Development of validated spectrophotometric method for routine analysis of an Ayurvedic formulation, Pancasama Churna

About Authors:
Megha Sharma,
M.Pharma (quality assurance)
Shoolini university of life sciences,
Bhajol, Solan, Himachal Pradesh.
*meghu35@gmail.com

ABSTRACT:
Traditional medicine, especially ayurveda, is gaining tremendous popularity in modern times due to several reasons. Though, there is still a lack of scientific tests to evaluate the quality, quantity, potency, safety and efficacy of ayurvedic formulations. Need of the hour is to develop simple routine analysis methods to eliminate the problems faced by both practitioners and patients due to sub standard medicines.^[4]

REFERENCE ID: PHARMATUTOR-ART-1307

INTRODUCTION:
Traditional medicine has seen a dramatic revival in recent times. Traditional medicine is also called alternative medicine or holistic medicine.

WHO defines traditional medicine as follows:

Traditional medicine can be defined as the knowledge, skills and practice of holistic health care, recognized and accepted for its role in maintenance of health and treatment of disease. It is based on indegenious theories. Beliefs and experiences handed down from generation to generation^[2].

                                                        OR

Traditional medicine is sum total of knowledge, skills, practices based upon the theories, beliefs, experiences indegeneous to different cultures that are used to maintain health as well as to prevent, diagnose, improve or treat illness^[2].

Ayurveda : the backbone of traditional medicine

“Ayurveda is the most ancient, continuously practiced health care discipline in the world.” ^[3] 

World Health Organization, Quality Control Methods For Medicinal Plants Materials, Geneva.(1998)^[2] had listed the benefits and limitations of ayurvedic formulations as under : Benefits of ayurvedic formulations

1.      Less side effects –If used properly, they have fewer side effects as compared to their synthetic pharmaceutical counterparts.

2.      Mother nature advantage-Since they are natural, body responds to them better.

3.      Age old practice-They are thousand years old and have a long track record of successful, safe and effective use. Whereas, modern medicines are relatively new.

4.      More effective-In some cases they prove to be more effective than their synthetic pharmaceutical counterparts. For example, successful use of plant Artemesia in malaria.

5.      Grass root treatment-Modern medicine focuses on treating symptoms, whereas ayurveda focuses on treating the disease itself.

Limitations of ayurvedic formulations

1. No side effects is a half truth-If used improperly, plenty of plants are poisonous and deadly. Various side effects may appear if –

· Poor quality herbs are being used.

· Ayurvedic formulation is used in conjunction with other incompatible drug

· Dose or dosage regimen is improper.

2. Delayed action-They take longer time to act as compared to their pharmaceutical synthetic counterparts.

Types of Ayurvedic formulations:^[5]
Ayurvedic medicines are formulated in various dosage forms as follows :
· Churnas – powder form
· Vati – tablet form
· Arishta and Asava – liquid fermented form
· Kwaath – concentrated semi liquid form
· Gutika – pill form
· Ghrita- medicated ghee form
· Kashayam- decoction form

Out of above mentioned dosage forms, churnas are the most common.

Ayurvedic churnas
Churna is a fine powder of a drug or drugs which is prepared by mixing clean, finely powdered, and sieved drugs

 Ayurvedic churnas are tasty and delicious powders taken after or before meals.They are taken-

1.      With water
2.      With milk
3.      With honey
4.      Alone

Churnas are oldest form of ayurvedic formulations .Churnas are most common as well as they have various advantages like

· since they are in powder form, disintegration step is bypassed and drug is absorbed faster than in tablet form (vati and gutika).
· Churnas are easier to be swallowed therefore they are a preferred choice for children and elderly patients
· Their formulation is comparatively simple.

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Pancasama Churna : An Ayurvedic polyherbal formulation
Pancasama churna shows its effects mainly on gastrointestinal tract. It increases peristaltic movements of GI tract. It is used as antiflatulent , antirheumatic. It is also used in the treatment of abdominal disorders.^[6]

Pancasama churna (PSC) consists of 5 ingredients viz.,

Piperine:

Category:  Alkaloid

Structure: 

IUPAC Name:
1-[5-(1,3-benzodioxol- 5-yl)-1-oxo-2,4-pentadienyl]piperidine

Therefore, due to the presence of conjugation in the structure of Piperine, Ultra- Violet Spectroscopy is a good choice for its analysis for the puropse of Quality check.

Why should the use of Piperine be emphasized as a biomarker for standardisation of polyherbal formulations containing Piper longum fruits:

Many clinical as well as pharmacological studies have revealed that apart from promoting digestive function_[15] , that explains its use in Pancasma churna, it has activities like analgesic, antipyretic, CNS depressant, anti-inflammatory, antioxidant, anticonvulsant, anti-bacterial, anti-tumor and hepatoprotective _[16]. Certain studies show that Piperine enhances the bioavailability of drugs like Sulfadiazine, Rifampicine, Pyrazinamide, Isoniazid, Ethambutol, Tetracycline, Phenytoin and Streptomycin_[17]. Therefore due to the diverse pharmacological properties of piperine, it can be used as a biomarker for standardisation of polyherbal formulations containing Piper longum fruits.

Why strict analysis of ayurvedic churnas?^[7] 
churnas require strict quality control measures especially because of their limited shelf life of usually six months.^[7]

 There are innumerable variables affecting the quality of Ayurvedic formulations.

 Major ones are-

1. Place of origin and time of collection of raw material

2. Specific part and quality of raw material

3. Size and shape of furnace

4. Type and quantity of fuel used in the preparation

5. Specification about the place of manufacturing.

6. Time and duration of process

7. Characterstic of finished material

8. Shelf life

9. Presence of –
·         Heavy metals
·         Pesticides
·         Mycotoxins
·         Microbes
·         Ash content

10. Extrinsic factors-
·         Growing
·         Harvesting
·         Drying
·         Storage

11. Intrinsic factors-
·         Physicochemical properties of herbal ingredients.
·         Presence of secondary metabolites

12.  Need of strict quality regulations of Ayurvedic formulations:
Due to the lack of strict quality measures, following questions become complex-
1. The analysis and the structure of the finished product.
2. Safety and side effects of the formulations.
3. Correct doses and duration of the treatment.
4. Mode of action.

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MATERIALS AND METHODOLOGY:

A.) Materials:

1. Procurement of  drug:

Pancasama churna (PSC) consists of 5 ingredients viz.,^[2]

All these ingredients were procured from local market and authenticity waschecked and confirmed.

2. Preparation of the formulation PSC:
Three marketed formulation of Pancasama churna from different manufactures (designated as PSC-A,PSC-B and PSC-C) and three laboratory batches of  Pancasama churna wereprepared in university laboratory and werenamed as PSC-I ,PSC-II, PSC-III.^[1],[9]-[14]

3. Procurement of Chemicals:
All the chemicals and solvents used wereof A.R. grade, standard piperine wasprocured from International Scientific and Surgicals, Solan, Himachal Pradesh.

4. Preparation of piperine extract of Pancasama Churna:
The powdered Pancasama churna(1gm) was refluxed with 60 ml ethanol for 1 hour. Then extract was filtered and refluxed the marc left with 40 ml of methanol for another 1 hour. Again filtered and combine the filtrate. Then concentrated the methanol extract under vacuum till the semisolid mass was obtained and dissolved the residue in 75 ml methanol and filtered through sintered glass funnel (G-2) by vacuum filtration assembly. The filtrate was centrifuged at 2000 rpm for 20 minutes, the supernatant wascollected in 100ml volumetric flask and volume wasmade with methanol. The same procedure wasperformed for each batch of Pancasama churna and separately powdered fruits of piperlongum (Pippli) and solution (100ml)of their piperine extract wasprepared. Only Pippali fruits are separately analyzed for piperine estimation because no other ingredient contains piperine, so none of those is included.^[1],[9]-[14]

5. Preparation of standard solution of piperine:
An accurately weighed piperine (100mg) wasdissolved in methanol and volume was made up to 100ml with methanol in volumetric flask. Two ml of this solution was diluted with methanol up to 100ml in volumetric flask to give 20μg/ml piperine solution.^[1],[9]-[14]

B.) Methodology:
U.V. absorbances were measured using Systronics Ultraviolet Double beam Spectrophotometer, 2202.

1. Development of method by calculating absorption maxima of piperine:
The solution of piperine was prepared in distilled water at the concentration of 10μg/ml. This was scanned in the wavelength range of 200-400 nm. Data were recorded at an interval of 5nm. The wavelength selected for piperine analysis was 342.5 nm.

2. Calibration curve of standard piperine:
Calibration curve from standard solution of piperine wasprepared and with the help of this curve the content of piperine from Pancasama churna was estimated. The method wasvalidated for precision and accuracy. .^[1],[9]-[14]

3. Calibration curve of piperine in PancasamaChurna:
A series of calibrated 10ml volumetric flask weretaken and appropriate aliquots of the working standard solution of piperine werewithdrawn and diluted up to 10ml with methanol. The absorbance was measured at absorption maxima 342.5 nm, against the reagent blank prepared in similar manner without the piperine. The absorption maxima and Beer’s law limit  will berecorded and data that prove the linearity and obey Beer’s law limit will benoted. The linear correlation between these concentrations(x-axis) and absorbance(y-axis) wasgraphically presented and slope(b), intercept(a), and correlation coefficient (r2) werecalculated for the linear equation (Y=bx+a) by regression using the methanol of the least square.^[1],[9]-[14]

4. Estimation of piperine in PancasamaChurna:
The appropriate aliquots from piperine extract of each batch of PancasamaChurna and separately powderd fruits of piper longum (Pippli) werewithdrawn in 10ml volumetric flask separately absorbance for aliquots of each wasnoted at 342.5 nm. the corresponding concentration of piperine against respective absorbance value wasdetermined using the piperine calibration curve.^[1],[9]-[14]

5. Validation Parameters:
Validation is the process of proving that an analytical method is acceptable for its intended purpose. For pharmaceutical methods, guidelines from the United States Pharmacopoeia (USP), International Conference on Harmonization (ICH), and the Food and Drug Administration (FDA) provide a framework for performing such validations. 

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The method was validated for following parameters by performing the various studies.

*  Optical parameters :These are Absorption maxima , Beer’s law limit, Regression equation, Intercept, Slope and Correlation coefficient (r²) .

*  Accuracy:Accuracy is the closeness of test results obtained by that method to the true value.    In   case of assay of a drug substance accuracy may be determined by application of the analytical method to an analyte ( piperine in this case) of known purity (e.g. reference standard) or by comparison of the results of the method with those of a second well characterized method, the accuracy of which has been stated or defined.  Accuracy is calculated as the percentage of recovery by the assay of the known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with confidence intervals. The ICH documents recommended that accuracy should be assessed using a minimum of nine determinations over a minimum of three concentrations levels, covering the specified range (i.e., three concentrations and three replicates of each concentration).

*   Precision:Precision is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogenous sample.  Precision of an analytical method is usually expressed as the standard deviation (SD) or relative standard deviation (RSD) or (coefficient of variation) of a series of measurements. Precision may be measure of either the degree of reproducibility or of repeatability of the analytical method under normal operating conditions.

*  Specificity: The instruments ability to measure or identify or measure the analyte without any interference from sample matrix, impurities, precursors or degradation products.

*   Detection Limit: Lowest amount of analyte in a sample that can be detected, but not necessarily quantities as an exact value, under the stated experimental conditions.  The detection limit is usually expressed as the concentration of analyte (e.g., percentage parts per million) in the sample.

For instrumental and non-instrumental methods detection limit is generally determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected.

LOD = 3 * SD / slope of calibration curve
SD = Standard deviation of intercepts

*  Quantitation Limit : It is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. Quantitation limit is expressed as the concentration of analyte (e.g. percentage, parts per billion) in the sample.

For instrumental and non-instrumental methods, the quantitation limit is generally determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be determined with acceptable accuracy and precision.

LOQ = 10 X SD / slope of calibration curve
SD = Standard deviation of intercepts

*   Linearity and Range: Linearity of an analytical method is its ability to produce results that are directly proportional to the concentration of analyte in samples.  The range of the procedure is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable with acceptable precision, accuracy and linearity.

These characteristics are determined by application of the procedure to a series of samples having analyte concentration spanning the claimed range of the procedure. When the relationship between response and concentration is not linear, standardization may be provide by means of a calibration curve.  ICH recommends that for the establishment of linearity a minimum of 5 concentrations normally used.

CONCLUSION:
Development and validation of spectrophotometric method for the estimation of piperine in pancasamaChurnacould be used in routine analysis as a valuable analytical tool, to check the variations in different batches. After the approval of the drug is achieved, pharmaceutical validation and development of analytical markers for the finger printing are necessary to ensure that the drug product will meet set pharmaceutical standards for identity, strength, quality, purity, stability, safety and efficacy. In simple words, estimation of piperine can be used as an appropriate analytical marker for the finger printing assuring the uniformity and integrity of the batch of product being manufactured.

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