Research Article ISSN: 0974-6943

A.Sathya et al. / Journal of Pharmacy Research 2010, 3(5),984-987

Available online through www.jpronline.info Pharmacognostical, Phytochemical and GC-MS Investigation of Successive extracts of Gymnema syvestre R.Br.
A.Sathya, *R.Ramasubramaniaraja, P. Brindha
Centre for Advaced Research and Indian System of Medicine (CARISM), SASTRA University, Thirumalaisamudram, Thanjavur - 613 401.Tamil Nadu, India. *SCBT, SASTRA University, Thanjavur - 613 401.Tamil Nadu, India.

Received on:25-02-2010; Revised on: 16-03-2010; Accepted on:12-04-2010 ABSTRACT Gymnema sylvestre R.Br is a very effective antidiabetic plant in Indian System of Medicine. It is used in treating ailments of throat, eye, heart, stomach and dental. It has to be ensured that only genuine and uniform raw materials need to be used in the preparation of herbal formulations. The present research describes the simplest methods for identification of phytochemicals like terpenoids, glycosides, and alkaloids from the successive leaf extracts of Gymnema sylvestre R.Br (Asclepiadaceae), a medicinal plant widely used in India. The Pharmacognostical details of Gymnema sylvestre have also been analysed scientifically. By using gas chromatography coupled to mass spectrometry (GC-MS), terpenoids, gylcosides, Saturated and unsaturated fatty acids and alkaloids were identified. These are reported for the first time in Gymnema sylvestre R.Br. Keywords: Gymnema sylvestre., GC-MS Terpenes, Phenolic compounds,fatty acids, Alkaloids INTRODUCTION Gymnema sylvestre R. (Br.) (Asclepiadaceae) is a woody climber, widely distributed in Southern China, India, and Vietnam. The plant is called Gur-mar in India and is well known for masking sweet taste. It is widely used in indigenous system of medicine for treatment of diabetes mellitus. It also has stomachic, diuretic and cough suppressant activity (Kapoor, L.D and etal1990, Sastri, B. N and etal 1956). In view of the importance of G. sylvestre in traditional and modern system of medicines, it was thought worthwhile to develop quality standard for the same. As far as botany and chemistry of this plant is concerned, large number of scientific data is available but a systematic standardization study is still lacking. Hence, in the present investigation an attempt has been made to standardize the G. sylvestre leaves using Pharmacognostical physicochemical values, phytochemical Screening and investigation of plant leaves in successive extracts like Pet ether, Chloroform, Methanol Extracts of Gymnema sylvestre R.Br. using GS-MS. MATERIALS AND METHODS Plant material Macroscopic Characters and Physiochemical Parameters: The Macroscopic evaluation was carried out for shape, size, color, odor, taste and fracture of the drug.( Anonymous, 1996) Different physio-chemical values such as ash value, extractive values, loss on drying, foreign organic matter, Crude fiber content, were also determined. Preparation of successive Extracts The leaves were dried under shade, powdered and passed through 40 mm meshes and stored in closed vessel for further use. The dried powder material (500g) was subjected to soxhelt extraction with petroleum ether (40-600c),Chloroform and Methanol (in order of increasing polarity) for continuous hot extraction. The extracts were concentrated under reduced pressure to obtain the extracts solid residues. The percentage values (%w/w) were 7.7, 9.2, and 11.3 respectively. Phytochemical Evaluation

The leaves of Gymnema sylvestre R.Br. were collected from Preliminary Phytochemical screening of drug was carried out Mooligai Pannai, 7km away from Thanjavur (Tamilnadu) in the month of as per method described by Peach and Tracy et al, 1955 August 2009. The plant was identified by local people of that village and authenticated by Dr. M. Jegadeesan, Professor and Head, Depart- GC-MS investigation of three successive extract of Gymnema sylvestre ment of Environment and Herbal Science, Tamil University Thanjavur, R.Br. The Voucher specimen (TUH-126A) had been preserved in laboratory for future reference. A Volume of 1l of clear extract was injected into GC-MS (PerkinElmer Clarus 500) with a oven programming of 50C @10 C/min Chemicals to 150 C@8C/min to 280C (10min). The injector temperature was mainAll the reagents used were of analytical grade obtained from Merck- tained at 280C. The split ratio was set as 1:8. The carrier gas used in the India. analysis was helium which had the flow rate of 1ml/min. A 30 m Capillary column of elite 5ms, with a Column id of 250 m was used. The com*Corresponding author. pounds were detected in the range of 40- 450amu by matching with Dr.A.Sathya Msc., PhD.,, NIST library.( Massada Y ., and et al 1976, Adams R. P andet al 2004, Jack Asst Professor-III Carism, SASTRA University,Thirumalai Samudram,Thanjavur- 613 401.Tamil Cazes and et al 2004)
Nadu, India.
Tel.: + 91- 9003914122 Telefax: +91-04362-264346 E-mail:sathyaalbert@gmail.com

Journal of Pharmacy Research Vol.3.Issue 5.May 2010

984-987

A.Sathya et al. / Journal of Pharmacy Research 2010, 3(5),984-987 RESULTS AND DISCUSSION Macroscopic characters of G.sylvestre leaves The following macroscopic features were observed for the leaves; the leaves were Green in colour and were 2.2-2.0cm in length and 1.3-1.30cm in width. Their shape was even-pinnate or ovate, the taste was extremely bitter, and had a Characteristic odour. It had pubescence. The leaves were simple, opposite with acute apex, rounded to cordate base and had a reticulate venation and had a 5-13cm long pubescent petiole. Physiochemical parameters of G.sylvestre leaves Based on the polarity the extractive values of the leaves of were analysed. The extractive value was highest in water and was recorded to be 30.56%w/w, and methanol soluble extractive value was about 20.8%w/w .The different ash values and the different physiochemical parameters were screened and are presented in the table No.1. Phytochemical screening of Gymnema sylvestre leaves Successive extracts like pet ether, chloroform and methanolic extracts of G.sylvestre leaves were qualitatively screened and are presented in the table No: 2. Triterpenes, Phenolic groups and flavonoids were present in all the three extracts, Carbohydrates, sapponins and Tannins were present only in the methanolic extract of G.sylvestre. GC-MS investigation of three successive extract of Gymnema sylvestre R.Br. The results of the GC-MS analyses on Successive extracts like Pet ether, chloroform and methanolic extracts of the leaves of Gymnema sylvestre is presented in Table 3-12. A total of 19 compounds were identified from the Pet ether extracts of G.sylvestre. The identified compounds were of different types of Terpenes, and the highest % Peak area of 69.136 is limonene (Retention time 8.47), Saturated and unsaturated fatty acids, recorded the next highest % peak area of 14.867. Hexadecanoic acid ethyl ester(Retention time 23.11), Phenolic groups had a % Peak area of 1.175 and the name of the phenolic group was identified as Phenol, 2,4-bis(1,1-dimethylethyl)(Retention time 16.42), Hydrocarbons followed the order of % peak area of 0.414 and the name was identified as Undecane, 3,7-dimethyl-(Retention time 12.28). A total of 13 compounds were identified from the Chloroform extracts of G.sylvestre.Alkaloids identified recorded a % peak area of 0.186 and name has been identified as 2-Isopropylpiperazine (Retention time 3.96), hydrocarbons ranked next with a% Peak area of 1.957 and the peak had been identified as Tridecane (Retention time 8.74). Aldehyde groups followed the list with a % peak area of 0.125 and its name has been noted as Butanal, 3-hydroxy- ( Retention time 9.56), Saturated and unsaturated fatty acids followed the list with a % Peak area of 16.068 and the corresponding name has been recorded as 9-Octadecenoic acid (Z)-, methyl ester (Retention time is 24.49). A Total of 7 compounds had been identified from the Methanolic extract of G.sylvestre. The identified compounds include Terpenes recording the highest % Peak area of 52.101 and the respective name has been identified as Limonene (Retention time 8.50), Saturated and Unsaturated fatty acids with a % Peak area of 0.787 have recorded the name as 9-Octadecenoic acid (Z)-, methyl ester (Retention time 24.44).

Table: 1 Physiochemical parameters of Gymnema sylvestre leaves

S.N0 1. 2. 3. 4. 5. 6. 7. Parameters Hexane soluble extractive Pet ether soluble extractive Chloroform soluble extractive Acetone soluble extractive Ethanol soluble extractive Ethylacetatesoluble extractive Methanol soluble extractive %w/w 3.04 5.24 2.344 7.12 9.256 3.92 20.8 S.No 8. 9. 10. 11. 12. 12. 13. Parameters Water soluble extractive Foreign organic matter Loss on drying Crude fiber content Total ash Acid insoluble ash Sulphated ash %w/w 30.56 0.38 5.6 14.05 8.3 0.9 15.3

Table: 2 Preliminary Phytochemical screening of Gymnema sylvestre leaves Extracts

Phytoconstituents Alkaloids Aminoacids Anthaquinones Carbohydrates Catechins Flavonoids Phenolic groups Resins/gums Saponins Steroids Tannins Triterpenes Petroleum ether + + + + Chloroform + + + Methanol + + + + + +

Chromatogram of Pet ether Extract of G.sylvestre:

, 16-FEB-2010 + 11:22:01
petther extract 100

7.68 41

0 5.46 5.96 6.46 6.96 7.46 7.96 8.46 8.96 9.46 9.96 10.46 10.96

-Myrcene

Limonene

8.47 68

Scan EI+ TIC 1.38e10

Time 11.46

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100

11.44 57

Phenol, 2,4-bis(1,1-dimethylethyl)-

petther extract

Disulfide, bis(1-methylpropyl)

A.Sathya et al. / Journal of Pharmacy Research 2010, 3(5),984-987 Chromatogram of Pet ether Extract of G.sylvestre: Table No: 6List of most probable Hydrocarbons of Pet ether extract of , 16-FEB-2010 + 11:22:01 Gymnema sylvestre.
1-Hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene
Scan EI+ TIC 4.13e8

S.No

Compound name

Molecular formula C10 H 14 C14 H 30 C13 H 28 C16 H 34

Molecular weight 134 198 184 226

Retention time 8.90 11.04 12.28 17.70

%Peak Area 0.169 0.215 0.414 0.028

16.42 191

1. 2. 3. 4.
Hexadecane

Undecane, 3,7-dimethyl-

n-Nonadecane

Dodecanoic acid, ethyl ester

2,6-Dimethyl-1,3,5,7octatetraene, E,ETetradecane Undecane, 3,7-dimethylHexadecane

Tetradecane

Undecanoic acid

Hexadecane

11.96 175

Chromatogram of Chloroform Extract:

chloroform extract 100

Benzene, 1,3-bis(1,1-dimethylethyl)-

, 16-FEB-2010 + 10:39:32
Scan EI+ TIC 2.88e8

Hexadecane

12.28 57

1,6;3,4-Dianhydro-2-deoxy--d-lyxo-hexopyranose

12.43 44

13.09 44

14.61 44

15.38 44

Tridecane

11.47

11.97

12.47

12.97

13.47

13.97

14.47

14.97

15.47

15.97

16.47

16.97

17.47

Dodecane, 2,6,10-trimethyl-

Time

Oxalic acid, 2-ethylhexyl hexyl ester

12.29 71

Oxalic acid, allyl decyl ester

3-Dimethylsilyloxytridecane

Oxalic acid, allyl decyl ester

Butanal, 3-hydroxy-

Diglycerol

Decane

7.58 44

S.No

Compound name

Molecular formula C10 H 16 C10 H 16 C10 H 14 C13 H 20 O

Molecular weight 136 136 134 192

Retention time 7.68 8.47 8.90 9.38

%Peak Area 1.858 69.136 0.169 0.113

0

8.84 44

5.54 44

7.33 44

8.20 8.05 44 44

9.47 44

11.04 44 11.22 44

9.56 44

10.64 44

11.68 44

12.44 44

13.10 44 12.68 44 13.27 44

1. 2. 3. 4.

-Myrcene Limonene 2,6-Dimethyl-1,3,5,7octatetraene, E,E6,8-Nonadien-2-one, 6-methyl-5-(1methylethylidene)1,6-Octadien-3-ol, 3,7-dimethyl1-Hydroxy-1,7dimethyl-4-isopropyl2,7-cyclodecadiene Phytol

Hexadecane

Tridecane

Table No: 3List of most probable Terpene compounds of Pet ether extract of Gymnema sylvestre.

Decane, 2,4,6-trimethyl-

8.74 43

Undecane, 4,6-dimethyl-

Hexadecane

11.04 44

14.45 44

15.93 44

16.74 44

11.96 175

17.70 44 17.16 17.61 44 44

5.46

6.46

7.46

8.46

9.46

10.46

11.46

12.46

13.46

Time

Table No: 7 List of probable Alkaloid compounds Chloroform extract of Gymnema sylvestre;
S.No Compound name Molecular formula C7H16N 2 Molecular weight 128 Retention time 3.96 %Peak Area 0.186

5. 6.

C10 H 18 O C15 H 26 O

154 222

9.55 16.74

0.144 1. 0.353 2-Isopropylpiperazine

7.

C20 H 40 O

293

24.57

1.963

Table No: 8 List of probable hydrocarbon compounds Chloroform extract of Gymnema sylvestre.
S.No Compound name Molecular formula C15 H 34 OSi C13 H 28 C15 H 32 C10 H 22 C13 H 28 Molecular weight 258 184 212 142 184 Retention time 7.33 8.74 8.84 10.64 10.97 %Peak Area 0.663 1.957 0.468 0.228 0.115

Table No: 4 List of probable saturated and unsaturated fatty acid compounds Pet ether extract of Gymnema sylvestre.
S.No Compound name Molecular formula C11 H 22 O 2 C14 H 28 O 2 C16 H 32 O 2 C17 H 34 O 2 C18 H 36 O 2 C19 H 34 O 2 Molecular weight 186 228 256 270 284 294 Retention time 17.16 17.61 20.51 22.27 23.11 24.38 %Peak Area 0.287 0.078 0.195 1.311 14.867 1.083

1. 2. 3. 4. 5.

1. 2. 3. 4. 5. 6.

Undecanoic acid Dodecanoic acid, ethyl ester Tetradecanoic acid, ethyl ester Hexadecanoic acid, methyl ester Hexadecanoic acid, ethyl ester 9,12Octadecadienoic acid, methyl ester

3-Dimethy lsilyloxytridecane Tridecane Dodecane, 2,6,10trimethylDecane Tridecane

Table No: 9 List of probable Aldehyde Compounds of Chloroform sylvestre

S.No Compound name Molecular formula C5 H 10 O C4 H 8 O 2 Molecular weight 86 88 Retention time 9.38 9.56 %Peak Area 0.059 0.125

1. 2.

Pentanal Butanal, 3-hydroxy-

Table No: 5 List of probable Phenolic compounds Pet ether extract of Gymnema sylvestre.
S.No Compound name Molecular formula C17 H 24 O 2 Molecular weight 260 Retention %Peak time Area 9.07 0.087

Table No: 10 List of probable saturated and unsaturated fatty acid compounds Chloroform extract of Gymnema sylvestre
S.No Compound name Molecular formula C12 H 24 O 2 C17 H 34 O 2 C19 H 36 O 2 C20 H 36 O 2 C25 H 40 O 2 Molecular weight 200 270 296 308 372 Retention time 17.18 22.30 24.49 25.23 26.84 %Peak Area 1.199 8.056 16.068 5.891 1.968

1. 2. 3. 4.

1.

2.

Cyclohexanol, 5-ethyl-2-(1methylethyl)-, enzoate, [1R-(1,2,5)]Phenol, 2,4-bis(1,1dimethylethyl)-

C14 H 22 O

206

16.42

1.175

5.

Dodecanoic acid Hexadecanoic acid, methyl ester 9-Octadecenoic acid (Z)-, methyl ester 9,12-Octadecadienoic acid, ethyl ester 9-Octadecenoic acid (Z)-, phenylmethyl ester

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A.Sathya et al. / Journal of Pharmacy Research 2010, 3(5),984-987

Chromatogram of Methanolic extract of G.sylvestre:

, 16-FEB-2010 + 12:32:10
methanol extract 100

Table No: 11 List of most probable Terpene compounds of Methanol e x t r a c to f Gymnema sylvestre.
S.No Compound name Molecular formula C10 H 16 C10 H 16 C10 H 16 C20 H 40 O Molecular weight 136 136 136 296 Retention time 7.72 8.50 9.42 24.58 %Peak Area 3.061 52.101 0.036 0.243

Limonene

8.50 68

Scan EI+ TIC 1.58e10

1.
Bicyclo[3.1.1]heptane, 6,6-dimethyl-2-methylene-, (1S)-

Benzoic acid, 2-hydroxy-, methyl ester

2. 3. 4.

Bicyclo[3.1.1]heptane, 6,6dimethyl-2-methylene(1S)Limonene 4-Carene Phytol

Table No: 12 List of probable saturated and unsaturated fatty acid compounds Methanol extract of Gymnema sylvestre.
S.No Compound name Molecular formula C17 H 34 O 2 C19 H 34 O 2 C19 H 36 O 2 Molecular weight 270 294 296 Retention time 22.27 24.38 24.44 %Peak Area 0.376 0.321 0.787

11.21 120

7.72 41

1.
Time 8.39 8.89 9.39 9.89 10.39 10.89 11.39 11.89 12.39 12.89 13.39 13.89 14.39

0 7.39 7.89

2. 3.

, 16-FEB-2010 + 12:32:10
methanol extract 100
27.44 55

Scan EI+ TIC 2.90e9

Hexadecanoic acid, methyl ester 9,12-Octadecadienoic acid, methyl ester, (E,E)9-Octadecenoic acid (Z)-, methyl ester

Methyl ricinoleate

9,12-Octadecadienoic acid, methyl ester, (E,E)-

9-Octadecenoic acid (Z)-, methyl ester

Hexadecanoic acid, methyl ester

Hexadecanoic acid, ethyl ester

12-Methyl-E,E-2,13-octadecadien-1-ol

for the presence of Phytoconstituents using GC-MS. Finally the GC-MS analyses revealed that the three successive extracts of Gymnema sylvestre were mainly observed to contain Terpenes, Saturated and unsaturated fatty acids eluting at different retention times with varied percentage of peak area. REFERENCE:
27.55 59

Methyl ricinoleate

22.27 74

23.12 88

24.44 55 24.58 24.38 44 44

Phytol

1. 2. 3.

25.17 44

26.80 55

0 20.61

21.61

22.61

23.61

24.61

25.61

26.61

27.61

Time 28.61

4. 5. 6.

CONCLUSION: The Gymnema sylvestre R.Br. has been subjected to prelimiminary evaluation of macroscopical characters, Polarity based preparations of the successive extracts were scrutinized and screened

7.

Kapoor, L.D.: CRC Handbook of Ayurvedic Medicinal Plants: Boca Raton, FL, pp 200-201 (1990). Sastri, B. N.: The Wealth of India: Raw materials. Vol IV, CSIR, Delhi, pp 275 (1956). Anonymous; (1996) Pharmacopoeia of India. Govt. of India, Ministry of Health, Controller of publication, New Delhi. Peach, K., and Tracy, M.V. (1955), Modern Methods of Plant Analysis. SpringerVerlag. Heidelberg Massada Y., Analysis of Essential Oil by Gas Chromatography and Mass pectrometry, John Wiley and Sons, New York, U.S.A., 1976. Adams R. P., Identification of Essential Oil Component by Gas Chromatography/ .Quadrupole Mass spectroscopy, Allured publishing corporation, Illinois, U.S.A., 2004 Jack Cazes., Encyclopedia of chromatography, CRC Press, 2004, 101-103.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.3.Issue 5.May 2010

984-987