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RESEARCH ARTICLE

 

Development of HPLC Finger Printing Methods for Polyherbal Ayurvedic Formulation: Mrichyadi Vati

 

Mishra Kamlendra*,  Mahajan Suresh Chandra, Shukla Karunakar

Mahakal Institute of Pharmaceutical Studies, Behind Air Strip, Datana, Dewas Road, Ujjain 456-664

*Corresponding Author E-mail:

 

ABSTRACT:

Marichyadi Vati is an important ayurvedic formulation which is official in Ayurvedic Formulary of India is combination of ingredients (Piper nigrum, Piper longum, Hordeum vulgare, Punica granatum, Saccharum officinarum).The formulation is dispensed for the disorder of respiratory tract. It eliminates the aggravated kapha in the respiratory tract and in the digestive channels. It also regulates the path for vatta and helps minimize gas formation in the abdomen, being hot in nature. Therapeutically it is also used for the treatment of kasa (cough) and svasa (asthma). Piperine is one of the major constituent of Marichyadi Vati. Herbal formulations show the number of problems when quality aspect is considered. This is because of nature of the herbal ingredients and different secondary metabolites present therein. Mainly, variation in the chemical profile of the herbal due to intrinsic and extrinsic factors (growing, harvesting, storage and drying processes).

 

The developed method of estimation can be considered as the protocol for the evaluation of Marichyadi vati which will assist the regulatory authorities, scientific organizations and manufacturers in developing standards. The method used for evaluation is found to be precise and reproducible and help to produce uniform standard products, which will restore faith in Ayurvedic system. The developed method can also be applied to various polyherbal formulations for the quantitation of piperine and can be used as a routine quality control method in pharmaceutical industries.

 

KEY WORDS: Ayurvedic formulation, Marichyadi vati, Finger printing, HPLC.

 

 


INTRODUCTION:

Herbal formulations show the number of problems when quality aspect is considered. This is because of nature of the herbal ingredients and different secondary metabolites present therein. Mainly, variation in the chemical profile of the herbal due to intrinsic and extrinsic factors (growing, harvesting, storage and drying processes) [1-3].

 

 

 

 

 

 

 

 

 

Received on 26.06.2015          Modified on 05.07.2015

Accepted on 23.07.2015        © RJPT All right reserved

Research J. Pharm. and Tech. 8(7): July, 2015; Page 880-884

DOI: 10.5958/0974-360X.2015.00143.2

Ayurveda is practiced widely in India, Srilanka and other countries[4] and Ayurvedic preparations are either of herbal origin, mineral origin, animal origin or combination of them. The safety and efficacy of these formulations is closely correlated with the quality and the source of raw materials used in their production[5].

It is generally possible to estimate a phytochemical marker for the ingredient plant raw material using various analytical techniques like TLC, HPLC and HPTLC. It is possible to detect the presence of the marker phytochemical compound and also quantify it to ascertain the limits in the final formulation[4,6,7]

 

Chromatographic fingerprint have been suggested to check for authenticity or provide quality control of herbal medicine[8]. Chromatography has the advantage of separating a complicated System into relatively simple sub-systems and then presenting the chemical patterns of herbal medicine in the form of a chromatogram. The World Health Organization (WHO) accepts fingerprint chromatography as an identification and quality evaluation technique for medicinal herbs since 1991[9]. Fingerprints can be a unique identification utility for herbs and their different species)[10,11], and can be used for modeling pharmaceutical activities[12]. Now, chromatographic fingerprint technique plays an important role in controlling the quality of TCM for the systemic characterization of compositions of samples and focusing on the identification and assessment of the stability of the components[13]. The Patent proprietary Ayurvedic medicines are sold over the counter in pharmacies; these products appear to represent a major share of branded traditional medicine in India. Nevertheless systems like Ayurveda still need to gain an empirical support of modern medical sciences to make them credible and acceptable for all. An innovative research effort to define the advantage of traditional system of medicine with respect to their safety and efficacy could result in a better utilization of these complementary systems of medicine[14].

 

Marichyadi Vati is an important ayurvedic formulation which is official in Ayurvedic Formulary of India is combination of ingredients (Piper nigrum, Piper longum, Hordeum vulgare, Punica granatum, Saccharum officinarum).The formulation is dispensed for the disorder of respiratory tract. It eliminates the aggravated kapha in the respiratory tract and in the digestive channels. It also regulates the path for vatta and helps minimize gas formation in the abdomen, being hot in nature[15]. Therapeutically it is also used for the treatment of kasa (cough) and svasa (asthma) [16]. Piperine is one of the major constituent of Marichyadi Vati. Molecular formula C17H19NO3 density 1.193 g/cm3, melting point 130°C, in animal studies, piperine also inhibited other enzymes important in drug metabolisms [17].

 

MATERIAL AND METHODS:

Instrumentation and Chromatographic Conditions

The method developed involved the use of Yongline acme 9000 pump, Shimadzu system controller and Shimadzu UV-VIS 1700 detector. The column used was LiChrosorb NH2 (10 cm) and AUTOCHROM 3000 software was used as integrator. A flow rate of 1.5 ml/min was maintained. The optimized mobile phase was found to be Methanol: Water (69:31 v/v) was passed through a 0.22µm membrane filters and degassed by ultrasonication under vacuum before use. The injection volume was 20µL and the effluent was monitored for UV absorption at 343 nm was used for quantitative estimation. All separations were performed at ambient temperatures. The optimized method was then validated for limits of detection, linearity, range, precision and accuracy and specificity.

 

Procurement of Crude Drugs and Marketed Formulations.

The raw materials were purchased from the local market of Ujjain for the formulation of Marichyadi Vati, and marketed formulation was obtained as a gift sample (i.e. Marichydi Vati manufactured by Shree Baidynath Ayurved Bhavan  Pvt. Ltd. Bamhani, Gopalgang, Dist. Seoni M.P. Batch no. 08), from Mahakal Ayurveda Sansthan, Ujjain.

 

Authentication of Plant Material

The plants were identified by Dr. S. K. Billore (Professor and head of department), botany department of Madhav Science College, Ujjain (MP) and voucher specimen (MIPS/P/001/2009) of Piper longum Linn., (MIPS/P/002/2009) Piper nigrum Linn., (MIPS/S/001/2009) Saccharum officinarum Linn., (MIPS/P/003) Punica granatum Linn, (MIPS/H/001/2009) Hordeum vulgare Linn. were deposited in the of department of Pharmacognosy, Mahakal Institute of Pharmaceutical Studies, Ujjain (MP).

 

Preparation of Formulations15

A fine powder of Pippali, Marica, Yavakshar, Dadima (phala, tvak) was prepared by passing the required quantity through the sieve no. 80.

                          

Table 1: Ingredients used in the formulation of Marichyadi Vati (300 vati)

S.No.

Botanical name

Local name

Plant part used

Quantity (in gms)

1

Piper nigrum

Marica

Fruit

12

2

Piper longum

Pippali

Fruit

12

3

Hordeum vulgare

Yavakshar

Salt

06

4

Punica granatum

Dadima (Phalatvak)

Bark

24

5

Saccharum officinarum

Guda

Dried concentrated form of juice

96

 

Preparation of standard solution of Piperine

Standard solution was prepared by dissolving accurately weighed (100 mg) piperine in 100 ml methanol into a 100 ml volumetric flask (Stoke A) containing 1000 micro gram of peperine per ml of the solution and further dilutions were made from stoke A [18, 19].

 

Standard plot of Piperine

Serial dilutions containing 2, 4, 6, 8 and 10 μg/ml piperine in methanol were prepared from the stock solution of piperine (100 mg/100ml). Each dilution was chromatographed on HPLC and area under the peak of piperine recorded (Table I). Retention time of piperine was observed to be 7.7 min. A standard curve of piperine was prepared by plotting the actual amount of piperine present in different dilutions against the area under the peaks of piperine observed by injecting above serial dilutions. The intercept and the slope of the standard plot was observed to be 0.00 and 70.52 respectively, with coefficient of correlation as 0.999 (r2).

 

Sample Preparation:

Accurately weighed 4 gm of Marichyadi Vati was refluxed for 1 hr with 100 ml of methanol. The extract was filtered and concentrated till the semisolid mass was obtained and from that 100 mg was dissolved and diluted up to 100 ml with methanol in volumetric flask. Same procedure was used for the preparation of solution for estimation of piperine for marketed formulations.

 

The same procedure was performed separately for, the powdered crude drug of Piper longums (Pippali) and piperine extract. Each of the solutions was subjected to HPLC analysis and the area under the peak of piperine was recorded.

 

 

 

Figure: Calibration graph of standard peperine.

 

 

Figure: HPLC Chromatogram of isolated piperine from crude drug.

Figure: HPLC Chromatogram of extract of laboratory preparation.

 

 

Figure: HPLC Chromatogram of extract of marketed formulation.

 

 

 

Figure: Overlay chromatogram of crude drug, sample and standard piperine.

Figure: HPLC chromatogram of piperine (Standard solutions) showing the precision.

 

Validation Parameters

Selectivity and peak purity

Selectivity was checked by using prepared solutions of MV and available standards optimizing separations and detection. The purity of the peaks was checked by multivariate analysis. Three spectra corresponding to upslope, apex and down slope of each peak were computer normalized and superimposed. Peaks were considered pure when there was a coincidence between the three spectra (match factor was =98%).

 

Linearity, limits of detection and quantification

The linearity of the detector response for the prepared standards was assessed by means of linear regression regarding the amounts of each standard, measured in mg, and the area of the corresponding peak on the chromatogram. Linearity was also confirmed for MV prepared sample solutions. After chromatographic separation, the peak areas obtained were plotted against concentrations by linear regression. Limits of detection and quantification were determined by calculation of the signal-to-noise ratio. Signal-to-noise ratios of approximately 3:1 and 10:1 were used for estimating the detection limit and quantification limit, respectively of the methods.

 

Precision

The repeatability of the injection integration was determined for both standard piperine and the content of piperine in MV. A standard solution containing reference compounds and prepared sample solutions was injected. MV samples were also prepared 3 times to evaluate the repeatability of the process. The mean amount and S.D. values were calculated. The precision was calculated at two different concentrations high and low tested in the concentration range. For standardization, sample was injected at six different concentrations and linearity was noted.

 

Accuracy

The accuracy of the method was determined by analyzing the percentage of recovery of the piperine in MV. The samples were spiked with two different amounts (50,100 mg) of standard compounds before sample preparation. The spiked samples were extracted by triplicate and analyzed under the previously established optimal conditions. The obtained average contents of the target compounds were used as the “real values” to calculate the spikes recoveries.

 

Robustness

For the determinations of the method’s robustness a number of chromatographic parameters, such as column package and size, mobile phase composition and gradient ratios, flow rate and detection wavelength, were varied to determine their influence on the quantitative analysis. Inter day and intraday variability was studied for the sample, by injecting the same concentration of the sample on three different days and the standard error mean was calculated.

 

Statistics

When applicable one-way or two-way analyses of variance was used to assess the observed differences in the piperine content. Differences were considered to be statistically significant when the P-value was <0.05.

 

Table: Analytical parameters of HPLC procedure for the piperine estimation.

S.No.

Parameters

Value

1.

Absorption maxima

343 nm

2.

Beer’s law limit

2-10 mg/ml

3.

Regression equation(y=mx + c)

y = 70.89x+2.843

4.

Intercept (a)

2.843

5.

Slope (b)

70.89

6.

Correlation coefficient (r2 )

0.999

7.

LOD

0.6

8.

LOQ

2.0

9.

Precision(n=3,% RSD)

0.35

10.

Accuracy (%)

99.3

 

                               

Table:  Piperine content.

 S.No.

Name

Piperine content ((mg/ml)

% RSD

1.

Crude

0.99±0.10

10.18

2.

MKT

13.52±0.60

4.47

3.

Lab

1.32±0.10

1.29

Mean ± SD of three determinations.

 

 

Table: Data of recovery

S.No.

Amount of piperine (mg/ml)

Recovery %

In sample

Added

Estimated

1

10

8

7.5

98.75

2

10

10

7.9

 98.80

 

 

RESULTS AND DISCUSSION:

The formulation follows Lambert-Beer’s law limit in the concentration range of 2- 10 µg/ml with λmax at 343 nm having piperine content as 13.52±0.60 and 1.32±0.10 %w/w in MKT and Laboratory formulations respectively.

 

The HPLC chromatogram of formulation has retention time as 7.7 minutes. The HPLC method was validated by defining the linearity, peak purity, limit of quantification and detection, precision, accuracy, specificity and robustness. For the qualitative purpose the method was evaluated by taking into account the precision in the retention time, peak purity, and selectivity of piperine elutes. A high repeatability in the retention time was obtained with (% R.S.D.) value lower than 1.5% for both standard and samples even at higher concentration. The peak purity was studied in the major peaks. Linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision were evaluated for quantification purposes. Thus LOD and LOQ found to be 0.6 and 2 mg/ml respectively which suggest full capacity for quantification of piperine content in different laboratory batches of formulation. R2 value for the regression equation of the piperine is: 0.9978 this confirms the linearity of the method. The recovery was performed at two levels by adding known amounts with pre-analyzed sample of formulation found to be close to 98.75, 99.45% and a higher repeatability indicate a satisfactory accuracy in the proposed methods .Finally the robustness of the method was also assessed. Minor modification of the initial mobile phase gradient had no effect on the peak resolution of the compound. This confirms that the formulation is properly standardized as per WHO guidelines and all parameters are found within the limits.

 

CONCLUSION:

Most of the Ayurvedic formulation is lacked in their defined quality control parameters and methods of its evaluation. WHO has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standareds.

The developed method of estimation can be considered as the protocol for the evaluation of Marichyadi vati which will assist the regulatory authorities, scientific organizations and manufacturers in developing standards.The method used for evaluation is found to be precise and reproducible and help to produce uniform standard products, which will restore faith in Ayurvedic system.The developed method can also be applied to various polyherbal formulations for the quantitation of piperine and can be used as a routine quality control method in pharmaceutical industries.

 

 

 

REFERENCE:

1.       AH Gilani, A Rahman. 2005. J. Ethno pharmacology. 100: 43- 49.

2.       WHO. 2002 Traditional Medicine Strategy 2002-2005.World Health Organization; Geneva.

3.       C Zidorn, Schubert B, Stuppner H. 2005. Biochem Sys and Eco. 33: 855-72.

4.       Shrikumar S., Ravi T.K.2007.  Approaches towards Development and Promotion of Herbal Drugs. Pharmacognosy Reviews, 1(1).

5.       Li S., Han Q. and Qiao C., et al, 2008. Review Chemical markers for the quality control of herbal medicines: an overview: Chinese Medicine. 

6.       Natalie J. Lazarowych and Peter P. 1998, Use of fingerprinting and marker compounds for identification and standardization of botanical drugs. strategies for applying pharmaceutical     HPLC analysis to herbal products: Drug Information Journal, 32: 497–512.

7.       Liang Y., Xie P., and Chan P. 2004, Review Quality control of herbal medicines. Journal of Chromatography B, 812 : 53–70.

8.       T Layloff. 1991. Scientific Fingerprinting: A Pharmaceutical Regulatory Tool Pharm.Technol. 15: 146-148.

9.       WHO 1991. Guidelines for the Assessment of Herbal Medicine, Munich, World Health Organization.

10.     CHMP Guideline on quality of herbal medicinal products/traditional herbal medicinal products. Committee for medicinal products for human use, European Medicines Agency Inspections, July 21, 2005, http://www.emea.eu.int

11.     WJ Welsh, WK Lin, SH Tersigni, E Collantes, R Duta, MS Carey. 1996. Anal. Chem. 68: 3473.

12.     YB Ji, QS Xu, YZ Hu, Y Vander Heyden. 2005. J. Chromatogr. A 1066: 97.

13.     AH Liu, YH Lin, M Yang, H Guo, SH Guan, JH Sun, DA Guo. 2007. J. Chromatogr. B 846 :32–41

14.     Government of India, Ayurvedic formulary of India, , Ministry of Health and family Planning, Department of Indian System of Medicine and Homeopathy, Delhi Part-1, 2nd, edition 2003. (check for marichyadi vati).

15.     Ayurvedic Formulary of India, Ministry of Health and Family Welfare. Govt. of India, New Delhi, Controller of Publications, 1978:181.

16.     Government of India, Ayurvedic formulary of India, , Ministry of Health and family welfare, Department of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homoeopathy, New Delhi Part-II, First edition, 2007:178

17.     Merck Index, 11th Edition, 1989:7442.

18.     Mukherjee K Pulok, Analytical Profiles of Selected Medicinal Plants. II edition, Quality control of herbal drugs, New Delhi, Horizon press; 2002: 755-767.

19.     Shukla KK, Saraf S, Saraf S. Development of Quality Control Parameters of Bhaskar Lavan Churna:  A traditional Ayurvedic Formulation. Taiwan Pharmaceutical Journal; 2007; 59: 47-56.