Pharmacognostic Evaluation of Chlorophytum orchidastrum Lindl

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1 Available online at Annals of Biological Research, 2010, 1 (4) : ( ISSN CODEN (USA): ABRNBW Pharmacognostic Evaluation of Chlorophytum orchidastrum Lindl V. N. Patil* and S. S. Deokule Department of Botany, University of Pune, Pune (MS) India. ABSTRACT Chlorophytum orchidastrum Lindl. 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 drug part is usually used as the white tuberous roots of this plant. The tuberous roots of other species of Chlorophytum, Asparagus, Bombax and Orchids are also known as safed musali. Because of its controversial name safed musali, there is a lot of scope for adulteration in the bazar drugs. Therefore to remove the controversy and for correct botanical standardization, the detailed pharmacognostic study on the tuberous roots of Chlorophytum orchidastrum has been carried out. It includes macroscopic, microscopic characters, histochemistry and phytochemistry. The Phytochemical and histochemical test includes starch, protein, saponins, sugar, tannins, glycosides and alkaloids. Percentage extractives, ash and acid insoluble ash and fluorescence analysis and HPTLC. Key Words: Chlorophytum orchidastrum, Pharmacognostic standardization, phytochemical analysis, HPTLC. INTRODUCTION Chlorophytum orchidastrum Lindl. belongs to family Liliaceae. In India, it is found to be growing in rain fed areas. The plant generally grows along the forest margins, grassy slopes and rocky places along valleys (between m) (1). The plant body is erect up to ft height with sheathing leaf base acute to acuminate with entire margin. The root system is an adventitious with tuberous root. The tuberous roots are long, slender cm long and each about 1 cm in diameter (2). The tuberous roots are medicinally important known as safed musali in trade. Literature survey indicated that the species Asparagus, Bombax and Orchids are also known as safed musali (3, 4, 5). There are 17 species of Chlorophytum recorded in India out of these 11 species of Chlorophytum are found to be growing in Maharashtra (6). For the present investigation Chlorophytum orchidastrum is selected as, it is being sold in the market under the common name safed musali. The drug part is usually used as the white tuberous roots. The drug safed musali is useful as an aphrodisiac and galactogogue (3, 4, 7). Review of literature revealed 126

2 that the safed musali is used as a nutritive and health promoting properties as well as an immunoenhancing, hepatoprotective and antioxidants (8, 9, 10, 11, 12). MATERIAL AND METHODS Collection and Identification of Plant Materials: The plant materials were collected from in and around Pune district of Maharashtra during rainy season for correct botanical identification. Efforts were made to collect the plants in flowering and fruiting condition for the correct botanical identification. It was identified with help of Flora of The Presidency of Bombay (2). The herbariums were prepared and finally authenticated from Botanical Survey of India, Western Circle, Pune (India). The voucher specimen number is PAVICH6/ Microscopic and Macroscopic evaluation: Thin (25µ) hand cut sections were taken from the fresh tuberous roots, permanent double stained and finally mounted in Canada balsam as per the plant microtechniques method of Johansen (13). The macroscopic evaluation was studied by the following method of Trease and Evans (14) and Wallis (15). 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 Krishnamurthy (16). Phytochemical evaluation: Some materials were dried under the shade so as to avoid the decomposition of chemical constituents, powdered in blender and finally stored in dry air tied containers for phytochemical screening. Ash and percentage extractives were accomplished by following standard pharmacopoeal techniques of Anonymous (17). Fluorescence analysis was carried out as per Chase and Pratt (18). Qualitative phytochemical test were carried out by standard methods of Harborne (19) and Trease and Evans (14). Quantitative phytochemical analysis were determined for proteins, carbohydrates and saponins by the methods of Lowry et al., (20); Nelson (21) and Obadoni and Ochuko (22) respectively. The phytochemical screening was also detected by the High Performance- Thin Layer Chromatography (HPTLC). HPTLC study was carried out 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). For HPTLC studies, an extract of methanol (25% GR) solvent system was used and after development, plate was scanned at 254 and 366 nm (23, 24). RESULTS AND DISCUSSIONS Macroscopic evaluation (Figure I and II) Herb: 2-3 ft. in height. Roots: Cylindrical, tuberous roots are measuring cm long, cm diameter. Leaves: 6-12 (6-9 in literature) in number, membranous, cm. acute, strongly nerved, elliptic, lanceolate, glabrous and shining on both side, winged petiole, 6-9 cm long, Scape: Naked 1ft. long, thick, unbranched (Scape with ½ brats) Flower: White, lax panicles, 1-2 cm. long. Bract: Large lanceolate, acute or sub-acute, cm long Pedicels: Ascending, geminate, cm long, jointed about the middle. 127

3 Perianth: Segments cm, sub-acute, 5 nerved, oblong, lanceolate Stamen: 6 in number, cm long, anther- 0.5 cm long Style: 1, cm long Capsule: cm long, depressed globose, deeply lobed at the apex, broader than longer, transversely veined. Seeds: Dull black, solitary flattened, broader than long, orbicular, minutely papillose. Microscopic characters The transverse section of root shows a circular in outline. The outermost layer is epidermis. Epidermis consists of uniseriate trichomes. This is followed by a very large zone of cortex. The outermost layer of the cortex just below the epidermis consists of cells which are mostly rectangular, appearing much longer than wide. The rest of the cortical cells are rounded to polygonal parenchymatous and have no intercellular spaces. The innermost layer of the cortex is single layered endodermis. The stellar structure shows that the endodermis is followed by the pericycle layer. Xylem is exarch type. The phloem is group in between the xylem along with the parenchyma. The centre region is occupied by large pith. These are mostly polygonal in shape (Figure III). Histochemical Screening Histochemical screening showed the presence of starch, protein, fat, saponins, tannin, sugars and alkaloids (Table I). Table No. I: Histochemical study of C. orchidastrum Lindl. Test Reagents Color Tissue Starch I 2 KI Blue Epi, Peri, Xy, Phlo. Protein Potassium Ferrocyanide + water + Blue Epi., Cort., xy., pith. acetic acid + 60% alcohol + FeCl 3 Tannin Acidic FeCl 3 Light brown Xy, Phlo. Saponin Conc. H 2 SO 4 Yellow Epi., hairs, endo., Peri., Phlo., xy. Fat Sudan III Pink Epi., hairs, endo., Peri., Phlo., xy. Sugar 20% aq. NaOH Yellow Hair, Epi., xy. Phlo. Glycosides Guignard s Test Brown Epi., hairs, endo., Cort Alkaloids: Mayer s Reagent Colorless Hair, Epi., Cort xy. Phlo.. Wagner s Reagent Orange Epi., Peri., Phlo., xy. Dragendorff s Orange to dark Epi., Cort. Peri., Phlo., xy. Reagent brown Tannic acid Buff color Cort. Xy., Phlo. Hager s Reagent Yellow Cort. Abbreviations: I 2 KI: Potassium iodide, FeCl 3 : Ferric chloride, Conc. H 2 SO 4 : 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 FeCl 3 Table No. II: Ash and Acid Insoluble Ash of C. orchidastrum Lindl. Parameter Total Ash Acid Insoluble Ash Results 13.5 % dry wt. 4.2 % total ash 128

4 Table No. III: Percentage extractives of C. orchidastrum Lindl Solvent Used Extract (%) Distilled Water 3.75 % Absolute Alcohol % Petroleum ether % Benzene % Chloroform % Diethyl ether % Acetone % Table No. IV: Fluorescence analysis of C. orchidastrum Baker at 230 nm Treatments Powder as such Powder as such in UV-light Powder + Nitrocellulose Powder + 1 N NaOH in Methanol Powder + 1 N NaOH in Methanol dry for 30 min. + Nitrocellulose Color Emits Yellowish brown Grayish yellow Gray Grayish brown Grayish black Phytochemical Study It contains the total ash 12.6% and acid insoluble ash is 5.6% (Table II). The values of percentage extractives were higher in chloroform and lower in benzene solvent (Table III). Fluorescence analysis was carried out to check the purity of the drug. The powder drug was observed in visible light as yellowish brown in color. The powder was then observed in ultraviolet light. It was treated with reagent like nitrocellulose, 1 N sodium hydroxide, 1 N sodium hydroxide in nitrocellulose and dry for 30 minutes and then it was observed under ultraviolet light and it emits the color as shown in (Table IV). Qualitative analysis of the root drug indicated the presence of proteins, reducing and non-reducing sugars, saponins, fats, tannin, glycoside and alkaloids in the plant (Table V). The quantity of proteins is higher than saponins and carbohydrates (Table VI). 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 galactogogue (3, 4, 6, 25). 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 nm (23, 24, 26). Analytical studies (Saponins) The HPTLC analysis showed that, the saponins from the C. orchidastrum 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. The table indicates the starting Rf values and end Rf values (Figure IV; Graph I- III; Table VII- IX). 129

5 Table No. V: Phytochemical study of C. orchidastrum Lindl. Compound Reagents Results Water Extracts Starch I 2 KI + ve Protein Millon s reagent + ve Tannins Acidic FeCl3 + ve Saponin Distilled water + ve Steroids Liebermann Burchard s + ve Test Salkowski Test + ve Anthroquinone s Benzene + 10% NH 4 OH - 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 No. VI: Quantitative estimation of C. orchidastrum Lindl. Quantitative estimation (mg / g) Protein 3.84 Reducing Sugar 1.84 Non - Reducing Sugar 2.16 Starch 3.15 saponins 2.24 Figure I: Habit of C. orchidastrum Figure II: Tuberous roots of C. orchidastrum 130

6 Hair Epi Exo. Cort. Endo. Peri. Protoxy. Metaxy. Phlo. Pith Figure III: Transverse section of root of Chlorophytum orchidastrum (10x X 3.3x) (Epi= Epidermis; Exo= Exodermis; Cort= Cortex; Endo= Endodermis; Peri= Pericycle; Protoxy= Protoxylem; Metaxy= Metaxylem; Phlo= Phloem) Figure IV- Detection of saponins by HPTLC techniques In Visible Light Image at 254 nm Image at 366 nm (Before derivatization) (After derivatization) 131

7 Figure - V -Detection of stegmasteroids by HPTLC techniques In Visible Light Image at 254 nm Image at 366 nm (Before derivatization) (After derivatization) Graph I- showing the peak for saponins for 20 µl plant extract Table VII: showing the peak values for saponins for 20 µl plant extract 132

8 Graph II- showing the peak for saponins for 10 µl plant extract Table VIII: showing the peak values for saponins for 10 µl plant extract Graph III- showing the peak for saponins for 5 µl plant extract 133

9 Table IX: showing the peak values for saponins for 5 µl plant extract Graph IV- showing the peak for stegmasteroids for 20 µl plant extract Table X: showing the peak values for stegmasteroids for 20 µl plant extract 134

10 Graph V- showing the peak for stegmasteroids for 10 µl plant extract Table XI: showing the peak values for stegmasteroids for 10 µl plant extract Graph VI- showing the peak for stegmasteroids for 5 µl plant extract Table XII: showing the peak values for stegmasteroids for 5 µl plant extract 135

11 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. The tables indicate the Rf values for all the peaks scanned by WINCATS software (Figure V; Graph IV-VI; Table X- XII). CONCLUSIONS The plant C. orchidastrum showed the correct taxonomy which is helpful for the standardization of drug. Findings of the present investigation will be useful for the correct botanical identification and authentication of the drug. After getting the overall results of C. orchidastrum and if, these are comparable with other species of safed musali, it will be used as a substitute for them. Acknowledgements Both the authors would like to express a sincere thank to Head, Department of Botany, University of Pune, Pune for encouragement and necessary laboratory facilities and The first author is grateful to authorities of Pune University for providing financial assistant. REFERENCES [1] H. Hara; The Flora of Eastern Himalaya, Tokyo University Press, Japan. 1966, 407. [2] T. Cooke; Flora of Presidency of Bombay, B.S.I. Calcutta, 1958, 3, [3] A. K. Nadkarni; K. M. Nadkarni s Indian Materia Medica. Popular Book Depot., Lamington Road, Bombay, 3 rd Edtn. 1927, 1, [4] R. N. Chopra, S. L. Nayer, I. C. Chopra; Glossary of Indian medicinal Plants CSIR. New Delhi. 1956, 218. [5] 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 (Ca-Ci), [6] W. Marais, J. Reilly; Kew Bulletin, 1978, 32 (3), [7] R. Govindarajan, M. Vijayakumar, P. Pushpangadan; Journal of Ethnopharmacology, 2005, 99, [8] Anonymous; Medicinal Plants more on Safed Musali. Agriculture and Industry Survey. 2001, [9] J. N. Dhuley; Journal of Ethnopharmacology, 1997, 58, [10] C. S. Nergard, D. Diallo, T. E. Michaelsen, K. E. Malterud, H. Kiyohara, T. Matsumoto, H. Yamada, B. S. Paulsen; Journal of Ethnopharmacology, 2004, 91,

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