PHARMACOGNOSTICAL STUDIES OF LEAVES OF SPINACIA OLERACEA LINN.

PHARMACOGNOSTICAL STUDIES OF LEAVES OF SPINACIA OLERACEA LINN.

Payal R. Dande*, G. M. Sharma, R. M. Sharma and G. S. Chakraborthy

SVKM’s, NMiMS, SPTM, Shirpur Campus, Maharashtra, India

ABSTRACT

The paper presents the pharmacognostical studies of leaves of Spinacia oleracea Linn. (Amaranthaceae) commonly known as Palak, a traditional medicinal plant with high nutritional value, used as anti diabetic, anti bacterial and hepatoprotective agent. Extensive literature survey revealed that no reports were available on organoleptic, microscopic and physicochemical properties of Spinacia oleracea Linn. The present study was undertaken to perform pharmacognostical research which can support the identification and characterization of the plant.

INTRODUCTION: A high intake of fruit and vegetables is well known to have positive effects on human health and has been correlated to a decreased risk of most chronic diseases such as cardiovascular disease, diabetes and several forms of cancer^{1, 2}. Spinach has a high nutritional value and is extremely rich in antioxidants, especially when fresh, steamed, or quickly boiled. It is a rich source of vitamin A (lutein), vitamin C, vitamin E, vitamin K, magnesium, manganese, folate, iron. Spinacia oleracea is an edible flowering plant in the family of Amaranthaceae. It is an annual plant (rarely biennial), which grows to a height of up to 30 cm. Spinach may survive over winter in temperate regions ³. Apart from having nutritional value, it has been also credited with various biological activities like virus inhibitor ⁴, anthelmentic ⁵, antioxidant, ⁶ hepatoprotective ⁷ and reducing risk of breast cancer ⁸. Our objective was to identify and characterize Spinacia oleracea.

MATERIALS AND METHODS: The Fresh leaves of Spinacia oleracea were collected in the month of February, 2010 from Shirpur, Dist Dhule (M.H.) India. These were identified, and authenticated by the scientists of Botanical Survey of India, Pune, India. A voucher specimen of the leaf itself is deposited in department for future reference. Collected fresh leaves were washed and used for study of organoleptic and microscopic characteristics. The powder of shade dried leaves was used for the determination of ash values, extractive values and phytochemical investigations. All chemicals and reagents used for testing were of analytical grade obtained from SD Fine Chemicals and Loba Chemicals, Mumbai (India).

Microscopy: Fresh leaves of Spinacia oleracea were selected for the microscopical studies. Microscopic sections were cut on a microtome and by free hand sectioning. Numerous temporary and permanent mounts of the microscopical sections of the leaf specimen were made and examined ⁹. Histochemical reactions were applied with various reagents to reveal lignified elements; Photomicrographs of the microscopical sections were taken with the help of MOTIC photomicroscope provided with MOTIC IMAGES PLUS 2.0 software. A powder characteristic, preliminary examination, behavior of powder with different chemical reagents and microscopical examination was carried out ¹⁰.

Micrometry: The measurements of different cells and cell contents were done with the help of calibrated ocular micrometer and MOTIC IMAGES PLUS 2.0 software.

Physicochemical parameters: Percentage of total ash, acid-insoluble ash, water soluble ash and sulphated ash were calculated as per the Indian Pharmacopoeia ¹¹. The total ash of the powdered leaf was tested for different inorganic constituents. Different extracts of the leaves were prepared for the study of extractive values. Fluorescence analysis of powdered leaf was carried out by standard methods ^12-13.

Preliminary phytochemical analysis - For the preliminary phytochemical analysis, 5g of powdered drug was extracted with petroleum ether, chloroform, methanol, ethyl acetate, ethanol, acetone and water successively. The extracts were dried and weighed. The presence or absence of different phytoconstituents viz. proteins, alkaloids, sugars, tannins, glycosides etc. were detected by usual prescribed methods ^14-15.

RESULTS:

Macroscopy: The leaves are alternate, simple, and ovate to triangular-based, too variable in size from about 2-30 cm long and 1-15 cm broad, with larger leaves at the base of the plant and small leaves higher on the flowering stem. Apex was oval in shape, base are symmetrical in shape vein lets are AlterNet. Upper surface of leaf are smooth while lower surface was ruff.

Microscopic Features: The transverse section of the leaf showed following characters:

• The leaf is generally ovate to triangular based. The lamina portion consisted of upper and lower epidermis. The midrib consisted of spiral vascular bundles namely lignified xylem and non lignified phloem. Below the vascular region spongy parenchyma cells were observed.
• Transverse section of leaf:

FIG. 1: TRANSVERSE SECTION OF LEAF

1. Powder characteristics: The powder was characterized on its morphological features as;

Color: Green,

Odor: Odorless,

Taste: Palatable

Microscopical examination of drug powder revealed the presence of anomocytic stomata, with lignified xylem, non lignified phloem and spongy parenchyma cells.

FIG. 2: STOMATA

FIG. 3: VASCULAR BUNDLES

FIG. 4: SPONGY PARANCHYMA

TABLE 1: HISTOCHEMICAL COLOR REACTIONS

Physicochemical Parameters: The percentage presence of acid insoluble ash, water soluble ash, water insoluble ash, and loss on drying are tabulated in table 2.

TABLE 2: ASH VALUES OF SPINACIA OLERACEA LEAF

Extractive value: The results revealed the highest extractability of powdered leaf drug was found in methanol and water. Extractive values of different solvent for the extractability are shown in table 3.

TABLE 3: EXTRACTIVE VALUES WITH DIFFERENT SOLVENTS

TABLE 4: LEAF CONSTANTS FOR SPINACIA OLERACEA

Florescence Analysis: The extracts were prepared by the maceration, further they were treated with various reagent and the color changes were observed under ultra-violet light. All the results are shown in table 5.

TABLE 5: RESULT OF FLUORESCENCE ANALYSIS OF ALCOHOLIC EXTRACT

TABLE 6: RESULT OF FLUORESCENCE ANALYSIS OF ACETONE EXTRACT

TABLE 7: RESULT OF FLUORESCENCE ANALYSIS OF CHLOROFORM EXTRACT

TABLE 8: RESULT OF FLUORESCENCE ANALYSIS OF PET ETHER

TABLE 9: RESULT OF FLUORESCENCE ANALYSIS OF ETHYL ACETATE

TABLE 10: RESULT OF FLUORESCENCE ANALYSIS OF AQUEOUS EXTRACT

Preliminary Phytochemical Analysis: The ethanolic, methanolic, aqueous, petroleum ether, chloroform, ethyl acetate, and acetone extract was used for the preliminary phytochemical analysis for their presence of the constituents. It showed the presence of Alkaloids, tannins, proteins and carbohydrates in all the extract except in petroleum ether.

DISCUSSION: The pharmacognostical study is one of the major criteria for identification of plant drugs. The present study on pharmacognostical characteristics of Spinacia oleracea leaf will provide useful information for its correct identity. Powder test and extractive value are performed as per stander method; LOD and all the result are in the specific criteria.

CONCLUSION: The current study will serve to become a ready reference for its identification and standardization on the basis of microscopy and chemical analysis. The preliminary phytochemical investigation will further help in isolation of important compounds in future.

REFERENCES:

1. Fuhrman J: Fruits and vegetables provide only modest protection from cancer, available at http://www.diseaseproof.com/archives/cancer-fruits-and-vegetables-provide-only-modest-protection-from-cancer.html 2010 (cited on April 2010)
2. Rutherford D: Fruits and Vegetables available at http://www.netdoctor.co.uk/focus/nutrition/facts/oxidative_stress/fruitvegetables.htm 2007 (cited on April 2010)
3. S. Department of Agriculture, Agricultural Research Service. 2005. USDA National Nutrient Database for Standard Reference, Release 18. Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/foodcomp (cited on April 2010)
4. Adam G, Mundry KW and Straub P: Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.) Journal of Phytopathology 2008; 115: 357 – 367.
5. Patil UK, Dave S, Bhaiji A, Baghel US, Yadav SK and Sharma VK: In-vitro Anthelmintic Activity of Leaves of Spinacia oleracae International Journal of Toxicological and Pharmacological Research 2009; 1, 1, 21 – 23.
6. Verma RK, Sisodia R. and Bhatia AL: Role of Spinacia oleracea as Antioxidant: A biochemical study on mice brain after exposure of gamma radiation. Asian Journal of Experimental Sciences 2003; 17, 51-57.
7. Gupta RS and Singh D: Amelioration of CCl4-induced hepatosuppression by Spinacia oleracea leaves in wistar albino rats. Pharmacologyonline 2006; 3: 267-278.
8. Longnecker MP, Newcomb PA, Mittendorf R, Greenberg R and Willet W: Intake of Carrots, Spinach, and Supplements Containing Vitamin A in Relation to Risk of Breast Cancer. Cancer Epidemiology, Biomarkers & Prevention 1997; 6: 887-892.
9. Wahi AK and Geetha M: Pharmacognostical studies on leaves of Barleria prionitis Indian Journal of Natural Products 2002; 16:16-19.
10. Wallis TE: Practical Pharmacognosy. J. and A. Churchill Ltd., London, 1953:139.
11. Anonymous: Pharmacopoeia of India. Govt. of India, Ministry of Health, Controller of publication, New Delhi.1996: II, A-54
12. Pratt RJ and Chase CR: Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. Journal of American Pharmaceutical Association 1949; 38: 324 -333.
13. Kokoski J, Kokoski R and Salma FJ: Fluorescence of powdered vegetable drugs under ultraviolet radiation. Journal of American Pharmaceutical Association 1958; 47: 715-717.
14. Kokate CK, Purohit AP, Gokhale SB: Pharmacognosy. Nirali Prakashan, Pune, thirtieth Edition, 2004: 593-597.
15. Harborne JB: Phytochemical methods. Chapman and Hall, London, Edition 3, 1998:90, 203.