecies has become threatened and is listed inInternational Union for Conservation of Nature (IUCN)red data book (Shailasree et al., 2012).
Gymnema sylvestre is a slow growing, perennial woodyclimber (Shrivastava and Singh, 2011). Seeds lose viabilityin a short period of storage (Reddy et al., l998).Conventional propagation methods are hampered due toits poor seed viability, low rate of germination and poorrooting ability of vegetative cuttings (Komavali and Rao,2000). The propagation of plant through seed results inless survivability under natural conditions (Anonymous,1950). Therefore, to fulfill the increasing demand of thispotent medicinal plant and population, in vitro cultureand micropropagation could be an alternative method toaid in its conservation. Therefore, the propagation ofthis plant species by alternative methods is needed.
The present study of Gymnema sylvestre has been takenup to carry out qualitative phytochemical analysis foralkaloids, flavonoids, tannins, saponins, phenols,steroids, terpenoids and glycosides present in leavesand quantify the flavonoids and phenols and in vitropropagation through direct organogenesis using nodalsegments as explants.
Materials and MethodsMaterial: Gymnema sylvestre plants were collected fromHerbal garden, N. G. Ranga Agricultural University atHyderabad and planted in the Botanical Garden atDepartment of Botany, Osmania University, Hyderabad.Gymnema sylvestre. is a slow growing, perennial woodyclimber of tropical and subtropical regions with a twiningwoody stem and opposite petiolate leaves, entire, smoothshiny, varying in shape and size according to their age.Flowers are small, in axillary sessile racemes. The root islong, rigid and cylindrical. These plants were subjectedto phytochemical analysis (qualitative and quantitative)for the presence of important secondary metabolitecompounds. Further, a good protocol formicropropagation was developed to aid in itsmultiplication and conservation.
Qualitative analysis:Preparation of plant extract:The plant extract was prepared by grinding 0.5 gm of theplant part (leaf, shoot or root etc…) in 100 ml distilledwater. This extract was filtered through a fine mesh into atest tube. This crude extract was used for the qualitativetests given below (Karthikeyan et al., 2009, Lozoya et al,1989) and the tests were carried out in triplicate.
Test for identification of Alkaloids: About 0.5 gm ofmethanol extract was taken in a test tube and was dilutedand homogenized with 10 ml distilled water, dissolved in20 ml dilute HCl solution and clarified by filtration. Thefiltrate was tested with Drangendroff’s and Mayer’sreagent. The treated solution was observed forprecipitation of white or creamy colour.
Test for identification of Flavonoids: About 0.5 gm ofextract was introduced into 10 ml of ethyl acetate in a testtube and heated in boiling water for 1 min. The mixturewas then filtered. About 4 ml of the filtrate was shakenwith 1 ml 1% aluminium chloride solution and incubatedfor 10 min. Formation of yellow colour in the presence of1 ml dilute ammonia solution indicated the presence offlavonoids.
Test for identification of Phenols: About 0.5 gm of extractwas taken in a test tube, mixed with 100ml distilled waterand heated gently. Ferric chloride solution of 2 ml volumewas added and observed for the formation of green orblue colour.
Test for identification of Saponins: About 0.5 gm ofmethanol extract was taken in a test tube and 5 ml distilledwater was added. Persistent froth was observed uponvigorous shaking of solution. 3 drops of olive oil wasadded to the frothing and shaken vigorously after whichit was observed for the formation of an emulsion.
Test for identification of Steroids: About 0.5 gm ofmethanol extract was taken in a test tube and 2 ml ofacetic anhydride was added to it and 2 ml of sulphuricacid was added along the sides of the test tube andobserved for the colour change to violet or blue green.
Test for identification of Tannins: Five grams of theground powder was extracted with 10 ml ammonicalchloroform and 5 ml chloroform. The mixture was filteredand the filtrate was shaken with 10 drops of 0.5 Msulphuric acid. Creamish white precipitate was observedfor the presence of tannins.
Test for identification of Terpenoids: 5 ml of the methanolextract was mixed with 2 ml of chloroform and 2ml
concentrated sulphuric acid. The layer interface wasobserved for reddish brown coloration which indicatesthe presence of Terpenoids.
Quantitative analysis:Quantitative analysis was carried out to estimate totalphenols and total flavonoids.
Determination of total phenols: Folin Ciocalteu reagentmethod (Mc Donald et al., 2001) with some modificationswas adopted for total phenolic content determination.The root extract (1.0 ml) was mixed with Ciocalteu reagentand allowed to stand for 15 min and 5 ml of saturatedNa2CO3 was added. The mixture was allowed to stand for30 min at room temperature and the total phenols weredetermined spectrophotometrically at 760 nm. Gallic acidwas used as a standard. Total phenol values areexpressed in terms of gallic acid equivalent (mg/g ofextracted compound).
Determination of total flavonoids: Aluminium chloride -colorimetric method (Chang et al., 2002) with somemodifications was used to determine flavonoid content.1.0 ml root extract was mixed with 1.0 ml methanol, 0.5 mlaluminium chloride (1.2 %) and 0.5 ml potassium acetate(0.1176%). The mixture was allowed to stand for 30 min atroom temperature. Later the absorbance was measuredat 415 nm. Quercetin was used as standard. Flavonoidcontent is expressed in terms of quercetin equivalent (mg/g of extracted compound).
Micropropagation studies:Gymnema sylvestre plants were subjected to tissueculture and a good protocol for micropropagation wasdeveloped to aid in its multiplication and conservation.The micropropagation studies comprised the culture ofnodal explants on defined culture media under standardgrowth conditions. The nodal explants were collectedfrom mature and healthy field grown plants. They werewashed under running tap water for 20 min followed bysoaking in 0.1 % (v/v) liquid detergent Tween-20 for 5min and then subsequently washed with tap water. Theexplants were then soaked in 70% ethanol for 5 minfollowed by washing with water. Finally the explants weresurface sterilized with 0.1% solution of mercuric chloridefor 3 to 5 min followed by thorough rinsing in steriledistilled water. A total of thirty explants were inoculatedin culture tubes containing MS medium (Murashige andSkoog, 1962) augmented with 2 % sucrose and 0.8 %agar and different combinations and concentrations ofvarious plant growth regulators. The experiment wascarried out in triplicates. Prior to that, the medium wasadjusted to pH 5.8, autoclaved at 121o C for 15 lbs / cm2
for 15 min and allowed to cool before inoculation. Theculture media comprised of the following: MS + BAP(0.5, 1.0, 1.5 and 2.0 mg/l) and MS + BAP (0.5, 1.0, 1.5 and2.0 mg/l) + IAA (0.5 mg/l). All the inoculated cultureswere incubated in sterile growth room under controlledconditions of 22 ± 1oC temperature, 75 % humidity and2000 lux illumination of 16 hr / 8 hr L/D cycle. The 2 cmlong regenerated shoots were transferred to rootinducing media comprising half MS mediumsupplemented with IBA (0.5, 1.0 and 1.5 mg/l). Theregenerated plantlets were later transplanted to potscontaining a mixture of soil and vermicompost in the ratioof 2:1. The plantlets were gradually acclimatized on thelaboratory bench by covering with a plastic bag withholes (to maintain high humidity), which were openedup gradually over a period of one week. The plants in thepots were moved to the glasshouse to a shaded area andgradually acclimatized.
Table – 1: Qualitative analysis of the plant extracts of Gymnema sylvestre to screen for the presence of phytochemicals.
+ve= present, -ve= absent
S. No Plant extract Phytochemicals Average Estimated value (mg/gm) (Mean ± S.E)
1. 2.
Flavonoids Phenols
Leaf Leaf
17.98 ± 0.55 40.22 ± 0.74
Table - 2: Quantitative analysis of the methonol extracts of Gymnema sylvestre for estimation of Flavonoids andPhenols.
* Phenols are expressed as Gallic acid equivalent (GAE) and Flavonoids are expressed as Quercetin equivalents(QE) in mg/100 gm
Culture medium No. of explants with shoot induction
Percentage of shoot induction* (Mean±S.E)
MS + BAP (0.5 mg/l) 56 3.14±1.00 MS + BAP (1.0 mg/l) 60 3.58±1.79 MS + BAP (1.5 mg/l) 68 4.23±2.01 MS + BAP (2.0 mg/l) 77 4.80±1.81 MS + BAP (0.5 mg/l) + IAA (0.5 mg/l) 51 1.55±0.22 MS + BAP (1.0 mg/l) + IAA (0.5 mg/l) 52 1.76±0.45 MS + BAP (1.5 mg/l) + IAA (0.5 mg/l) 54 2.00±0.89 MS + BAP (2.0 mg/l) + IAA (0.5 mg/l) 58 2.15±0.92 *The value was calculated as the percentage of nodal explants that have produced shoots out of a total number of
inoculated explants (90).
Table – 3: Efficiency of shoot regeneration and production of multiple shoots from nodal explants of Gymnemasylvestre, on different culture media
Culture medium No. of shoots with root induction
Percentage of root induction* (Mean ± S.E)
MS + IBA (0.5 mg/l) 50 8.82±0.043 MS + IBA (1.0 mg/l) 60 10.00±0.124
MS + IBA (1.5 mg/l) 80 12.93±0.116
Table – 4: Percentage of root induction from multiple shoots regenerated from nodal explants of Gymnema sylvestre.
*The value was calculated as the percentage of shoots with root induction out of a total number of inoculated shoots.
Figure 1 A-F: A. Gymnema sylvestre plant, B. Shoot regeneration from nodal explants, 12 days after inoculation, C.Multiple shoots, 25 days after inoculation, D. Rooting from regenerated shoot, 15 days after inoculation of shoot, E.Acclimatization of regenerated plantlet, F. Regenerated plant transferred to the field.
Results and discussionThe present study contributes valuable information ofbioactive compounds in G. sylvestre. Qualitative analysisof plant extract was carried out for Alkaloids, Flavonoids,Saponins, Phenols, Tannins, Steroids, Terpenoids andGlycosides. All of the phytochemicals like AlkaloidsFlavonoids, Phenols, Tannins, Terpenoids andGlycosides were present in Gymnema sylvestre exceptSaponins and Steroids (Table-1) which is similar to thereports of Vaghasiya et al. (2011) and Han et al. (2007).The plant extracts were quantitatively analyzed forFlavonoids and Phenol (Table-2). Whereas, our studyreports the absence of Saponins, Kalidas et al. (2010)and Ajaiyeoba (2000) indicated that Saponins werepresent in G. sylvestre in the aqueous extract. Severalmedicinal properties have been attributed to Saponins(Gopinath, 2012) and Kalidas et al. (2010) but surprisingly,Saponins were not found in the present study. Flavonoidsand Phenol are however reported in the present studywhich agrees with the findings of Vaghasiya et al. (2011)who has attributed antidiabetic, anti-aging anti-inflammation and bactericidal effects.
An efficient micropropagation protocol was developedwith a high percentage of shoot regeneration and multipleshoots (fig.1-A to C). The highest response of 4.80±1.81for production of multiple shoots was recorded with MS+ BAP (2.0mg/l) followed by 4.23±2.01 in MS + BAP (1.5mg/l) (Table-3). The explants proliferated by 5-8 days andshoot regeneration was observed by 10-15 days (fig.1-D). Shoots of about 2 cm with 2-3 nodes were producedby 25 days. These were cultured on root induction mediacontaining different concentrations of IBA (0.5, 1.0, 1.5mg/l) to induce roots. The higher concentration of IBA(1.5 mg/l) produced better rooting efficiency of12.93±0.116 (Fig.1-E; Table-4). The regenerated plantswere transferred to the glasshouse for acclimatization(fig.1-F). Out of a total of 720 explants (pooled fromtriplicates) inoculated, 476 explants could regenerateshoots and 190 shoots were inoculated on rooting mediafor root induction out of which 152 shoots could developroots to enable 121 plants to be transplanted out of which96 plants survived in pots. In the present study, differentconcentrations of BAP and BAP with IAA were used toinduce regeneration. Komalavalli N & Rao M.V (2000)however, reported the regeneration of Gymnema sylvestrethrough the use of BAP and KN individually andcombined with NAA. The present results agree well withthe above report with supplementation of BAPindividually or in combination but a higher frequency ofregeneration was obtained with BAP (2.0 mg/l) presently.Manonmani and Francisca, (2012) reported the plantregeneration of Gymnema sylvestre from nodal explantson MS medium supplemented with BAP (1.0 mg/l) andNAA (2.0 mg/l) whereas, in our present report use ofBAP individually produced the highest shootregeneration frequency without any additionalsupplementation of NAA. In the present study it wasobserved that MS + IBA combination produced efficientrooting compared to other reports where they achievedrooting on MS medium supplemented with NAA. Thisefficient high frequency plant regeneration protocoldeveloped presently can be taken up for large scalemicropropagation for its multiplication and conservation.Further, the information of phytochemical analysis wouldbe useful to the pharmaceutical industry.
ConclusionIt is concluded that Gymnema sylvestre is a plant with avariety of ethnic medicinal uses. The qualitative andquantitative analysis of G. sylvestre shows the presenceof bioactive compounds such as Alkaloids Flavonoids,Phenols, Tannins, Terpenoids and Glycosides. This isvaluable information for preparation of drugs inpharmaceutical industry and stress the need for moreintensive research since they play a great role inhealthcare. The present study describes the successfuldevelopment of rapid micropropagation protocol ofGymnema sylvestre. This protocol provides a successfultechnique for multiplication and conservation of thevaluable medicinal plant which is used in treating variousdisorders. The protocol developed presently can be takenup in large scale to produce the planting material fordevelopment of medicinal plant cultivation programmesand it can also help the pharmaceutical industry.
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