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Int J Pharm Biomed Sci 2010, 1(3), 37-40 ISSN No: 0976-5263

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Preliminary phytochemical studies on the leaves of Hardwickia binata Roxb. (Fabaceae)

The present study deals with the detailed preliminary phytochemical and GSG. Gunaselvi*, V. Kulasekaren, MS studies on leaves of Hardwickia binata Roxb (Fabaceae). The physico chemical G. Magalakshmi, V. Gopal
Department of Pharmacognosy, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, Gorimedu, Pondicherry, India *Correspondence: Ms. G. Gunaselvi Tel: +91 9791682233 Email: gunampharm27@gmail.com

parameters such as foaming index, crude fiber content moisture content, ash value, extractive value and histological parameters presented in this paper may be proposed as parameters to establish the authenticity of Hardwickia binata Roxb (Fabaceae) and can possibly help to differentiate the drug from its other species. The preliminary phytochemical examination of various extracts and GC-MS studies were used to detected the presence of compounds in leaves of Hardwickia binata Roxb (Fabaceae). Key words: Preliminary phytochemical, GC-MS studies, Physico chemical parameters, Leaves extracts.

Received: 15 Nov 2010 / Revised: 10 Dec 2010 / Accepted: 12 Dec 2010 / Online publication: 15 Dec 2010

1. INTRODUCTION Hardwickia binata Roxb (Fabaceae) is commonly known as anjan synonym is Hardwickia trapeziformis R. Grah. Hardwickia binata is a moderate-sized to large tree, up to 24-30 m tall, girth 1.8-3.0 m with a clean cylindrical bole up to 12-15 m; graceful, drooping slender branches; crown conical in early life, becoming broader later. Leaves small, 2-6 cm long by 2-3 cm wide, alternate, pinnate, almost kidney shaped and greyish-green. The natives of Chhattisgarh Plains used this leaves for headache. The leaves are collected and by crushing it in water, an aqueous paste is prepared. This paste is applied externally on painful parts as treatment [1]. The natives of Kanker region used leaves for purgative and can be used in treatment of constipation. Medicinal plants are an important therapeutic aid for various ailments. To the best of our knowledge, there are few pharmacognostical studies on the leaves of Hardwickia binata Roxb. (Fabaceae) has been reported in the literature. Therefore, the present investigation was planned to study the preliminary phytochemical, physico chemical parameters and GC-MS studies on leaves of Hardwickia binata Roxb. (Fabaceae). 2. MATERIALS AND METHODS 2.1 Collection of plant material

The plant leaves of Hardwickia binata Roxb(Fabaceae) were collected from foot of the Arunachala hills, Tiruvannamalai, Tamil Nadu and the collected plant materials were botanically identified and confirmed by B.R.RAMESH, Director Of Research, French institute of Puducherry. 2.2 Preparation of extracts The collected leaves were chopped into small pieces separately, shade-dried, and coarsely powdered using a pulverizor. Then, the powder was passed through sieve no. 22 and stored in tightly closed amber color bottles at room temperature away from sunlight. The coarse powders were subjected to successive extraction with organic solvents by Soxhlet method [2]. Each time before extraction with the next solvent, the powdered material were dried in hot air oven at below 50oC. Finally extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvents was removed in vacuo. The resulted extracts were used for further studies. 2.3 Histochemical color reactions The Histochemical color reactions were carried out on the leaf transverse sections by the reported methods with various chemical reagents [3-7]. The result was recorded in Table 1.
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G. Gunaselvi et al., Int J Pharm Biomed Sci 2010, 1(3), 37-40 Table 1 Histochemical color reactions Reagent Phloroglucinol + HCl Weak iodine solution Conc. H2SO4 Dragendorffs reagent 5% Aq. KOH Color observed Pink Blue Green No Light orange No Greenish Inference Due to Lignin Due to Starch Due to Cellulose Absence of Alkaloids Absence of Glycosides

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2.5 Preliminary phytochemical screening The various extracts of leaves of Hardwickia binata Roxb (Fabaceae) were subjected to preliminary phytochemical tests by standard methods [13-17]. These results were recorded in Table 4. 2.6 Fluorescence analysis

Table 2 Physico-Chemical Analysis S. No. 1 2 3 Physico chemical parameters Foaming index Loss on drying Crude fiber content Value (% w/w) Less than 100. 8.7 27.6

The analysis of powder and various extracts of the leaves of Hardwickia binata Roxb (Fabaceae) under day and under near and far UV [13,14,18]. These results were recorded in Table 6 and Table 7. 2.7 GC-MS studies The chemical composition of the petroleum ether, chloroform and ethanolic extracts of the leaf of Hardwickia binata Roxb (Fabaceae) was determined by a GC-MS analysis. Interpretation of mass spectrum GC-MS was conducted using the data base of National Institute of Standard And Technology (Nist version year 2005 Inlet line) having more than 62,000 patterns. The spectrum of unknown compounds was compared with the spectrum of the known compounds stored in the Nist library. The name molecular weight and structure of the compounds of the extract were ascertained. The results of the compounds identified in the GC-MS study on the petroleum ether, chloroform and ethanolic extracts of leaves of Hardwickia binata Roxb (Fabaceae) given in Table 9 and Table 10. 3. RESULTS AND DISCUSSION 3.1 Histochemical color reaction Histochemical color reaction was studied to detect the presence of phytoconstituents. Lignin, cellulose and starch were found to be present and alkaloids & glycosides were found to be absent. 3.2 Physico Chemical Parameters The physico chemical parameters such as foaming index, crude fiber content moisture content, ash value, and extractive value of the selected drug were determined to establish the pharmacognostical parameters of the selected drug. The determination of foaming index was carried out to estimate the saponins content of the drug .The crude fibre content was also estimated. The percentage of chemical constituents in selected drugs is mentioned on air dried basis, the moisture content of the drug was also determined. The moisture content of a drug should be minimum in order to prevent decomposition of the drug either due to chemical changes or microbial contamination. The ash value was determined by three different methods, which measured total ash, acid-insoluble ash, and water-soluble ash.
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Table 3 Ash Values Sl. No. 1 2 3 4 Ash values Total ash Water soluble ash Acid insoluble ash Sulphated ash Yield (% w/w) 5.47 4.35 3.89 4.97

Table 4 Extractive Values Solvents Batch process Petroleum ether Acetone Benzene Carbon tetrachloride Chloroform Ethanol Hexane Methanol Water Successive method Petroleum ether Chloroform Ethyl Acetate Ethanol Extractive values (%w/w) 9% 14 % 8% 9% 11 % 13 % 9% 17 % 16 % 4.3 % 6.3 % 7.3% 11.3 %

2.4 Physico-chemical analysis Physico-chemical analysis on leaves of Hardwickia binata Roxb (Fabaceae) was carried out with the WHO guidelines. The various parameters considered were Foaming index [8], Loss on drying [9], Ash values (Total ash, Water soluble ash, Acid insoluble ash and sulphated ash) [9], Extractive values [4,9,11,12] and qualitative and quantitative estimation of inorganic elements was carried out by using WD-XRF spectrometry to detect the amount of various inorganic elements. The result was recorded in table: 2.and the crude fibre content of the powdered plant material was carried out by Dutch method [10]. These results were as recorded in Table 2-5.
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G. Gunaselvi et al., Int J Pharm Biomed Sci 2010, 1(3), 37-40 Table 5 Qualitative & quantitative estimation of inorganic elements by using WDXRF spectrometer Sl. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Inorganic elements Bromine Calcium Chlorine Copper Iron Potassium Magnesium Manganese Phosphorus Sulphur Silicon Strontium Zinc Vanadium Chromium Arsenic Rubidium Nickel Zirconium Barium Cerium Yield 1.08% 78.90% 0.834% 0.121% 0.729% 8.740% 2.18% 0.272% 1.15% 2.997% 2.62% 0.204% 0.177% 5 ppm 5 ppm 8 ppm 8 ppm 4 ppm 8 ppm 296 ppm 10 ppm

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The extractive values are indicative of approximate measures of the chemical constituents present. Taking into consideration the diversity in chemical nature and properties of contents of drugs, various solvents are used for determination of extractive values. These experiments were repeated thrice in order to arrive at standard values. The results showed greater extractive values (almost double) in water, methanol and ethanol extraction, indicating the effect of chemical compounds present in the plant. Qualitative and quantitative estimation of inorganic elements was carried out by using WD-XRF spectrometry to detect the amount of various inorganic elements. The result indicated that the presence of 21 elements. The results indicate that the leaves are rich in calcium and potassium. These parameters would help to identify and to authenticate the plant Hardwickia binata Roxb (Fabaceae). 3.3 Fluorescence analysis Fluorescence analysis is an important qualitative diagnostic tool for detecting the presence of chromophore in crude powdered drug and extracts. Fluorescence properties of powdered leaves and various extracts were studied and the powder showed fluorescence with various chemical reagents. The Ethanol, Hexane, Isopropanol, Methanol and N-butanol extracts showed fluorescence at 320-400nm. These experiments indicate the presence of chromophore in the plant. 3.4 Preliminary Phytochemical Screening Qualitative preliminary Phytochemical screening of extracts were performed initially with different chemical reagents to detect the Phytoconstituents present in each extract. The extracts showed the presence of saponins, tannins, steroids, terpenoids, phenolic groups, coumarin, flavanoids and carbohydrate. These experiments were repeated thrice in order to arrive at standard values.These standard values will be useful in the quality control of leaves of Hardwickia binata Roxb (Fabaceae). 3.5 GC-MS studies

Table 6 Fluorescence analysis of various extracts Extracts Florescence Day light Acetone Benzene Ccl4 Chloroform Ethanol Hexane Isopropanol Methanol N-butanol Petroleum ether Water Yellow Yellow Pale yellow Pale yellow Yellow Pale yellowish green Brownish yellow Yellow Yellow Pale green Yellow UV light 254 nm Pale brown Pale brown Pale brown Colorless Pale brown Pale brown Brown Brown Pale brown Green Pale brown 366 nm Florescent green Pale brownish green Pale yellow Bluish green Florescent brownish green Florescent green Florescent brownish green Florescent brown Florescent green Florescent green Florescent green

Table 7 Fluorescence analysis of powder Chemical reagents Florescence Day light Powder as such Sulphuric acid Hydrochloric acid Nitric acid Ferric chloride Ammonia Sodium hydroxide Potassium hydroxide Iodine Glacial acetic acid Sandal Pale yellow Pale brown UV light 254 nm Brown Colorless Colorless 366 nm Sandal Florescent yellow Pale florescent yellow Florescent bluish green Florescent brown Brown Brown Brown Florescent blusih green Pale bluish green

Florescent yellow Pale yellow Brown Pale brown Brown Deep red Pale brown Brown Dark brown Dark brown Dark brown Colorless

Yellowish brown Colorless

The chemical composition of the leaf of Hardwickia binata Roxb (Fabaceae) was determined by a GC-MS analysis. GC-MS studies were carried out on the pet.ether, chloroform and ethanolic extracts. The chemical composition of the leaves extracts were presented in Table 8 and Table 9. From the GC-MS study the plant is found to be Cyclopentaneoctanoic acid, Dioxapentacyclo [14.2.2.0(1,14).0(2,11).0(5,10)] icos-13-en-8-one,6-(3-furyl)16-methoxy-5,15,15-trimethyl and Yohimban-16-carboxylic acid, 17-oxo-, methyl ester, (16). From the phytochemical screening of the leaf of Hardwickia binata Roxb (Fabaceae) among the all extract the ethanolic extract showed the presence of maximum chemical constituents.
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G. Gunaselvi et al., Int J Pharm Biomed Sci 2010, 1(3), 37-40 Table 8 GC-MS Studies on Petroleum Ether Extract of leaves of Hardwickia binata Roxb (Fabaceae) Sl. No. 1 2 3 Name of the compound Cyclopentaneoctanoic acid, 3,5-bis(acetyloxy)--(methoxyimino)-2-(3-methoxy-3-oxopropyl)-, methyl ester Dioxapentacyclo[14.2.2.0(1,14).0(2,11).0(5,10)]icos-13-en-8-one, 6-(3-furyl)-16-methoxy5,15,15-trimethyl Yohimban-16-carboxylic acid, 17-oxo-, methyl ester, (16)Retention time 10.32 12.35 20.55 Area 6432344 2440792 83186304

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% Area 6.90 2.61 90.48

Table 9 GC-MS Studies on Ethanolic Extract of leaves of Hardwickia binata Roxb. (Fabaceae) Sl. No. 1 2 3 4 5 6 7 8 9 Compound Name Retention time Cyclopentaneoctanoic acid,3,5-bis(acetyloxy)--(methoxyimino)-2-(3-methoxy-3-oxopropyl)- 11.4 methyl ester. 7,17-Dioxapentacyclo[14.2.2.0(1,14).0(2,11).0(5,10)]icos-13-en-8-one, 6-(3-furyl)-1613.98 methoxy-5,15,15-trimethyl 9,10-Anthracenedione,1,8-bis(1,3-dihydro-1,3-dioxo(2H)isoindol-2-yl)15.57 4-[[7-Chloro-4-quinolinyl]amino]-2-[[diethylamino]methyl]-6-(p16.4 trifluoromethylphenyl)phenol 3-[18-(3-Hydroxy-propyl)-3,3,7,12,17-pentamethyl-2,3,22,24-tetrahydro-porphin-2-yl]propan- 17.01 1-ol Salicylic acid, 3-amino-5-iodo18.34 Dibenzo[fg,ij]pentaphene 19.00 3-[18-(3-Hydroxy-propyl)-3,3,7,12,17-pentamethyl-2,3,22,24-tetrahydro-porphin-2-yl]propan- 19.57 1Yohimban-16-carboxylic acid, 17-oxo-, methyl ester, (16)20.72 Area 139015864 26850336 48225800 17738288 54637388 48522776 77313976 62068932 69273816 % Area 25.47 4.92 9.02 3.25 10.01 8.89 14.16 11.37 12.69

4. CONCLUSIONS Pharmacognostical studies on leaves of Hardwickia binata Roxb. (Fabaceae) such as preliminary phytochemical and physico chemical parameters have been reported. REFERENCES
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