Multivitamin plant: pharmacognostical standardization and phytochemical profile of its leaves

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1 Research Article ISSN: Available online through Multivitamin plant: pharmacognostical standardization and phytochemical profile of its leaves Sorabh Kumar Agrawal, Venkatachalam Karthikeyan*, Periyasamy Parthiban, Ramraj Nandhini Associate Professor, Department of Pharmacy, The Tamil Nadu DR. M.G.R Medical University, Chennai, Tamil Nadu, India. Received on: ; Revised on: ; Accepted on: ABSTRACT To explore the micro morphology and physio chemical parameters of the leaves of Sauropus androgynus L. Merrill. (Euphorbiaceae). Macroscopy, microscopy, physicochemical analysis, preliminary phytochemical screening and other WHO recommended parameters for standardizations were performed. Leaves: Stipules lanceolate or linear-lanceolate, 1.5-3mm long. Petioles 2-4 mm long; leaf blade sub membranous or thin-papery, ovate-lanceolate, oblong-lanceolate or lanceolate, 3-10cm long, cm wide, base cuneate, rounded or truncate, apex acuminate; lateral veins 5-7 pairs, flattened adaxially, elevated abaxially, reticular nerves obscure. Microscopic evaluation revealed the Midrib of the leaf is flat on the adaxial side and triangular with conical abaxial part. The adaxial epidermis of the midrib is fairly distinct with squarish cells and abaxial epidermis has small radially oblong cells. The vascular bundle is single broadly top shaped and collateral. The xylem elements are several parallel lines they are thick walled and angular in outline phloem occurs in wide band beneath the xylem. The lamina has bifacial symmetry of the mesophyll and the midrib. The lamina has smooth and even surface. The adaxial epidermis has dilated rectangular cells and small squarish cells. The abaxial epidermis also has circular or spindle shaped dilated cells. The adaxial epidermis is apostomatic. The cuticle layer is fairly thick, smooth and highly waxy. The cells are random in orientation. The abaxial epidermal is stomatiferous. They are paracytic type with two unequal, parallel subsidiary cells for each stoma. The epidermal cells are amoeboid with highly wavy anticlinal walls. Vein islet numbers, vein termination numbers, stomatal number, stomatal index and other physico chemical tests like ash values, loss on drying, extractive values were determined. Preliminary phytochemical screening showed the presence of alkaloids, proteins and aminoacids, tannins, flavonoids, carbohydrates, sterols and absence of mucilage, saponin, glycosides, volatile oil and fixed oil. Microscopic analysis was informative and provides useful information in the botanical identification, standardization for purity & quality and immense value in authentication of the leaf. KEY WORDS:Sauropus androgynus, Euphorbiaceae, Multivitamin Plant, Microscopical evaluation, physicochemical analysis. 1.INTRODUCTION Sauropus androgynus L. Merrill. is a perennial shrub belonging to the family Euphorbiaceae. It is also known as katuk, star gooseberry, or sweet leaf. In India it also known as Multivitamin Plant as it contains an excellent source of vitamins A, B, C, carotenoid and also it has high nutritive value and contains phytochemicals which can act as antioxidant. It is most popular in South Asia and Southeast Asia. The dark-green leaves provide a rich source of chlorophyll which is a valuable blood building element, cell rejuvenator and beneficial to the circulation 1-4. Leaf juice was also widely advertised as natural diet good for weight reduction 5, 6. Decoction is given in stricture of the bladder and in fevers; used as a diuretic. The leaves contain protein 6.8, carbohydrates 11.6, mineral matter 3.4, Ca 0.57, phosphorus *Corresponding author. Venkatachalam Karthikeyan Assistant Professor, Department of Pharmacy, The Tamil Nadu DR. M.G.R Medical University, Chennai, Tamil Nadu, India. 0.20, iron 28mg, carotene (as vitamin A), 9510IU, thiamine 0.48, riboflavin 0.32, nicotinic acid 2.6, and vitamin C 247mg/g 7. Sauropus androgynus is commonly used as an effective medicinal herb in the treatment of diabetics, cancer, inflammation, microbial infection, cholesterol and allergy due to its antioxidant effect 8. The leaves are traditionally used as antibacterial 9, analgesic and anti-inflammatory 10, antihypertensive 11 and wound healer 12. It was reported that leaves contain 1,14-Tetradecanediol, 1- Octadecyne, 1-Hexadecyne, Decanoic acid, ethyl ester, Phytol, Pyrene, hexadecahydro, 2(1H) Naphthalenone, 3,5,6,7,8,8a-hexahydro- 4,8adimethyl-6-(1-methylethenyl), Azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4- dimethyl- 7-(1-methylethenyl)-, [1-methylethenyl)-Squalene 13. In spite of the numerous medicinal uses attributed to this plant, limited report available on its pharmacognostical information. The objective of the present study was to establish various Pharmacognostic standards, important microscopical feature (Midrib and Lamina) and to evaluate preliminary phytochemical and physicochemical analysis of

2 S.androgynus that can facilitate identification and assist in the preparation of monograph of this plant. 2. MATERIALS AND METHODS 2.1: Chemicals Formalin, acetic acid, ethyl alcohol, chloral hydrate, toludine blue, phloroglucinol, glycerin, hydrochloric acid and all other chemicals used in this study were of analytical grade. 2.2: Plant collection and authentication The plant specimen was identified and authenticated as Sauropus androgynus (Euphorbiaceae) by TNAU, Botanical survey of India, Coimbatore, Tamil Nadu, India and Dr. P. Jayaraman, Director of Plant Anatomy Research Institute, Tambaram, Chennai, Tamil Nadu, India. 2.3: Macroscopic analysis Macroscopic observation of the plant was done. The shape, size, surface characters, texture, colour, odour, taste etc was noted 14. It is a technique of qualitative evaluation based on the study of morphological and sensory profiles of herbs. branchlets green slender, angled at upper part when young, terete at age (Fig 1). Leaves: Stipules lanceolate or linear-lanceolate, 1.5-3mm long. Petioles 2-4 mm long; leaf blade sub membranous or thin-papery, ovate-lanceolate, oblong-lanceolate or lanceolate, 3-10cm long, cm wide, base cuneate, rounded or truncate, apex acuminate; lateral veins 5-7 pairs, flattened adaxially, elevated abaxially, reticular nerves obscure (Fig 2). 2.4: Microscopic analysis Transverse section midrib region of fresh leaf pieces were cut and fixed in FAA and then dehydrated by employing graded series of ethyl alcohol and tertiary butyl alcohol 15. Sections were taken using microtome. Permanent mount was prepared using saffranin fast green double staining technique 16. In order to supplement the descriptive part the photomicrographs in different magnifications of all necessary cells and tissues were taken with NIKON Coolpix digital camera and Labphot2 microscopic unit. Figure 1: Habit of Sauropus androgynus L 2.5: Physicochemical analysis Total ash, acid insoluble ash, water soluble ash, loss on drying, extractive values was performed as per standard procedure 17, : Leaf constants Leaf constants such as vein islet numbers, vein terminal number, stomatal number and stomatal index, palisade ratio were determined : Preliminary phytochemical screening Preliminary phytochemical screening was carried out to find out the presence of various phytoconstituents using standard procedure 20, RESULTS 3.1 Macroscopy: Sauropus androgynus L. Merill (Euphorbiaceae) commonly known as Multivitamin Plant is an erect glabrous perennial shrub 1-3m tall, Figure 2: Leaves of Sauropus androgynus L 3.2 Microscopy of the leaf: Transverse section (T.S) of the leaves through the midrib with lamina showed the following tissue systems (Fig 3). The midrib is flat on the adaxial side and triangular with conical abaxial part. The adaxial epi-

3 dermis of the midrib is fairly distinct with squarish cells and abaxial epidermis has small radially oblong cells. The palisade zone is horizontally transcurrent in between the adaxial epidermis and the vascular bundle. The conical abaxial part has compact parenchymatous cells. The vascular bundle is single broadly top shaped and collateral. The xylem elements are several parallel lines they are thick walled and angular in outline phloem occurs in wide band beneath the xylem. T.S showing Epidermis and Stomata: The abaxial epidermal is stomatiferous. They are paracytic type with two unequal, parallel subsidiary cells for each stoma. The epidermal cells are amoeboid with highly wavy anticlinal walls (Fig 5). Venation pattern: The lateral veins are thin and less conspicuous. The vein islets are not distinct; when distinct they are wide and irregular in outline. The vein terminations are thin and delicate. They are long, wavy, simple and occasionally forked (Fig 6). La- Lamina, AdS- Adaxial side, X- Xylem, GT- Ground Tissue, Cr- Crystal, MR- Medullary Ray. Figure 3: T.S through Midrib of Sauropus androgynus L. Leaves Lamina: The lamina has bifacial symmetry of the mesophyll and the midrib. The lamina has smooth and even surface. The adaxial epidermis has dilated rectangular cells and small squarish cells. The abaxial epidermis also has circular or spindle shaped dilated cells. The mesophyll tissue is distinctly separated into palisade cells and spongy parenchyma. The palisade cells are single layered and they are cylindrical and spongy parenchyma have four layers of spherical or lobed, loosely arranged cells. The epidermal cells are made of parenchyma cells. The adaxial epidermis is apostomatic. The cuticle layer is fairly thick, smooth and highly waxy. The cells are random in orientation (Fig 4). St-Stomata, EC- Epidermal Cell, SC- Subsidiary Cell Figure 5: T.S showing Epidermis and Stomata Figure 6: Venation Pattern 3.3 Physicochemical analysis: Table 1: Extractive value of Sauropus androgynus L. Leaves AdE- Adaxial Epidermis, PM-Palisade Mesophyll, AbE- Abaxial Epidermis, SM-Spongy Mesophyll Figure 4: T.S of Lamina- Sauropus androgynus L. Leaves S.no Solvent Extractive value %w/w 1 Ethanol 23 2 Chloroform Water Petroleum ether Benzene 16.41

4 Table 2: Ash value of Sauropus androgynus L. Leaves S.no Ash type Ash value (%w/w) 1 Total ash Acid insoluble ash Water soluble ash Sulphated ash 6.21 Loss on drying 1.7%w/w and foreign organic matter was nil. 3.4: Leaf constants Leaf constants were as follows Vein islet number 20-24, Vein termination number 24-27, Stomatal number (Lower Epidermis) 80-84, Stomatal index (Lower epidermis) ±2 and Palisade Ratio : Preliminary phytochemical screening DISCUSSION Sensory evaluation plays a key role in determining the suitability or denunciation of a crude drug. Organoleptic testing of a crude drug is mainly for qualitative evaluation based on the observation of morphological and sensory profile 22. In this report, various morphological, microscopical, physicochemical standards have been developed. Hence we have undertaken this study to serve as a tool for developing standards for identification, quality and purity of Sauropus androgynus leaves. Adulteration and misidentification of crude drugs can cause serious health problems to consumers and legal problems for the pharmaceutical industries. It can be conducted via a variety of techniques, namely macro and microscopic identification and chemical analysis especially description of microscopic botanical aspects to determine definitively the proper species of plant material while it is still in its non extracted form. The observation of cellular level morphology or Table 3: Preliminary phytochemical screening of various extracts of Sauropus androgynus L. Leaves S.No Chemical Test Extracts Ethanol Chloroform Benzene Pet ether Water I. ALKALOIDS Mayer s reagent Dragondroff s reagent Hager s reagent Wagner s reagent II. CARBOHYDRATES Molisch s test Fehling s test Benedict s test TANNINS Ferric chloride Lead acetate test III. IV. PROTEINS AND FREE AMINO ACIDS Millon s test Biuret test Ninhydrin test STEROLS Salkowski test LibermanBurchard s test V. FLAVONOIDS Shinoda test Alkali test VI. GLYCOSIDES Anthroquinone glycosides Borntrager s test Modified Borndrager s test Cardiac glycosides Keller Killiani test Raymond test Legal test Cyanogenetic glycosides Coumarin glycosides VII. TERPENOIDS

5 anatomy is a major aid for the authentication of drugs. These characters are especially important for identification of powdered drugs, because in these cases most of the morphological diagnostic features are lost 23. Microscopic evaluation is one of the simplest and cheapest methods for the correct identification of the source of the materials 24. The macroscopic and organoleptic characters of the leaf can serve as diagnostic parameters 25. Previously reported study 26 only mentioned stomata and not reported adaxial epidermis apostomatic and other important feature of the microscopic character of the leaves. All the observations are included in this study and moreover clear microscopical plates are displayed. Microscopic evaluation revealed the Midrib of the leaf is flat on the adaxial side and triangular with conical abaxial part. The adaxial epidermis of the midrib is fairly distinct with squarish cells and abaxial epidermis has small radially oblong cells. The vascular bundle is single broadly top shaped and collateral. The xylem elements are several parallel lines they are thick walled and angular in outline phloem occurs in wide band beneath the xylem. The lamina has bifacial symmetry of the mesophyll and the midrib. The lamina has smooth and even surface. The adaxial epidermis has dilated rectangular cells and small squarish cells. The abaxial epidermis also has circular or spindle shaped dilated cells. The adaxial epidermis is apostomatic. The cuticle layer is fairly thick, smooth and highly waxy. The cells are random in orientation. The abaxial epidermal is stomatiferous. They are paracytic type with two unequal, parallel subsidiary cells for each stoma. The epidermal cells are amoeboid with highly wavy anticlinal walls. The ash values are particularly important to find out the presence or absence of foreign inorganic matter such as metallic salts and or silica (earthy matter) 27. Acid insoluble ash provides information about non-physiological ash produced due do adherence of inorganic dirt, dust to the crude drug. Increased acid insoluble ash indicates adulteration due do dirt, sand (or) soil. The extractive values are primarily useful for the determination of exhausted or adulterated drug and helpful in the detection of adulteration 28. Phytochemical evaluation and molecular characterization of plants is an important task in medicinal botany and drug discovery 29. Preliminary phytochemical screening showed the presence of alkaloids, proteins and aminoacids, tannins, flavonoids, carbohydrates, sterols and absence of mucilage, saponin, glycosides, volatile oil and fixed oil. CONCLUSION The study of Pharmacognostical features of Sauropus androgynus L. had shown the standards which will be useful for the detection of its identity and authenticity. 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