STANDARDIZATION STUDY OF GHRITAS

Abstract. The standardization of ghritas such as amritaprasa ghrita, brahmi ghrita, chagalyadi ghrita and phala ghrita has been studied. These ghritas are the important Ayurvedic formulations used for peri-natal care of mother and child health. Standardization of ghritas were achieved by organoleptic study, physico-chemical analysis, qualitative analysis, thin layer chromatography (TLC), UV - visible spectrophotometry and high performance liquid chromatographic (HPLC) fingerprint studies. Qualitative analysis of alcoholic extracts of all the four ghritas shows the presence of glycosides and hexane extracts shows the presence of glycosides and steroids. TLC study of ghritas was carried out in toulene-ethyl acetate solvent system. Hexane extracts of ghritas were used for UV- visible spectrophotometry and qualitative HPLC fingerprint study.


Introduction Introduction
Ghrita is one of the Ayurvedic drugs that contain ghee as the base to dissolve or extract or hold the active therapeutic principles from the ingredients.Ghritas are medicated ghee preparations containing the fat-soluble components of the ingredients used in these preparations.The principle of preparation is the protracted boiling of ghee with prescribed kashayas (decoctions) and kalkas (a fine paste of the drug/drugs) to dehydration or near dehydration thereby effecting the transference of the fat soluble principles to the ghrita, from the drug ingredients or kashayas or swarasas as the case may be according to the formulation. 1][3][4][5] The present communication deals with the standardization of ghritas such as amritaprasa ghrita, brahmi ghrita, chagalyadi ghrita and phala ghrita which are used for the perinatal care of mother and child health.In Ayurveda, amritaprasa ghrita is therapeutically indicated for nastasukra, ksataksina, vyadhikarsata, kasa, hikka, jvara, svasa, daha, trsna, raktapitta, etc., brahmi ghrita for unmada, kustha, apasmara, vandhyatva, etc., chagalyadi ghrita for kasa, uroroga, parsvasula, urakasta, ksaya, etc., phala ghrita for balaroga, balagraha, sukravikara, yonivikara, vandhyatva, garbhiniroga, etc.A few analytical standard values have been prescribed for amritaprasa ghrita, brahmi ghrita, chagalyadi ghrita and phala ghrita.

Materials and Methods
The authentic ingredients were procured from the local market of Hyderabad, Andhra Pradesh and were botanically identified.Amritaprasa ghrita, brahmi ghrita, chagalyadi ghrita and phala ghrita were prepared as per the procedure described in Ayurvedic Formulary of India.

Analytical study
The prepared samples were analyzed for the parameters such as organoleptic study, moisture content, total ash, acid insoluble ash, alcohol soluble extractive, hexane soluble extractive, qualitative organic analysis, free fatty acid, acid value, saponification value, iodine value and refractive index. 5in layer chromatography TLC plates were prepared as per the procedure described by Stahl. 8 The 4% hexane extracts of the samples were prepared by soaking them for 18h in absolute hexane.Hexane extracts were filtered and concentrated.Respective concentrated hexane extracts were redissolved in toluene-ethyl acetate (93:7) and about 100µl was loaded on the TLC plate and eluted in toluene-ethyl acetate (93:7) solvent system. 9The plates were sprayed with vanillin-sulphuric acid reagent and the spots were detected after heating at 110°C for 30min.Rf value of each spot was calculated.

Sample preparation
The 4% hexane extracts of the samples were prepared by soaking them for 18 h in hexane.The extracts were filtered through Whatman filter paper number 1 using high-pressure vacuum pump.The samples were used for UV-visible spectrophotometric and HPLC fingerprint study.

UV-visible spectrophotometric analysis
The samples were scanned over a range of 200-800nm using ELICO (SL-159) UV-visible spectrophotometer equipped with quartz cuvettes of 10mm path length and UV-visible spectrasoft software.Hexane was used as a reference.

HPLC Analysis
An isocratic HPLC (Shimadzu HPLC Class VP series) with two LC-10 AT VP pumps (Shimadzu), variable wave length programmable photo diode array detector SPD-M10A VP (Shimadzu), CTO-10AS VP column oven (Shimadzu), SCL-10A VP system controller (Shimadzu) and normal phase Luna 5µ Silica (2) Phenomenex column (250mm X 4.6mm) was used.The HPLC system was equipped with software Class VP series version 6.1 (Shimadzu).The mobile phase components hexane-isopropanol were filtered through 0.2µ membrane filter before use and pumped from the solvent reservoir to the column at a flow rate of 2ml/min which yielded a column back pressure of 180 kgf/cm 2 .The column temperature was maintained at 27°C.20µl of sample was injected by using Rheodyne syringe (Model 7202, Hamilton).
The variation in analytical values of ghritas may be due to several factors such as heat induced changes due to protracted boiling, oxidation changes due to open heating or due to presence of drugs during heating for a long time, changes brought about by the incorporation of fat soluble fractions from drugs, changes brought about by the mixing in of fats or oils other than ghee, changes caused by substitute drug ingredients. 7The data of qualitative organic analysis of ghritas is summarized in Table -   The qualitative HPLC fingerprint profiles of amritaprasa ghrita, brahmi ghrita, chagalyadi ghrita and phala ghrita are shown in Figure -1 to 4 respectively.The HPLC chromatogram of amritaprasa ghrita showed four peaks at a retention time of 1.515min, 1.749min, 2.005min and 2.251min with an area percentage of 71.54, 17.93, 2.23 and 4.71; brahmi ghrita showed two peaks at a retention time of 1.461min and 1.941min with an area percentage of 89.41 and 9.76; chagalyadi ghrita showed two peaks at a retention time of 1.461min and 2.051min with an area percentage of 91.09 and 5.2; phala ghrita showed four peaks at a retention time of 1.451min, 1.621min, 1.931min and 2.048min with an area percentage of 79.91, 11.59, 5.51 and 2.17 at a wavelength of 220 nm.

Conclusion Conclusion
The analytical data, TLC, UV-visible spectrophotometric and HPLC fingerprint profiles evolved can be considered as viable parameters which will go a long way for prescribing a dependable standard to these preparations.

FIGURE 4
FIGURE 4 FIGURE 4. HPLC CHROMATOGRAM OF PHALA GHRITA 2 The data of TLC study of ghritas is summarized in Table -3 The UV-visible spectrophotometric data of ghritas is tabulated in Table-4.