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Shambaditya Goswami

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

PRELIMINARY PHYTOCHEMICAL SCREENING AND STANDARDISATION OF LEAVES OF CATHARANTHUS ROSEUS (L.) G. DON
Shambaditya Goswami Department of Pharmacy, ITM, Gorakhpur, UP, India Email:shamba_mpharm@rediffmail.com ABSTRACT Catharanthus roseus (L.) G. Don (Periwinkle) belongs to the family Apocyanaceae, is a bushy perennial herb and evergreen shrub. More than 100 alkaloids and related compounds have so far been isolated and characterized from the plant. Main alkaloids are vincristine and vinblastine which are responsible for anticancerous activity. The present study deals with phytochemical screening, standardization and transverse section of the plant leaf. The dried leaves of Periwinkle were subjected for different standardization parameters like Total ash (0.4%), Acid insoluble ash (0.68%), Water soluble ash (1.68%), Sulphated ash (4.12%), Water soluble extractive (6.34%), Alcohol soluble extractive (4.8%), Moisture content (10.09%), Loss on drying (5.01%), Foaming index (0.9cm height) and Swelling index (0.8g) for their purity and strength as per WHO guideline. The Preliminary phytochemical screening report reveals the presence of Alkaloids, Glycosides, Terpenoids, Flavonoids, Phenols, Tannins, Carbohydrates, Saponins, Phytosterols, Protein and amino acids in ethanolic extract of the leaf of Catharanthus roseus (L.) G. Don . The transverse section of the plant exhibits small layer of rectangular cells covered with thick cuticle, uni-cellular covering trichome and vascular bundle present in the middle of midrib region and cruciferous stomata. This phytochemical standardization study will improvise the further research on this plant. Key Words: Catharanthus roseus (L.) G. Don, Apocyanaceae, Standardization, WHO guideline, Phytochemical Screening, Transverse section INTRODUCTION Periwinkle or Catharanthus roseus (L.) G. Don (Family Apocyanaceae), commonly known as Nayantara or Sadabahar, is an erect bushy perennial herb and evergreen shrub. The species was formerly known as Vinca rosea. The native of Periwinkle is mainly Madagaskar. This plant is grown commercially for its medicinal uses in Australia, Africa, India and Southern Europe. Except the highly alkaline or water logged soil, Periwinkle does not require any special conditions of soil. It favourably grows in light sandy soil, rich in humus. The rainfall of about 100 cm is most suitable for it. The leaf is simple, opposite, exstipulate, petiolate (Kokate C K, 2009). More than 100 alkaloids and related compounds have so far been isolated and characterized from the plant. The alkaloid contents in different parts show large variations as roots 0.14-1.34%, stem 0.074-0.48%, leaves 0.321.16%, flowers 0.005-0.84%, fruits 0.40%, seeds 0.18% and pericarp 1.14%. Dry leaves contain vinblastine (vincaleucoblastine or VLB) 0.00013-0.00063%, and vincristine (leurocristine or LC) 0.0000003-0.0000153% which have anticancerous activity (PP Joy, 1998). Vinblastine and vincristine (the antileukemic agents) were isolated, in a pure form, from Catharanthus roseus by several chromatographic techniques. All the isolated mixtures were evaluated by HPLC and HPTLC analyses (Khaled A Shams, 2009). Phytochemical studies have attracted the attention of plant scientists due to the development of new and sophisticated techniques. These techniques played a significant role in the search for additional resources of raw material for pharmaceutical industry (phytochemicals) (Arvind J Mungole, 2008). The present study deals with the macroscopy, microscopy, standardization for purity and strength as per WHO and preliminary phytochemical screening of the leaf of Catharanthus roseus (L.) G. Don. MATERIALS AND METHODS Collection of plants:The plant leaves of Catharanthus roseus (L.) G. Don were collected from the area of ITM, campus (Gorakhpur, Uttar Pradesh). The plant was authenticated and deposited in Department of Pharmacy, ITM, GIDA. Macro morphology of leaf: The parameters studied were form, shape and surface characters of drug (Pulok K. Mukherjee, 2008). Fluorescence analysis: Many herbal drugs when exposed to illumination emit light of different color. The fluorescence analysis helps to identify the drug with specific fluorescence. It also helps to detect fluorescent impurities. This method can be used as a diagnostic tool for testing adulteration (Madhavan V, 2009) This method has been done by treating the leaf powder along with 1N HCl, 1 N NaOH, 50% HCl, 50% H2SO4, 50% HNO3 and Methanol was observed under the short UV light (254nm) and long UV light(365nm). Transverse section of root: The leaves were taken cleaned and fixed in formalin, acetic acid and ethanol. After 24 hours of fixing the specimens were dehydrated with graded series of tertiary butyl alcohol. The sections were stained with toluidine blue, safranin, fast green and iodine. Temporary preparations of the slide were made by mounting on glycerin.

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Shambaditya Goswami

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Powder microscopy: Shade dried leaf of the plant was powdered with the help of a electric grinder till a fine powder was obtained .This fine powder was subjected to powder microscopy using different staining reagents as mentioned above. Determination of physico chemical parameter: The dried leaves of Catharanthus roseus were subjected for different standardization parameters for their purity and strength. Different parameters like Ash Value, Moisture content Loss on Drying has been performed according to WHO guideline, 1998. Extraction: The leaf powder (250g) was packed in a soxhlet apparatus and was subjected to successive extraction using increasing order of polarity solvents like petroleum ether, acetone, chloroform, ethanol and water. The temperature was maintained 20-250c and 24 hrs to 72 hrs time has been consumed to complete each extraction under ideal condition. After completion of extraction, the solvent was removed by distillation. All the extracts were then concentrated except petroleum ether. The residues were then stored in dessicator. Preliminary phytochemical Screening: All the extracts of Catharanthus roseus (L.) G. Don were subjected to qualitative tests for the identification of various active constituents by different chemical tests. The tests for carbohydrates, glycosides, fixed oil and fat, proteins & amino acids, saponins, tannins, alkaloid, phenolic compounds and flavonoids have been performed. RESULT AND DISCUSSION Macro morphology of leaf: Morphology of leaf implies that the leaves are simple, petiolate, ovate and oblong. The colour, odour, shape, margin and appearance is shown in Table 1. Fluorescence analysis: The fluorescence analysis of the leaf powder of Catharanthus roseus (L.) G. Don is given in Table 2. Transverse section of leaf: TS of the fresh leaf of Catharanthus roseus (L.) G. Don shows that the presence of Epidermis which is single layered and covered with thick cuticle. The trichomes are unicellular and covered. Parenchyma layer which consists of Palisade and Spongy Parenchyma, is present just below upper epidermis and it is single layered, elongated and closely packed. Xylem and Phloem are present in the middle of midrib region. The diagram by observing TS has been drawn and photomicrographs of TS are shown in Figure 1 and 2 respectively. Powder microscopy: Powder microscopy reveals that there is presence of cruciferous stomata and unicellular covering trichome. Calcium oxalates are absent. (Figure 3 and 4) Physicochemical parameter: All the physicochemical parameters like ash value, water and alcohol soluble extractive, moisture content, loss on drying, foreign matter, foaming index, swelling index are within the limit prescribed by WHO and has been tabulated. (Table 3) Extractive Value: Successive extractive values were reported in Table 4, which implied that ethanolic extract has greatest values than aqueous one. Preliminary Phytochemical Screening: All the extracts were subjected for different chemical tests and ethanolic extracts showed the presence of most chemical constituents. Alkaloids, Glycosides,Terpenoids, Flavonoids, Phenols, Tannins, Carbohydrates, Saponins, Phytosterols, protein and amino acids were showed their presence in ethanolic extract. Also it has been reported that in winter season total phenolic and flavonoid showed more content in the plant and that total flavonoids and total phenolics in this plants increased by increasing pollution loads across the sites (Qayoom A, 2009). Table 1: Macromorphology of the leaf of Catharanthus roseus (L.) G. Don Properties Inference Colour Green Odour Characteristic Taste Bitter Shape Petiolate, Ovate or Oblong Margin Centric Appearance Glossy Apex Acute Table No.2: The fluorescence analysis of the leaf powder of Catharanthus roseus (L.) G. Don Reagents Used Day light LowerUV (320-400nm) Short UV(280-320nm)
Powder Drug Powder+1N HCl Powder+1 N NaOH Powder+ 50% HCl Powder+ 50% H2SO4 Powder+50% HNO3 Powder+ Methanol Light Green Pale Yellow Red Pale Yellow Brownish Black Reddish Orange Yellowish Orange Dark Green Pale Green Greenish Yellow Pale Green Brown Dark Green Yellowish Green Black Dark Green Pale Green Greenish Blue Dark Brown Pale Green Pale Green

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Shambaditya Goswami

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Table 3: Physico-chemical constants Parameters Value obtained Total Ash 0.4% w/w Acid insoluble ash 0.68% w/w Water soluble ash 1.68% w/w Sulphated ash 4.12% w/w Solubility Water soluble extractive 6.34% w/w Alcohol soluble 4.8% w/w extractive Moisture content 10.09% w/w Loss on drying 5.01% w/w Foaming index 0.9cm height Swelling index 0.8 g

Table No 4: Extractive Values Extraction Method Values w/w Petroleum Hot 0.3% ether Percolation Acetone Hot 1.2% Percolation Chloroform Hot 1.05% Percolation Ethanol Hot 3.45% Percolation Aqueous Cold 2.1% Maceration

Test Alkaloid Glycoside Terpenoids, Flavonoids Phenols Tannins Carbohydrates Saponins Phytosterols protein and amino acids Fixed oil and fats Resin

Table 5: Phytochemical screening Petroleum ether Acetone Chloroform + + + + + -

Ethanol + + + + + + + + + + -

Aqueous + + + + + + + -

Figure1: Diagram of TS of Catharanthus roseus (L.) G. Don

Figure 2: Photomicrograph of TS of Catharanthus roseus (L.) G. Don

Figure 3: Trichome CONCLUSION Volume 1 Issue 1 www.ijrpb.com

Figure 4: Cruciferous Stomata

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Shambaditya Goswami

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

The phytochemical screening and standardistion study is not only an important tool for identification of plant part but also it informs quality control and product formulation development. As Catharanthus roseus (L.) G. Don is an medicinally important plant which possesses anticancerous (El-Sayed A and Cordell GA, 1981) (ElSayed A, 1983) antidiabetic (both ethanolic and aqueous) (Singh SN, 2001), anti-inflammatory (Chattopadhyay RR, 1992), antioxidant (Zheng W and Wang SY, 2001) activity, this study will improve the identification criteria and research validation scheme which finally will be useful in alternative medicine. REFERENCE Arvind J. Mungole, Ravi Awat Alka Chaturvedi and Prakash Zanwar, Preliminary Phytochemical screening of Ipomoea obscura (L): A hepatoprotective medicinal plant, International Journal of PharmTech Research, 2(4), 2008, 2307-2312. Chattopadhyay RR, Sarkar SK, Ganguli S, et al. Antiinflammatory and acute toxicity studies with leaves of Vinca rosea Linn in experimental animals, Indian J Physiol Pharmacol, 36, 1992, 291292. El-Sayed A, Cordell GA, Catharanthus alkaloids, Catharanthamine, a new antitumor bisindole alkaloid from Catharanthus roseus J Nat Prod, 44(3), 1981, 289-293. El-Sayed A, Handy GA, Cordell GA, Catharanthus alkaloids, Confirming structural evidence and antineoplastic activity of the bisindole alkaloids leurosine-N'b-oxide (pleurosine), roseadine and vindolicine from Catharanthus roseus, J Nat Prod, 46(4) (1983), 517-27 Khaled A Shams, Naglaa M Nazif, Nahla S Abdel Azim, Khaled A Abdel Shafeek, Mostafa M El-Missiry, Shams I Ismail, and Medhat M Seif El Nasr,Isolation and Characterization of Antineoplastic Alkaloids from Catharanthus Roseus L. Don. Cultivated in Egypt, Afr J Tradit Complement Altern Med, 6(2), 2009, 118122. Kokate C K, purohit A P, Gokhale S B, A Textbook of Pharmacognosy, Nirali Prakashan, 2009, 27- 28. Madhavan V, Hema Basnett, Gurudeva MR, Yogonarasimhan SN, Pharmacognostical evaluation of Drosera BurmanniiVahl(Droseraceae). Indian journal of traditional knowledge, 8 (3), 2009, 326-333. P. P. Joy, J. Thomas, Samuel Mathew , Baby P. Skaria ,Medicinal Plants, Kerala Agricultural University, Aromatic and Medicinal Plants Research Station Odakkali, Kerala, India , 1998 Pulok K. Mukherjee, Quality control of Herbal Drugs- An approach to evaluation of Botanicals, New Delhi: Business Horizons, 2008. Qayoom A. Mir, T. Yazdani, S. Ahmad and M. Yunus, Total flavonoids and phenolics in Catharanthus roseus L. and Ocimum sanctum L. as Biomarkers of Urban Auto Pollution, Caspian, J. Env. Sci, 7, 2009, 9-16. Quality control methods for medicine plant materials, World Health Organisation, Geneva, 1998. Singh SN, Vats P, Suri S, Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in streptozotocin induced diabetic rats, J Ethno pharmacol, 76, 2001, 269-277. Zheng W, Wang SY, Antioxidant Activity and Phenolic Compounds in Selected Herbs, J Agric Food Chem, 49, 2001, 5165-5170.

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