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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN 2230 – 8407 Available online http://www.irjponline.com Research Article

PHARMACOGNOSTIC AND PHYTOCHEMICAL STUDY OF

CHLOROPHYTUM LAXUM R. BR. Patil Vishal Narayan1*, Abyari Mohammad2 and Deokule Subhash Sadhu1

1Department of Botany, University of Pune, Pune-411 007 (MS) India 2Department of Biology, Urmia University, Urmia, West Azarbayjan, Iran

Article Received on: 06/04/2011 Revised on: 16/05/2011 Approved for publication: 19/06/2011

*Patil Vishal Narayan, Department of Botany, University of Pune, Pune-411 007 (MS) India Email: vnpatil85@gmail.com ABSTRACT Chlorophytum laxum R. Br. belongs to family Liliaceae and is being used in the indigenous systems of medicine as a galactogogue and aphrodisiac. It is being sold in the market under the common name “safed musali”. The white tuberous roots of this plant are the medicinally useful parts. The tuberous roots of other species of Chlorophytum, Asparagus, Bombax and Orchids are also sometimes called safed musali leading to confusion. In order to ensure correct botanical standardization, the detailed pharmacognostic study on tuberous roots of Chlorophytum laxum has been carried out in this study. KEY WORDS Chlorophytum laxum, Pharmacognostic standardization, phytochemical analysis. INTRODUCTION Chlorophytum laxum R. Br. Baker belongs to family Liliaceae. In India, it is found in rain fed areas. The plant generally grows along the forest margins, grassy slopes and rocky places along valleys (between 1300-2800 m)1,2. This is an erect plant growing up to a height of 1 ft with sheathing leaf base acute to acuminate with entire margin. Tuberous root are cylindrical and are measuring 10-14 cm long and 1-1.4 cm diameter3. The tuberous roots are medicinally important and are known commonly as safed musali. Safed musali is used as an aphrodisiac and galactogogue4,5,6 as well as for its nutritive, health promoting properties and immunoenhancing, hepatoprotective and antioxidants activities7,8,9,10,11. The tubers are also used in fever, leucorrhoea and also as an aphrodisiac12. The species Asparagus, Bombax and Orchids are also known as safed musali4,5 in literature. It is therefore important to define specifications that will allow correct identification of the plant that is being sold as “safed musali”. In addition there are 17 species of Chlorophytum recorded in India out of these 11 species of Chlorophytum are found to be growing in Maharashtra13. We chose Chlorophytum laxum for the present investigation as it is being sold widely in the market under the common name safed musali because of its white tuberous roots.

MATERIAL AND METHODS Collection and Identification of Plant Materials The plant materials were collected from in and around Pune district of Maharashtra during the rainy season for correct botanical identification. Efforts were made to collect the plant in flowering and fruiting condition for the correct botanical identification. It was identified with help of Flora of The Presidency of Bombay3. Herbarium specimen were prepared and authenticated from Botanical Survey of India, Western Circle, Pune (India). The voucher specimen number is PAVICH5/ 2009. Microscopic and Macroscopic evaluation Thin (25µ) hand cut sections were taken from the fresh tuberous roots, permanently double stained and finally mounted in Canada balsam as per the plant microtechnique method of Johansen14. The macroscopic evaluation was studied by the method of Trease and Evans15 and Wallis16. Histochemical study The thin transverse sections of fresh root were taken (about 25µ). It was treated with respective reagent for the detection and localization of chemicals in the tissues as per the method of Krishnamurthy17. Phytochemical evaluation Some roots were dried under the shade so as to avoid the decomposition of chemical constituents, powdered in a blender and finally stored in dry air tied containers for phytochemical screening. Ash and percentage extractive content was measured by following standard

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pharmacopoeal techniques18. Fluorescence analysis was carried out as per Chase and Pratt19. Qualitative phytochemical tests were carried out by standard methods of Harborne20 and Trease and Evans15. Quantitative phytochemical analysis were determined for proteins, carbohydrates and saponins by the methods of Lowry et al., 21; Nelson22 and Obadoni and Ochuko23 respectively. The phytochemical screening was also done by High Performance- Thin Layer Chromatography (HPTLC). HPTLC study was carried out on Linomat 5 for application using Densitometer- TLC Scanner 3 with “WINCATS” software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). For HPTLC studies, an extract of methanol (25% GR) solvent system was used and after development, plate was scanned at 254 and 366 nm24, 25, 26. RESULTS AND DISCUSSIONS Macroscopic evaluation (Figure 1 and 2) The details of the macroscopic examination are mentioned in Table 1 and illustrated in Figures 1 and 2. Microscopic characters The transverse section of the root had a circular outline. The outermost layer is the epidermis consisting of uniseriate trichomes followed by a very large zone of the cortex. The outermost layer of the cortex just below the epidermis consists of cells which are mostly rectangular, appearing longer than wide. The rest of the cortex are rounded to polygonal parenchymatous cells and have no intercellular spaces. The innermost layer of the cortex is a single layered endodermis. The stellar structure shows that the endodermis is followed by the pericycle layer. The xylem is exarch variety and the phloem is in between the xylem along with the parenchyma. The central region is occupied by large pith mostly polygonal in shape (Figure 3). Histochemical Screening Histochemical screening showed the presence of starch, protein, fat, saponins, tannin, sugars and alkaloids (Table 2). Phytochemical Studies The tuber had a total ash content of 11.5 %, the acid insoluble ash being 3.7% (Table 3). The values of percentage extractives were higher in chloroform and lower in benzene solvent (Table 4). Fluorescence analysis was carried out to check the purity of the drug. The powder drug was observed in visible light as Brownish yellow in color. The powder was treated with nitrocellulose, 1 N sodium hydroxide, 1 N sodium hydroxide in nitrocellulose and dried for 30 minutes. After this it was observed under

ultraviolet light and it emits the color as shown in (Table 5). Qualitative analysis of the root indicated the presence of proteins, reducing and non-reducing sugars, saponins, fats, tannin, glycoside and alkaloids (Table 6). The quantity of proteins is higher than saponins and carbohydrates (Table 7). Saponins are the important chemical and justify the use of tubers of this plant and are used as a well known health tonic, aphrodisiac and galactogogue4,5,6,27,28. In HPTLC study, the methanolic extract is ultrasonic for 15 minutes and filtered. The filtrate is used as an application for saponins and stegmasteroids. For each application 20 µl, 10µ and 5µl extracts were used and loaded on instrument comprising of Linomat 5 for application using Densitometer- TLC Scanner 3 with “WINCATS” software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). The plates were scanned at 254 and at 366 nm24, 25, 26, 29. Analytical studies (Saponins) The HPTLC analysis showed that, the saponins from the C. laxum root samples gave light yellow bands in visible light and blue bands after derivatization in fluorescence light. The plates were scanned at 254 and 366 nm. When images were compared with the graph and table values, it showed maximum area 20.47% at 366nm after derivatization. The table also indicates the starting Rf values and end Rf values (Figure 4; Graph 1, Table 8). Analytical studies (Stegmasteroids) In HPTLC analysis, stegmasteroids revealed white bands in visible light. After derivatization in fluorescence light it showed the dark blue bands. The plates were scanned at 254 and 366 nm. It was covered the area 23.02% at 254 nm. The tables also indicate the Rf values for all the peaks scanned by “WINCATS” software (Figure 5; Graph 2, Table 9). CONCLUSION The plant C. laxum showed the correct taxonomy which is helpful for the standardization of drug. The morphological characters and histochemical study with double staining of the root, percentage extractives, fluorescence and ash analysis and the phytochemical screening of the plant. As in case of saponins and stegmasteroids, the peaks are denoted by the Rf values. These investigations will be useful for the correct botanical identification and authentication of the drug. After getting the overall results of C. laxum and if data is comparable with the above mention species of safed musali, it can be used as a substitute for them. ACKNOWLEDGEMENT Both the authors would like to express a sincere thank to Head, Department of Botany, University of Pune, Pune-

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411 007 for encouragement and necessary laboratory facilities and The first author is grateful to authorities of Pune University for providing financial support in the form of research stipend. REFERENCES 1. Hara H. The Flora of Eastern Himalaya. Tokyo University Press,

Japan; 1966. p. 407. 2. Patil VN, Deokule SS. Pharmacognostic standardization of

Chlorophytum bharuchae Ans., Ragh. & Hem. International Journal of Current Research 2010; 3: 27- 32.

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7. Anonymous. Medicinal Plants more on Safed Musali. Agriculture and Industry Survey; 2001. p. 38-39.

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10. Kirtikar KR, Basu BD. Indian Medicinal Plants. Liliaceae: Chlorophytum. In Kirtikar KR; Basu BD (Eds.) L.M. Basu Publishers. Allahabad, India;1975. p. 2508-2509.

11. Sreevidya N, Kumar V, Kumar S, Sikarwar RLS. Utilization, depletion and conservation of Safed Musali (Chlorophytum spp.). Journal of non- Timber Forest Products 2003; 10: 155-157.

12. The Wealth of India- A dictionary of Indian raw materials and industrial products, Revised Edn. Publication and Information Directorate, CSIR. Dr. R.S. Krishnan Marg, New Delhi,1992; 3: 482-483.

13. Marais W, Reilly J. Chlorophytum and its related Genera (Liliaceae). Kew Bulletin1978; 32 (3): 653-663.

14. Johansen DA. Plant Microtechnique, McGraw-Hill Book Co. Inc. New York; 1940. p. 151-154, 182-203.

15. Trease GE, Evans WC. Trease and Evans Pharmacognosy, 15th Ed. W. B. Saunders Edinburgh London, New York, Philadelphia St. Louis Sydney Toronto; 2002. p. 3-4, 528-533, 538-547.

16. Wallis TE. A Text Book of Pharmacognosy, reprinted edition, Churchill, Livingstone. London; 1967. p. 578 – 617.

17. Krishnamurthy KV. Methods in the Plant Histochemistry, Viswanadhan Pvt. Limited, Madras; 1988. p. 1 – 77.

18. Anonymous. Pharmacopoeia of India, Government of India, Ministry of Health Manager, Publications Delhi, 1st Edn.; 1955. p. 370 & 864.

19. Chase CR, Pratt R. Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. Journal of Amer. Phar. Asso. 1949; 38: 324-330.

20. Harborne JB. Phytochemical Methods, Chapman and Hall International Edition, London. 2nd edition; 1973. p. 5-8.

21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. Jour. of Biol. Chem. 1951; 193: 265-275.

22. Nelson N. A photometric adaptation of the Somogyi method for the determination of Glucose. Jour. of Biol. Chem. 1944; 153: 375-380.

23. Obadoni BO, Ochuko PO. Phytochemical studies and comparative efficacy of crude extracts of some homeostatic plants in Edo and Delta state of Nigeria. Global Jour. Pure Appl. Sci. 2001; 8: 203-208.

24. Patil VN, Deokule SS. Pharmacognostic Evaluation of Chlorophytum orchidastrum Lindl. Annals of Biological Research 2010; 1 (4): 126-137.

25. Wagner H, Baldt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas. Springer – Verlag, Berlin; 1996. p. 129, 144, 155, 157, 176, 178, 206.

26. Reich E, Schibii A. High Performance- Thin Layer Chromatography for the analysis of medicinal plants, Thieme medical publishers. Inc.; 2007.p. 129-160, 206-210, 224-240.

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29. Patil VN, Deokule SS. Pharmacognostic and Phytochemical Investigation of Chlorophytum breviscapum Dalz. International Journal of Current Research and Review 2010; 2 (12): 11 – 24

Table 1 Macroscopic examination of C. laxum R. Br.

Herb Slender, up to 1 ft. in height. Roots Tuberous roots are slender with pendulous ellipsoid tubers (actually fusiform i.e. tapering towards both

the end) and are measuring 6-10 cm long, 1-1.5 cm diameter. Leaves:

Green grass like, 6-12 in number, linear or linear lanceolate, 22.5 - 45 ´ 1.2 – 2.5 cm. acute with 15 – 20

distinct immersed veins. Scape: Naked, slender, flexuous, 1 – 3 in. long, winged.

Flower: Few, racemose, greenish white up to 1 in. apart, branched or unbranched.

Bract: Ovate lanceolate, 0.8 – 1.2 cm long at the base of the branches when forked, acuminate. Pedicels: Short, jointed about the middle. Perianth: Segments hyaline, 3 nerved, oblong, obtuse. Stamen:. Alternately short and long, longer 0.8 cm and the shorter 0.6 cm long, anther- 0.5 cm long green.

Style: 1, 0.5 cm long. Capsule: Up to 1 cm longer, broadly obchordate, 3 winged, obtusely trigonous.

Seeds: 0.7 – 1cm black, angular.

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Table 2 Histochemical study of C. laxum R. Br.

Test Reagents Color Tissue Starch I2KI Blue Epi, Peri. Endo. Protein Potassium Ferrocyanide + water + acetic acid +

60% alcohol + FeCl3 Blue Epi., Cort., Xy. Pith.

Tannin Acidic FeCl3 Light brown Epi., Peri., Phlo., xy. Saponin Conc. H2SO4 Yellow Cort., Xy., Phlo.

Fat Sudan III Pink Hair, Epi., Pith.

Sugar 20% aq. NaOH Yellow Epi., Peri., Xy. Phlo. Glycosides Guignard’s Test Brown Epi., Xy., Phlo. Alkaloids: Mayer’s Reagent Colorless Hair, Epi., Cort xy. Phlo.

Wagner’s Reagent Orange Endo., Peri., Xy. Dragendorff’s

Reagent Orange to dark

brown Hairs, Epi., Endo., Peri., Phlo.,

xy. Tannic acid Buff color Hairs, Epi., Cort.

Hager’s Reagent Yellow Cort. Abbreviations I2KI: Potassium iodide, FeCl3: Ferric chloride, Conc. H2SO4: Concentrated sulphuric acid, NaOH: Sodium hydroxide.

Epi: Epidermis, Endo: Endodermis, Peri: Pericycle, Cort: Cortex, Xy: Xylem, Phlo: Phloem + Sign indicates the addition of Potassium Ferrocyanide in water, then acetic acid , 60% alcohol and lastly FeCl3

Table 3 Ash and Acid Insoluble Ash of C. laxum R. Br.

Parameter Results Total Ash 11.5 % dry wt.

Acid Insoluble Ash 3.7 % dry wt.

Table 4 Percentage extractives of C. laxum R. Br.

Solvent Used Extract (%) Distilled Water 3.45 %

Absolute Alcohol 0.245 % Petroleum ether 0.22 %

Benzene 0. 175 % Chloroform 7.49 %

Diethyl ether 0. 305 % Acetone 0.36 %

Table 5 Fluorescence analysis of C. laxum R. Br. at 230 nm

Treatments Color Emits

Powder as such Brownish yellow Powder as such in UV-light Grayish green

Powder + Nitrocellulose Grayish yellow Powder + 1 N NaOH in Methanol Blackish green Powder + 1 N NaOH in Methanol dry for 30 min. + Nitrocellulose

Blackish gray

Table 6 Phytochemical study of C. laxum R. Br.

Compound Reagents Results

Water Extracts

Starch I2KI + ve

Protein Millon’s reagent + ve

Tannins Acidic FeCl3 + ve

Saponin Distilled water + ve

Steroids Liebermann – Burchard’s Test + ve

Salkowski Test + ve

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Anthroquinone’s Benzene + 10% NH4OH - ve

Sugars Benedict’s reagent + ve

Fats Sudan III + ve

Alcoholic extracts

Alkaloids

a Mayer’s Reagent + ve

b Wagner’s Reagent + ve

c Dragendorff’s Reagent + ve

d Tannic acid + ve

e Hager’s Reagent + ve

f Folin-Phenol Reagent + ve

Glycosides Benzene + ve

Table 7 Quantitative estimation of C. laxum R. Br. Quantitative estimation (mg /g )

Protein 2.82461 Reducing Sugar 0.86038

Non - Reducing Sugar 0.14608 Starch 1.90642

saponins 1.49682

Graph 1 showing the peak for saponins

Table 8 showing the peak values for saponins

Peak Start Rf Start Height

Max Rf

Max Height

Max % End Rf End Height

Area Area %

1 0.09 0.6 0.11 17.2 7.49 0.14 4.5 360.5 5.77 2 0.23 5.4 0.25 15.3 6.64 0.27 1.5 198.9 3.18 3 0.29 4.5 0.33 14.1 6.14 0.35 7.3 357.3 5.72 4 0.39 10.5 0.44 38.2 16.64 0.47 18.1 1279.7 20.47 5 0.56 19.9 0.59 40.4 17.59 0.62 21.9 1256.3 20.10 6 0.63 21.6 0.65 32.6 14.16 0.66 26.9 768.4 12.29 7 0.68 22.4 0.68 23.4 10.19 0.72 5.4 505.4 8.09 8 0.74 7.4 0.78 20.7 9.00 0.80 16.5 670.4 10.72 9 0.82 20.5 0.85 28.0 12.16 0.89 2.1 853.8 13.66

Graph 2 showing the peak for stegmasteroids

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Table 9 showing the peak values for stegmasteroids Peak Start

Rf Start

Height Max Rf

Max Height

Max % End Rf End Height

Area Area %

1 0.07 0.2 0.09 30.7 5.08 0.12 1.7 557.2 2.33 2 0.15 4.1 0.22 57.8 9.57 0.28 3.2 2591.8 10.84 3 0.30 2.3 0.33 13.3 2.20 0.34 10.6 291.8 1.22 4 0.38 31.1 0.44 62.3 10.31 0.49 1.1 3341.0 13.97 5 0.50 1.1 0.56 149.2 24.69 0.60 63.7 5502.5 23.02 6 0.60 64.3 0.63 120.7 19.98 0.67 20.6 3441.4 14.39 7 0.69 16.0 0.76 105.2 17.42 0.81 23.9 4835.8 20.23 8 0.84 28.0 0.88 64.9 10.74 0.95 0.1 3346.4 14.00

Figure 1 Habit of C. laxum R.Br Figure 2 Tuberous roots of C. laxum R. Br

Figure 3 Transverse section of root of Chlorophytum laxum R. Br. (10x X 3.3x)

Hair

Epi

Exo.

Cort.

Endo.

Peri.

Protoxy.

Metaxy.

Phlo.

Pith

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In Visible Light Image at 254 nm Image at 366 nm

(Before derivatization) (After derivatization)

Figure 4 Detection of saponins by HPTLC techniques

In Visible Light Image at 254 nm Image at 366 nm (Before derivatization) (After derivatization)

Figure 5 Detection of stegmasteroids by HPTLC techniques

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