Preliminary phytochemical and pharmacognostical evaluation of Carissa spinarum leaves

Beck Neeli Rose* and Namdeo Kamta Prasad

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidhyalaya, Bilaspur, C.G.

*Corresponding Author E-mail: neeli05011974@gmail.com, knamdeo@yahoo.com

ABSTRACT

Carissa spinarum Linn. is one of the medicinal plants. It has many medicinal uses such as to treat rheumatism, purgative etc. The present study has been carried out pharmacognostical and physico-chemical evaluations of its leaves. Preliminary Phytochemical investigations also have been carried out. Diagnostic pharmacognostic feature of Carissa spinarum leaves closed collateral vascular bundle, anomocytic stomata, covering and glandular trichomes, calcium oxalate crystal and prism are found. Physiochemical observation of leaves revealed total ash 14%, acid insoluble ash 5.3%, water insoluble ash 6.6%, sulphated ash 10.3%,water soluble extractive value 15.62% alcohol soluble extractive value 4.56%, chloroform soluble extractive value 4.3%, loss on drying3.52%.

Phytochemical screening of various extract of C.spinarum leaves revealed that phytoconstituents are alkaloids, tannin, glycoside, steroids and carbohydrates are present in plant.

KEYWORDS: Carissa spinarum, Karonda, Wild Karonda and Medicinal plant.

INTRODUCTION:

The roots of Carissa spinarum Linn. are reported to have many medicinal uses. It is belonging the family Apocynaceae. It is commonly known as Jangli Karonda in Jashpur District. The plant has been traditionally applied to kill worms in the wounds of cattle. It is also used in combination with the roots of some other medicinal plants to treat rheumatism by the Mundas (a tribe) of Chhota Nagpur. It is a strong purgative and is used as one of the ingredients in some purgative preparations. (Kirtikar and Basu, 1938).

In view of its medicinal uses and its insufficient literature the present investigation pharmacognostical and physico-chemical evaluations and Preliminary Phytochemical investigation of C.spinarum was carried out.

MATERIAL AND METHODS:

Plant collection and identification

The plant specimens were collected from forest of Jashpur district Chhattisgarh. The specimen was identified by Dr. Chandrama Prakash Upadhyay, Assistant Professor Guru Ghasidas Vishwavidhyalaya Bilaspur, C.G. Care was taken to select healthy plant for normal organs. Plants parts were washed with water and dried it in sunlight for one hour and then it were dried in shade.

The Voucher specimen (SLT/Med Plant/03/2009) was maintained in research laboratory for further reference in SLT Institute of Pharmaceutical Sciences. With the help of wood grinder the dried plant parts were powdered and passed through the sieve no 40 for powder analysis and coarse powder was used for phytochemical work.

Macroscopic characters

The macroscopic characters such as size, shape, margin, apex, surface, colour, odure, taste, nature, texture were studied for morphological investigation. (Kokate et al 1995 Wallis (1976) and Trease and Evans (2004).

Microscopic characters

For microscopical studies, free hand section of leaves were cut, cleared chlorophyll by heating with chloral hydrate solution and stained with phloroglucerol and hydrochloric acid (1:1) according to the prescribed methods. Stained section was mounted in 50% glycerin water and observed under microscope. (Kokate et al 1995, Wallis 1998 and Trease and Evans 1982). Powdered materials of leaves were cleared with sodium hydroxide and mounted in glycerin medium after staining of phloroglucerol and observed under the microscope. (Kokate et al 1995). Photographs of different magnifications were taken with Nikon Lab hot 2 microscopic Unit from Department of Pharmaceutical Sciences, Pt Ravi Shankar Shukla University, Raipur (Chhattisgarh). Different cell component were studied and measured.

Phytochemical evaluation-

The dried leaves powder (100gm) was extracted successively with petroleum ether, chloroform, alcohol and water using soxhlet extraction (48 hrs for each solvent). Finally the extracts were concentrated by under vacuum pressure and it is subjected to various phyto chemical tests for identification of different chemical constituents present in their respective extracts.

Physiochemical evaluation-

Physiochemical parameters carried out according to the official methods prescribed in Indian Pharmacopoeia. Physiochemical parameters such as ash values (total ash, acid insoluble ash, water insoluble ash) extractive values (Chloroform extractive value, water extractive value and alcohol extractive value), moisture content etc. (Kokate et. al 1995).

Thin layer Chromatography-

100gm of silica gel G was weighed and made into homogenous slurry with adding sufficient distilled water. Slurry was poured into TLC glass plates by spreading and made uniform silica gel layer on plate about 0.25mm thickness. The coated plates were allowed to dry in air and activated by heating in hot air oven at 100-105^oc for 1 hrs. Solvent system was selected by trial and error method. Detecting agent is Iodine vapour. The Rf values for different spots were calculated and recorded. (Wagner 1996)

RESULT AND DISCUSSION:

Macroscopic Characteristics of leaves (fig1)

Colour- Green

Odour- Odourless

Taste- Tasteless

Shape-ovate

Surface- smooth

Size-Length-3.3 cm, Wide 1.2cm

Margin – Entire

Apex- Acute

Veination –Reticulate pinnate

Base-Symmetrical

Texture-Brittle

Fig1 - Dry leaves of C.spinarum

Microscopic study of Carrisa spinarum leaf

Transverse section of Carrisa spinarum leaf showed in Fig 2. Histological characteristic of C. spinarum leaf are -

1. Upper epidermis and lower epidermis- Single layered rectangular cells, covering trichomes are seen.

2. Collenchyma-It is found below upper epidermis and above lower epidermis. Rest of midrib is filled with parenchyma. Collenchymas are thick walled cellulosic cells.

3. Spongy parenchyma- 4-6 layered and contains sclerenchymatous cell.

4. Vascular bundle-Arc shaped, xylems are lignified and phloem non lignified and closed collateral type.

5. Pericycle - Collenchymatous cells within endodermis.

6. Endodermis – Covers V.B. and contains starch grain.

Fig 2-Photomicrograph of T.S. of C. Spinarum leaf

Ct-Covering trichome, Uep- Upper epidermis, Mc-Microspinoid crystal, VB- Vascular bundle containing xylem and phloem, Sp-Spongy parenchyma, Pt- Palisade tissue, Lep- Lower epidermis

Powder morphology and microscopy – The macrocopical evaluation of powder of C. spinarum leaf revealed the green colour with odourless and tasteless. Powder microscopic images of C. spinarum leaves are presented in fig 3,and 4. The microscopic study of powder showed-

1. Epidermis: It is composed of sub rectangular to polygonal cells with straight, moderately thickened walls. Anomocytic types of stomata are abundant.

2. Fibres: The fibres have thick walls with few pits and fibres are cone shape.

3. Cluster crystals of calcium oxalate present are found numerous. They are prism shaped.

4. The fragments of thick walled parenchyma from the cortex of the midrib and the larger veins composed of longitudinally elongated cells.

5. Covering trichomes are unicellular conical shape. Glandular trichomes are less numerous, single cell stalk and a unicellular head.

Table 1

S/n	Test	Petroleum ether extract of Carrissa spinarum Leaves.	*Chloroform extract of Carrissa* spinarum leaves**	Ethanol extract of Carrissa spinarum leaves	Aq Ext of Carrissa spinarum leaves
1	Alkaloid	-	-	+	+
2	Tannin	-	+	+	+
3	Flavonoid	-	-	-	+
4	Saponin	-	-	-	-
5	Glycoside	-	+	+	-
6	Terpenoid	-	-	-	-
7	Carbohydrate and sugars	-	-	+	+
8	Fats and fixed oil	+	-	-	-
9	Protein and amino acid	-	-	-	-
10	steroids	-	-	+	+
11	Gums and mucilages	-	-	-	+

Note- (-) Negative, (+) Positive, Aq- Aqueous

St-stomata

Fig 3- Photograph of anomocytic type of stomata

Fig 4- Powder characteristic of C.spinarum leaves

Gt-Glandular trichome, Ct- Covering trichome, Fibre, Ca oxa- Crystal of calcium oxalate, Ec-Epidermal cell

Preliminary phytochemical evaluations

Preliminary phytochemical tests revealed the presence of alkaloids, tannins, flavonoids, glycosides, carbohydrates and sugars, steroids and fats and oils.

Table 2

S.No	Analytical parameter	Leaves %w/w
1	Total ash	14
2	Acid insoluble ash	5.3
3	Water insoluble ash	6.6
4	Sulphated ash	10.3
5	Alcohol soluble extractive	4.56
6	Water soluble extractive value	15.62
7	Chloroform soluble extractive value	4.3
8	Loss on drying	3.62

After trying several mobile phases, TLC profile of the leaf extracts were prepared. Solvent system is used by trial and error method. Benzene: Acetic acid: Water (16:3:1) for Petroleum ether extract of Carissa spinarum leaves, Chloroform: Acetic acid: Water (20:4:2) for chloroform extract, Chloroform: Methanol: Ammonia (20:8:2) for ethanolic extract, Acetone: Chloroform: Alcohol (3:5:5) for aqueous extract. Numbers of spots were produced after applying iodine vapour. Rf values according to their respective extracts are 0.84, 0.70, 0.98, 0.53, 0.80.

The present study establishes morphology and microscopic characteristics physiochemical values, phytochemical screening of Carissa spinarum leaves. The major diagnostic characteristics of the leaves of Carissa spinarum are seen in the T.S. views of leaves. Single layered epidermis cells. An arc shaped closed collateral type vascular bundle was present in the centre of mid rib portion. Covering trichomes are seen in epidermis. Powder characteristics of leaves of C. spinarum anomocytic type of stomata, covering trichome, cluster crystal of calcium oxalate are present.

Lastly it can be concluded that the pharmacognostical studies of the leaves of C. spinarum. Provide analytical parameters for both qualitative as well as quantitative. These can help to the identity and to determine the quality and purity of the plant materials for future studies. These parameters also will serve as standard data for quality control studies of pharmaceutical Ayurvedic preparations if C.spinarum added as an ingredient in any preparation.

REFERENCES-

1. Malik S, Gauttam V, Kalia AN. Pharmacognostical evaluation of Cassia obtusifolia leaves, Indian journal nat. product, 2011; 27(3): 3-17.

2. Harbone, JB. Method of extraction and isolation, Chapman andHall, London, 1998; 60.

3. Wagner, H. and Bladt, S. Plant Drug analysis. A thin layer chromatography atlas, 2^nd edition, Springer private limited, New Delhi, 1996; 232.

4. Wallis TE. A textbook of Pharmacognosy 3^rd edition J and A Churchill Ltd, London 1998; 60-66.

5. Kokate CK. Practical Pharmacognosy, Vallabh prakashan 1994; 4-29.

6. Kokate CK, Khandelwal KP, Pawar AP, Gokhale SB. Practical Pharmacognosy Nirali Prakashan 1995; 1: 11-19.

7. Anonymous: Macroscopic and Microscopic Examination: qualitative control methods for medicinal plants materials WHO Geneva 1998.

8. Trease E and Evans WC. Pharmacognosy William Charles Evans as edited in 15^thedition Saunders publisher London.2004; 137-440.

9. Gorwadiya HC, Savalia RM, Vachhani KV, Desai TR, Pandya DJ, Pharmacognostic study and establishment of quality parameters of leaves of Ficus racemosa Linn, Pharmacognosy journal 2010;2 15:15-20.

10. Abid M. Pharmacognostic studies of some Indigenous medicinal plants of Pakistan, Ethanobotanical leaflets.2005; 2005: 1: 20.

11. Khatoon S, Rai V, Rawat AKS, Mehrotra S. Comparative Pharmacognostic studies of three Phyllanthus species,2006;104: 1-2:79-86.

12. Krishnaveni A, Thaakur, Rani S, Pharmacognostical and preliminary phytochemical studies of Argyreia nervosa Burm, Ethnobotanicle leaflets.2009; 2: 1.

13. Al-said MS, Kadertaragan AHU, Hifnawy MS. Pharmacognosticle and preliminary phytochemical investigation of the fruit of Pergularia Tomentosa L. Pharmaceutical biology1988; 26, 1: 9-16.

14. Stahl E.Thin layer Chromatograpy A Laboratory Handbook Spring Verlog Berlin Heidenberg.

15. Siddiqui S, Verma A, Rather AA, Jabeen F, Meghvansi MK, Preliminary phytochemicals analysis of some important medicinal and aromatic plants Advances in Biological Research 2009;3 :(5-6): 188-1995.

16. Basu K. Indian medicinal Plants, Oriential enterprises 5^th Volume II ^nd Ed 1938; 1494-1499.

17. Ansari SH. Essential of Pharmacognosy, Birla Publications. 2004; 1-20.

Received on 20.01.2013 Accepted on 10.02.2013

Asian J. Pharm. Tech. 3(1): Jan.-Mar. 2013; Page 30-33