Comparative Powder Microscopic and HPTLC Studies on Stem barks of Symplocos racemosa Roxb . and Symplocos crataegoides Ham .

Introduction: Comparative powder microscopic and HPTLC studies were carried out on stem barks of Symplocos racemosa Roxb. and Symplocos crataegoides Ham. to differentiate its identity in Ayurvedic formulations. Method: Powder microscopic and HPTLC studies of these barks were undertaken on a comparative basis and results are reported in this paper. The authentic samples are cleaned, powdered and passed through sieve No. 80. A few mg of powder was stained with saffranin, Toluidine blue and Iodine and photographed under different magnifications with the help of NICKON Labphot -2. HPTLC studies were followed by Sethi and Wagner et al method. Results: The colour, taste, cork cells, fibres, sclereids, starch grains, calcium oxalate crystals, number of spots and Rf values of HPTLC are found to be differentiating diagnostic characters in powdered form of S. racemosa and S. crataegoides. Conclusion: The findings of the present study is believed to be helpful in standardization of Ayurvedic formulation containing stem bark of S. racemosa and S. crataegoides as ingredients in powder form. The study sets the specific microscopic protocol and HPTLC finger print of the two drugs and may lead to global acceptance and reputation of the Ayurvedic system.


MATERIALS AND METHODS Collection and Identification
The stem bark of Symplocos racemosa Roxb.was purchased locally from market and authenticated by Prof. P. jayaraman, Director, Plant Anatomy Research Centre, Chennai.The authentic stem bark of Symplocos crataegoides Ham. was supplied by Dr. G. C. Joshi, Research Officer, Regional Research Institute of Himalayan Flora, CCRAS, Thapala, Uttarakhand.The photos of the medicinal plants and its barks are given in Figure 1.The specimen vouchers of the stem barks of S. racemosa and S. crataegoides were deposited in CSMRIASDD Museum (K219/SB22 and C222/BSB26).

Powder microscopic studies
The samples were cleaned, shade dried, powdered and passed through sieve No. 80.A few mg of powder was analyzed microscopically after clearing them in Chloral hydrate solution.A few mg of powder was Preparation of extracts for TLC/HPTLC studies 5 g of drug of each sample were shade dried and coarsely powdered and defatted with hexane.The plant materials were packed in a Soxhlet apparatus and extracted successively with chloroform and ethanol for 5 hrs separately.The extracts were filtered by using Whatmann No.1 filter paper.The extracts were concentrated on water bath and made up to 10 ml volumetric flask.

Method for developing TLC/HPTLC
Instrument: CAMAG (Switzerland), sample applicator: Camag Linomat-IV applicator with N 2 gas flow, photo documentation system: Digi store-        of both stem barks were chromatographed using toluene: ethyl acetate (8: 2), toluene: ethyl acetate: formic acid (7 : 3: 0.1) respectively.The plate was developed upto a height of 8 cm, air dried, spots were observed under the UV light at 254 nm and 366 nm.The HPTLC finger print profiles were also recorded at 254 nm.Finally the plates were derivatized using vanillin sulphuric acid reagent heated at 105°C till colour spots appeared. 13,14

RESULTS
Powdered stem bark of Symplocos racemosa and Symplocos crataegoides were studied for microscopic point of view and given in Table 1 and Figure 2 and 3. Different compositions of the mobile phase for TLC and HPTLC analysis were tested in order to obtain high resolution and reproducible peaks.
The TLC profile of chloroform and alcohol extract of S. racemosa and S. crataegoides is shown in Figure 4 and 5.The corresponding Rf values of various spots for chloroform and alcohol extract is given in Table 2. HPTLC finger print profile of chloroform extract of S. racemosa and S. crataegoides showed 11 and 7 peaks respectively (Figure 6).
HPTLC finger print profile of alcohol extract of S. racemosa and S. crataegoides showed 7 and 11 peaks respectively (Figure 7).

DISCUSSION
Microscopic method of authentication is the first and fundamental step for standardization of herbal formulation.The findings of the present study is believed to be helpful in standardization of Ayurvedic formulation containing stem bark of S. racemosa and S. crataegoides as ingredients in powder form.HPTLC profile of chloroform and alcoholic extract provides a suitable method for monitoring the identity, purity and also standardization of the drug.The study sets the specific microscopic protocols of the two drugs and may lead to global acceptance and reputation of the Ayurvedic system.

CONCLUSION
The present study, analysed the powder microscopic characters of stem barks of Symplocos racemosa Roxb and Symplocos crataegoides Ham. and HPTLC fingerprint of chloroform and alcoholic extract of the same respectively.The results will be helpful in differentiating these barks in powdered form or in authentication/identification of the crude drug/raw drug and in standardization of Ayurvedic formulation Curna.

Figure 7 :
Figure 7: HPTLC profile of alcohol extracts of S. racemosa and S. crataegoides.