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R.Meera. et al. / International Journal of Biological & Pharmaceutical Research . 1(2); 2010: 1-4.

International Journal of Biological & Pharmaceutical Research

Journal homepage: www.ijbpr.com

IJBPR

PHYTO-PHYSICO CHEMICAL EVALUATION OF LEAVES OF RAPHANUS SATIVUS

Meera R1*., Devi P2., Muthumani P1., Jeya Sundari K.3, 1Ratnaji Chilakalapudi,
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Vijay kumar Thota, 1Duddy.V.D.Murthy

Dept of Pharmaceutical Chemistry, 2Dept of Pharmacognosy, Dept of Pharmacy practice, K.M. College of Pharmacy, Uthangudi, Madurai, Tamil Nadu, India

Abstract: The present study deals with the preliminary phyto physicochemical evaluation of a well Known folklore remedy for diuretic and urinary trouble. The standardization is carried out on the basis of physiochemical and phytochemical studies including parameters such as fluorescence characteristics and analysis of inorganic constituents. Thin layer chromatography has been carried out for the entire phytochemical constituent and the inference is noted. It was found that phytosterols, flavanoids, triterpenoids, alkaloids, present in the extracts. Key words: Raphanus sativus, Phyto-Physiochemical evaluation, Flavanoids. INTRODUCTION India has a rich cultural heritage of traditional Macroscopy medicines which chiefly comprised the two widely Stems simple, basal leaves long, lyrately pinnate, coarsely flourishing systems of treatments i.e Ayurvedic and Unani toothed, flowers in long terminal racemes, white or lilac systems since ancient times. The multiple therapeutic action with purple veins. Fruits inflated, long tapering beak, and uses of these drugs are sufficiently described in irregularly constricted and filled inside with white pink classical literature on indigenous medicines in many between the seeds. Seeds are globose, yellow or brown. medicinal plant books and pharmacopoeias MATERIALS AND METHODS: (Yoganarasimhan SN, 2000; The Wealth of India, 1959). Plant material It is an annual herb with rapiform roots. Fresh leaf Whole plants were collected from the hill regions of juice considered as laxative. Roots used in the treatment of Kodaikanal during the month of December. diuretic, urinary troubles, piles and gastrodynia. Seeds for Extraction expectorant and carminative. Further, the study will greatly The leaves of the plant was washed under running help in quality assurance of finished products of herbal tap water to remove adhered dirt, followed by rinsing with distilled water, shade dried and pulverized in a mechanical drugs. * grinder to obtain coarse powder. The dried powdered leaf Corresponding author: material (250g) defatted with petroleum ether and extracted with 3 times with 70% acetone at room temperature. The R.MEERA extract was concentrate under reduced pressure . The aqueous extract of the leaf material was refluxed with distilled water for 3 hours. Following filtration and Email ID: meeraharsa @yahoo.com. concentration under vacuum the residue was obtained (28g) which was preserved in a desicator for further use.

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R.Meera. et al. / International Journal of Biological & Pharmaceutical Research . 1(2); 2010: 1-4.

Physico- Chemical standards Physico- chemical parameters of the powdered drug such as ash value, extractive value, loss on drying and crude fiber content were performed according to the method (Anonyums 1; Anonyums 2; Khandalwal KR, 1999). Extracts were prepared by various solvents by standard methods and percentage of dry extract was calculated in terms of air-dried leaf powder. (Table 1, 2, 3) Fluorescence characteristics When physical and chemical parameters are inadequate as it often happens with the powdered drugs, the plant material may be identified from their adulterants on basis of fluorescence study (Pratt RJ, 1949; Kokoski J et al., 1958 ). (Table 4) Behaviour of leaf powder with different chemical reagents Behaviour of leaf of Raphanus sativus with different chemical reagents was performed to detect the occurrence of phytoconstituents along with colour changes under ordinary daylight by standard method (Singh VK and Govil Gurdip Singh, 2005). (Table 5) Quantitative standards Total carbohydrate content in leafs of Raphanus sativus by phenol- sulphuric acid method (Dubois M et al., 1956) was estimated similarly protein content (Haritee EP, 1972). (Table 6) Preliminary phytochemical investigation The qualitative chemical test of various extracts of Raphanus sativus was carried out using standard procedure (Trease GE and Evans MC, 1983; Kokate CK, 1996; Harbone JP, 1973; Plaisted and Philip H, 1958) Thin Layer Chromatography About 30gms of silica gel B was weighed out and it was

shaken with 100ml of water to form a homogenous suspension. The suspension was poured into a thin layer chromatography applicator which was adjusted to 0.25mm thickness. 20 to 40 Carrier plates (20.5cm) were laid down for air drying. The plates were kept in the hot air oven at 110C for one hour to activate the silica gel G. The plates were stirred in a dry atmosphere and used whenever required. By using the capillary tube the extracts are spotted on the T.L.C plates 2cm above the bottom and in the chromatogram in various solvent systems for different compounds. The spots are developed in solvent system were identified by means of different spraying reagents. (Table 7). HPTLC fingerprinting of different extracts of Raphanus sativus Butanolic extract 10l of 1mg/mL solution of butanolic extract in methanol was applied on the silica gel GF254 HPTLC plates (10x10). Chloroform: Acetone: Formic Acid (9:2:1) was used as the mobile phase. After development the plates were scanned in ultraviolet range at 254 nm and 366nm and then the plates were derivatized by using 20% ethanolic sulphuric acid. After spraying four spots were observed at Rf 0.22, 0.32, 0.36, 0.38 (Sethi PD, 1996). Petroleum ether extract 10l of 1mg/mL solution of Petroleum ether extract in acetone was applied on the silica gel GF254 HPTLC plates (10x10). Petroleum ether: ethyl acetate: acetone (9:0:5::0:5) was used as the mobile phase. Six spots at Rf 0.20,0.28, 0.32, 0.39,0.42,0.48 were observed under visible after spraying the plates with 20% ethanolic acid reagent (Sethi PD, 1996).

Table 1: Ash values

S. no

Type of ash

Results

1.

Total ash

12 % w/w

2.

Acid insoluble ash

0.75 % w/w

3.

Water soluble ash

1.5 % w/w

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R.Meera. et al. / International Journal of Biological & Pharmaceutical Research . 1(2); 2010: 1-4.

Table 2: Extractive value, Percentage yield and colour of extracts

Solvent used Petroleum ether Acetone Ethyl acetate Butanol Water Percentage yield (%) 2 6 5 6 5 Colour of extract Green Blackish green Dark green Dark yellow Brownish black

Table 3. Loss on drying

Loss on drying 5.4%

Table 4: Fluorescence characteristics of leaf extract of Raphanus sativus

Powder + Reagent Powder Powder+ 1N NaOH in methanol Powder+ 1 N NaOH in water Powder++ 1 N HCl Powder+50% HNO3 Powder+50% H2SO4 Powder+Methanolic NaOH.dried+ nitrocellulose in aceticacid Powder+ 1N NaOH + nitrocellulose in aceticacid Color observed in Ordinary light Green Greenish black Brownish green Brownish yellow Green Green Yellowish green Green Dark brown Color observed under UV (254 nm) Green Green Brown Green Green Light Green Green Dark Green Light Green Color observed under UV (365 nm) Slight brown Green Black Black Black Black Black Greenish Black

Table 5: Behaviour of leaf extract of Raphanus sativus

Reagent Powder + con. H2so4 Powder + aqueous Fecl3 Powder + Iodine solution Powder + Aqs. Hgcl2 Powder + picric acid Powder + Mg Hcl Powder + aqueous AgNo3 Powder + ammonia solution Powder + Aqs. KOH Powder + Aqs. Na nitrite Powder + Water by shaking Colour / ppt Brown Bluish black No black blue Colour change Black colour Precipitate is formed no colour no colour Red colour Foam is not produced Constitituent Carbohydrate present Tannin present Starch absent Alkaloids present Alkaloids present Flavonoids present Protein present Cardiac glycoside absent Cardiac glycoside absent Phytosterols present Saponins absent

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R.Meera. et al. / International Journal of Biological & Pharmaceutical Research . 1(2); 2010: 1-4.

Table 6: Results of Quantitative estimation of leaf extracts of Raphanus sativus

S.No 1 2 3 Estimation Total carbohydrates Total proteins Total crude fibers Results 3.5 % w/w 0.737 % w/w 3.2% w/w

Table7: Phytochemical constituents of Thin Layer Chromotography

S.No 1 2 Active constituens Aminoacids Triterpenes Mobile phase N butanol :Aceticacid : Water 4:1:1 Petroleum ether : Dichloroethane : Acetic acid 12:5:12.5:2 Hexane : Diethyl ether 32:1 Butanol : Aceticacid : Water : Ether 9:6:1:3 Spraying reagent Ninhydrin Chloro sulphonic acid reagent Stannic chloride reagent Phenol sulphuric acid Inference Light green Dark brown

3. 4.

Phytosteroles Carbohydrates

Orange brown round Greenish brown

RESULTS AND DISCUSSION The preliminary phytochemical screening of the extract showed the presence of tannins, alkaloids and flavanoids in the extracts of Raphanus sativus. The above studies provide information in respect of their identification, chemical constituents and physico chemical characters which may be useful standardization of herbal drugs of folk medicinal practice of present era and

enrichment of Ayurvedic Pharmacopoeia. CONCLUSION The present study on physicochemical characteristics and preliminary phytochemical screening of Raphanus sativus provide useful information. This may help in authenticating the genuine plant along with nature of phytoconstituents present in it.

REFERENCES
Anonyums 1. The Ayurvedic Pharmacopoeia of India , Part I, Vol 1 , 1st edn, Govt .of India, Ministry of Health and Family Welfare, Department of Indian Systems of Medicines and Homeopathy, NewDelhi, 1996; A-53-55. Anonyums 2. Pharmacopoeia of India,Vol .2, 4 th edn, Govt .of India, Ministry of Health, Controller of publication, NewDelhi, 1996; A-53-55. Dubois M, Gilles KA, Hamilton JK, Rebers PA and Smith F, Colorimetric method of determination of sugars and related substances. Analytical chemistry. 1956; 28: 350-356. Haritee EP, Determination of protein, A modification of Lowrys method, that gives a linear photometric response, Analytical Biochemistry. 1972; 48: 422-427. Khandalwal KR Practical Pharmacognosy, Nirali Prakashan, 6 th edn 1999;146-148. Kokate CK, Practical Pharmacognosy, 4th edn Vallabh Prakashan Pune, 1996; 107. Harbone JP, Phytochemical methods, a guide to modern technique of plant analysis (Chapmann and Hall, London). 1973; 1-271. Kokoski J, Kokoski R and Salma F.J. J.Am.Pharm.Ass. 1958; 47: 715. Plaisted, Philip H, Contributions from Boyee Thompron Institute. 1958; 9: 231,44. Pratt RJ, Chase CR. J.Am.Pharm.Ass, 1949; 38: 324-333. Sethi PD, HPTLC Quantitative Analysis of Pharmaceutical Formulations, 1st edn. CBS Publishers and Distributors, New Delhi. 1996; 73. Singh VK, Govil Gurdip Singh, Recent progress in medicinal plants, Vol I, SCI Tech publishing LIC Texas, 2005; 325. The Wealth of India, Raw Materials, Publication and Information Directorate (CSIR), New Delhi 1959; 8: 367 Trease GE and Evans MC, Textbook of Pharmacognosy, 12th edn. Balleire, Tindall, London .1983; 343-383. Yoganarasimhan SN, Medicinal Plants of India, 2000; 458