INTRODUCTION:

Herbal drugs have been used since ancient times as medicines for treatment of a wide range of diseases. In spite of the great advances observed in modern medicine in recent decades, crude drugs either of plant; animal or mineral origins still make an important contribution to health care. Ayurveda is unique holistic system, based on the interaction of body; mind and spirit In about 400 BC, the first Ayurvedic medical school was founded, its compendium of writings from around 100 AD and describes 341 plant medicines as well as medicines of animal and mineral origin; Among them are visnaga, Gotu kola and others.^[1]

Churnas are classical ayurvedic formulation. In Talisadi churna, Zingiber officinale is one drug, which contains 6-Gingerol as a major constituent.

Figure 1: [6]-Gingerol (5-hydroxy-1-(4′hydroxy-3′-methoxyphenyl)- 3-decanone)

The Talisadi churna is a well-known ayurvedic formulation included in Ayurvedic formulary containing ginger as the main ingredient, which is one of the most popular ayurvedic medicines and possess many safe and effective drugs, useful in various disorders. The Talisadi churna is useful in treatment of vomiting, flatulence with gurgling sound, cough, asthma, fever, anorexia, indigestion, diarrhoea, cachexia, splenic disease, malabsorption syndrome and anaemia.^[2]

Talisadi churna (as per Ayurvedic formulary of India) mainly contains the mixture of powdered crude drugs of Abies webbiana, Piper nigrum, Zingiber officinale, Piper longum, Bombusa bombos, Elettaria cardamomum, Cinnamomum zeylanicum and Sugar.^[2]

Standardization of herbal formulations in terms of quality of raw materials, manufacturing practices, and composition is important to ensure quality and optimum levels of active principles for their bio-potency. Recently, the concept of marker-based standardization of herbal drugs is gaining momentum. Identification of major and unique compounds in herbs as markers and development of analytical methodologies for monitoring them are the key steps involved in marker-based standardization.^[3]

A comprehensive and quantifiable identification method for fingerprint development; is able to reveal chemical information of herbal medicines with spectrogram and other graph by analytical and chemical techniques.^[4]

The identification of herbal medicines from various sources is crucial in order to ensure authenticity, quality, safety and efficacy. Now a day’s most of the herbal formulation are lacking in their defined quality control parameters. Therefore, they are not well accepted in global market. Hence, WHO has emphasized the need to ensure the quality of medicinal plant products by using modern analytical technique and applying suitable standards.^{[5, 6]}

MATERIALS AND METHODS:

Procurement of Crude Drug and chemicals

Crude drugs were procured from local market and identification was confirmed by macroscopic and microscopic characters. Reference 6-Gingerol was purchased from Sigma–Aldrich (Germany). All solvents used were of analytical grade and procured from Merck (Mumbai, India).

Preparation of the Formulation

Talisadi churna, two laboratory batches (named TLS-I, TLS-II) were prepared in laboratory according to reported method of Ayurvedic formulary of India. The available commercially brand TMS-A, TMS-B and TMS-C of Talisadi churna was procured from local Pharmacy.^[2]

Preparation of Standard Solution of 6-Gingerol

Accurately weighed 6-Gingerol (10 mg) was transferred in 100 ml volumetric flask and dissolved in and diluted to 100 ml with methanol. The final solution contained 100 mg of the Gingerol per ml of the solution.

Preparation of Calibration Curve for 6- Gingerol

Standard solutions of 6-Gingerol were pipetted into concentration range 10-60 mg/ml in a series of five 25 ml volumetric flask. The absorbance of the 6-Gingerol was measured at 282 nm against ethanol by Shimadzu 1700 UV-Vis spectrophotometer. The results are shown in Table 1, Figure 2, 3.

Figure 2: UV-Visible Spectra of 6- Gingerol

Table 1: Calibration data of 6- Gingerol

Conc.(mg/ml)	Absorbance
10	0.231
20	0.512
30	0.824
40	1.128
50	1.411
60	1.758

Figure 3: Calibration curve of 6- Gingerol

Method Validation

Precision

The intra-day precision of the spectroscopic method was validated with a standard solutions (concentrations 10, 30 and 60 mg/ml) of 6-Gingerol under the selected optimal conditions three times a day. For inter-day precision, measurements one time a day on three consecutive days were conducted. All of the measurements were expressed as relative standard deviations (RSD).

Repeatability

Six independently prepared sample solutions (concentrations 10-60 mg/ml) of 6- gingerol with the same amount were analyzed and the variations within six measurements were calculated for evaluation of repeatability. The processes of the measurements were in accordance with the “Preparation of sample solutions of 6-gingerol” in parallel.

Recovery

Standards of 6-Gingerol with the known amounts in solutions were spiked to the Talisadi churna solution of which the contents of 6-Gingerol had been determined before the addition of the standard chemical. Then, 6-Gingerol marker compound in Talisadi churna sample solutions were extracted, processed and quantified in accordance with the established procedures, and finally the recovery rates were calculated. Same procedures were applied for the three different marketed formulations.

Estimation of 6-Gingerol in the Formulations

6-Gingerol was isolated by the methods of Thresh and Garnett and Grier.1 gm of powdered ginger was exhausted with 95% alcohol and the percolate concentrated to a volume of one litre. An equal volume of water was added which caused the precipitation of a large part of the fats and resins, leaving most of the 6-Gingerol in solution. The solution was shaken out with petroleum ether and evaporated and the residue extracted by repeatedly boiling out with petroleum ether from which the 6-Gingerol was removed by shaking with 60% alcohol. On evaporating the alcohol 6-Gingerol was obtained as very pungent, yellow oil. The obtained extract was analyzed. The same procedure was applied for the estimation of 6-Gingerol in the marketed formulations. The analysis was repeated six times. The possibility of interference from other components of extract in the analysis was studied. ^[7]

RESULT AND DISCUSSION

The developed method was found to be reliable, accurate, precise and sensitive. The method involves absorbance measurement at 282 nm for 6-Gingerol corresponding to the absorption maxima of the herbal formulation Talisadi churna. The optical characteristic shows that 6-Gingerol obeys Beer Lambert’s law in concentration range 10-60 mg/ml at λ-max 282 nm. The correlation coefficient (r²), Regression equation, Precision and Accuracy were calculated for the spectrophometric method and results are summarized in table-2.

Table 2: Optical and regression characteristics of 6-Gingerol

Parameters	Observations
Absorption maxima	282 nm
Beer’s law limit (mg/ml)	10-60
Correlation coefficient (r²)	0.9993
Regression equation (y^*) Slope (a) Intercept (b)	Y = 0.3039 X -0.0863 0.03039 0.08630
Precision Repeatability (n = 6) Interday precision (n = 3) Intraday precision (n = 3)	0.823 ± 0.001414 0.230667 ± 0.001528 0.228333 ± 0.002517

y* = b + ac, where “C” is concentration in mg/ml and y is absorbance unit.

The r² value 0.9993 indicates the good linearity between the concentration and absorbance. The estimation of 6-Gingerol content of Talisadi churna (three laboratory and two marketed samples) and powder of crude drug Ginger (Zingiber officinale) was carried out separately. The concentration of 6-Gingerol present in raw material was found to be 0.771 ± 0.001643 w/w in Zingiber officinale. Content of 6-Gingerol in different batches of Talisadi churna (laboratories and marketed batches) was found to be 0.669 ± 0.001862 %, 0.667 ± 0.001169 %, 0.666± 0.001211 and 0.615 ± 0.000894 % and 0.611 ± 0.001095 % w/w respectively for TLS-I, TLS-II, TLS-III and TMS- A and TMS-B. Results are summarized in Table-3.

Table 3: Estimation of 6-Gingerol content (% (w/w))

Formulations		6-Gingerol Content (w/w)	Standard Deviation
Talisadi Churna	TLS-I	0.669	0.0018
	TLS –II	0.667	0.0012
	TLS-III	0.666	0.0012
	TMS-A	0.615	0.0009
	TMS-B	0.611	0.0011
Zingiber officinale (Ginger)		0.771	0.0016

In order to obtain precision and accuracy the recovery study were performed by adding known amount of 6-Gingerol with pre-analyzed sample of 6-Gingerol in Talisadi churna. The result shows 99.07 ± 0.0592 % recovery of 6-Gingerol which shows reproducibility of the result. Results are summarized in Table-4.

Table 4: Results of recovery of 6-Gingerol in Talisadi Churna

S No.	Conc. of sample (µg/ml)	Added Conc. (µg/ml)	Found Conc. (µg/ml)	Recovery %				SD	% CV
S No.	Conc. of sample (µg/ml)	Added Conc. (µg/ml)	Found Conc. (µg/ml)	I	II	III	Avg.	SD	% CV
1	10	5	14.76	98.4	98.40	98.30	98.37	0.0577	0.0588
2	10	10	19.86	99.3	99.20	99.40	99.30	0.1000	0.1007
3	10	15	24.89	99.56	99.54	99.52	99.54	0.0200	0.0200

CONCLUSION:

The present work was carried out for preparation, standardization and fingerprint development of Talisadi Churna. The results obtained were equally compatible with that of marketed formulation. The proposed method of quantitative determination of 6-Gingerol in the raw plant material, extract, and marketed formulations and characterized by high sensitivity, relatively small error, and good reproducibility.

So far since no attempt has been made to evaluate the polyherbal formulation, the present work is a small step towards the development of quality control methods for polyherbal preparation. This will also help to produce uniform standard products, which will restore the faith in Ayurvedic system.

ACKNOWLEDGEMENT:

The authors are highly grateful to FIST scheme, {F. No. (SR/FST/LS1-013/2010)} Govt. of India, New Delhi for supporting infrastructure and UGC, [F. No: 34-131/2008 (SR)] Govt. of India, New Delhi for financial assistance under major research project.

REFERENCES:

1. Mukherjee P. Quality Control of Herbal Drug: An Approach to Evaluation of Botanicals. New Delhi: Eastern Publishers; 2002.

2. Anonymous: Ayurvedic formulary of India (2003), Part-1, 2^nd edition, Government of India, Ministry of Health and family Planning, Department of Indian System of Medicine and Homeopathy, Delhi, pp.103-119.

3. Shukla SS, Saraf Swarnlata, Saraf S. Approaches towards standardization and quality assessment of herbals. J Res Educ Indian Med 2009;15(1):25-32.

4. Yu Rongmin, et al. Fingerprint analysis of fruiting bodies of cultured Cordyceps militaris by high-performance liquid chromatography–photodiode array detection. J Pharm Biomed Anal 2007;44:818–23.

5. Jain V, Saraf Swarnlata, Saraf S. Standardization of triphala churna, spectrophotometric approach. Asian Journal of Chemistry 2007;19(2):1406-10.

6. WHO. General guidelines for methodologies on research and evaluation of traditional medicine. Geneva, 2000. Available from: <http://whqlibdoc.who.int/hq/2000 /WHO_EDM_TRM_2000. 1.pdf>.

7. Tzucker Robert, Jordan CB. A study of the assay of ginger. J Am Pharm Assoc 1940;29(6):265-269.

Received on 11.10.2011 Modified on 22.11.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 138-140

Absorbance

Table 4: Results of recovery of 6-Gingerol in Talisadi Churna

SD

%

CV