INTRODUCTION:

In India, the Ayurvedic philosophy first emerged and evolved between 2500 and 500 BC. Ayurveda literally translates to "science of life," since the ancient Indian medical system placed a strong emphasis on understanding human health and illness.¹ Positive health is defined as having a balanced metabolism, as is mentioned. Ayurveda holds that diseases develop naturally as a result of outside influences. Its extensive Sanskrit literature covers every facet of medicine, pharmacy, and illness. The techniques for making various formulations described in traditional literature and included in Schedule I of the Drugs and Cosmetics Act. An Ayurvedic polyherbal composition called Avipattikar Churna (AVC) helps maintain the digestive tract's functioning and alleviate the symptoms of various ailments.² Avipattikara Churna is used in several clinical disorders stated, such as constipation, mutraghata (urine retention), arsha (piles), amlapitta (hyperacidity), agnimandya (digestion impairment), and prameha (diabetic mellitus).³^,⁴This medication contains natural herbs that reduce stomach acid production and shield the stomach mucosa from harm from the acidic release.⁵ When it comes to treating heartburn and GERD, or gastroesophageal reflux disease, Avipattikar Churna (AVC) is helpful. It lowers the amount of acid reflux into the mucosa of the oesophagus and stops cancer and esophageal ulcers from developing.⁶ Avipattikar Churna promotes the development of a healthy microbial flora in the gut, which improves digestive processes. Urinary tract issues such as infections and kidney stones can be treated with Avipattikar Churna. It has potent antimicrobial properties. It guarantees that the germs in the urinary system are destroyed and eliminated, which aids in the treatment of urinary infections.⁷

EXPERIMENTAL METHODS:

Collection of Plant Materials

Dried fruits of the following: Musta (Cyperus rotundus), Maricha (Piper nigrum), Pippali (Piper longum), Haritaki (Terminalia chebula), Vibhitaka (Terminalia bellerica), Ela (Amomum subulatum), Patra (Cinnamomum tamala), Lavanga (Syzgium aromaticum), Trivrit (Operculina terpethum), and Sharkara (Sugar candy). Every component was purchased with authentication certifications from reputable supplier of Raipur alog medication vendors.

Preparation of Avipattikar churna⁸

Using the 14 ingredients listed above, avipattikara churna was made in accordance with Ayurvedic Formulary of India Part-I, 7:2 (Bhaisajyaratnavali, Amlapittadhikara: 24-27). The drug ingredients were then thoroughly mixed and stored in an airtight glass container for future use. All of the marker chemicals were quantified using this formulation.

Table 1: Composition of Avipattikar churna

S.No.	Name of Drugs	Botanical Source	Part Used	Ratio
1.	Shunthi	Zingiber officinale	Rhizome	1 Part
2.	Maricha	Piper nigrum	Fruite	1 Part
3.	Pippali	Piper longum	Fruite	1 Part
4.	Haritaki	Terminalia chebula	Fruite rind	1 Part
5.	Vibhitaka	Terminalia bellerica	Fruite rind	1 Part
6.	Aamalaki	Emblica officinalis	Fruite pulp	1 Part
7.	Musta	Cyperus rotundus	Rhizome	1 Part
8.	Vidanga	Embelia ribes	Fruite	1 Part
9.	Salt	Vida Lavana	Vida salt	1 Part
10.	Ela	Ellateria cardamomum	Fruite	1 Part
11.	Patra	Cinnamomum tamala	Leaves	1 Part
12.	Lavanga	Syzgium aromaticum	Flower bud	11 Part
13.	Trivrit	Operculina terpethum	Root	44 Part
14.	Sharkara	Sugar candy	Sugar	66 Part

Physicochemical Study of Avipattikar churna⁹

Avipattikar churna was analysed in accordance with the physicochemical parameters of churna given in the Ayurvedic pharmacopoeia of India (API,2011) such as, loss on drying (%), Total ash (%), Acid-Insoluble ash (%), Loss on drying (%), Alcohol-soluble extractive (%), Water soluble extractive (%), and pH (10% aqueous solution).

Table 2: Physicochemical parameters of Avipattikar churna

S.N.	Parameters	Result (%½	API Limits (%½
1.	Particle fineness	Fine Powder	Fine Powder
2.	Loss on drying (%)	5.1	NMT – 7
3.	Total ash(%)	4. 37	NMT – 6
4.	Acid insoluble ash(%)	0.36	NMT – 0.5
5.	Alcohol soluble extractive(%)	22.12	NLT – 20
6.	Water soluble extractive(%)	53.22	NLT – 53
7.	pH (10% aqueous solution)	5.55	4 – 6

STUDY OF AVIPATTIKAR CHURNA USING HPTLC¹⁰^,¹¹^,¹²

Reagents and Chemicals

SD Fine Chem. Ltd., Mumbai provided analytical quality Toluene (B.N.-IE01600277), Ethyl acetate (LOT NO-1598131216), Methanol (B.N.- B245971710), Formic acid (B.N.- 3009741018), Chloroform (LOT NO-1585301216), and Glacial acetic acid (B.N.- A224441703). The standard marker of piperine (LOT NO.-MKBJ9209V) was acquired from Sigma Aldrich Bengaluru, while the pre-coated TLC aluminium sheets (B.N.-HX72235254) and silica gel 60F254 (20 x 10 cm, 0.2 mm thick) were acquired from Merck Ltd. in Mumbai. Gallic acid (MCR.-7951) was purchased from Molychem (Mumbai, India).

Sample preparation

One gramme of powder A sample of Avipattikar churna was taken in a 10-milliliter volumetric flask, extracted using 10-milliliters of methanol, and then sonicated for ten minutes. The filtrate was used for an HPTLC research after being filtered through membrane filter paper with a 0.45 µ.

Preparation of Standard Solution of Gallic acid and Piperine

Taken two separate volumetric flasks, each holding 10 millilitres capacity, were precisely weighed and filled with 10 milligrammes of piperine and gallic acid. After dissolving 5 millilitres of methanol individually and sonicating them for 10 minutes each, 10 millilitres of methanol were produced, yielding a standard stock solution with a concentration of 1000 μg/ml.

Chromatographic Conditions

Using a TLC applicator Linomat V, 10 mm from the bottom, a sample of methanolic extract of Avipattikar churna, Standard Gallic acid, and Piperine were spotted on a pre-coated TLC aluminium sheets silica gel 60 F254 (20x10cm, 0.2mm thickness) as 8mm broad band width. Every marker was utilised using the solvent system; The mobile phase consisted of toluene, ethanol, ethyl acetate, and formic acid (6:2:2:0.3 v/v/v/v) and was allowed to reach saturation in a twin trough chamber for 20 minutes. The plate was run from the place where the sample was applied up to a 70 mm solvent front. Following plate development, it was allowed to air dry before being photographed using CAMAG Visualizer 2 at 254 and 366 nm. The plate was scanned at 254nm for gallic acid and and at 366 nm for Piperine by using CAMAG Scanner IV.

Preparation of Calibration Curve

The standard solutions of piperine acid (1.0, 2.0, 3.0, μL/spot) and gallic acid (1.0, 2.0, 3.0, 4.0, and 5.0 μL/spot) was applied in TLC plate, which were then developed and scanned in accordance with the previously stated chromatographic conditions. The peak areas were recorded. Calibration curve of Gallic acid and Piperine was prepared by plotting graph against peak area and concentration

Quantification of Gallic acid and Piperine in Avipattikar churna

The 5µL of the methanolic extract of Avipattikar churna, together with varying dilutions of Gallic acid and Piperine, were applied in triplicate to a TLC plate and developed in a solvent system including Toluene, Ethyl Acetate, Ethanol, and Formic acid (6:2:2:0.3 v/v/v/v). On a TLC plate, the sample and marker were applied in the shape of an 8 mm band spaced at least 2 mm apart and 10 mm from the plate's lower edge. The amount of gallic acid and piperine was determined using the densitometric method and the corresponding calibration curve after the plates were scanned at 254 and 366 nm.

Figure – 01 : Image of Developed TLC plate at white light, 254 nm and 366

Figure – 02 : HPTLC Chromatogram of Avipattikar Churna with Standard Marker Gallic acid and Piperine

HPTLC METHODS VALIDATION¹³^,¹⁴^,¹⁵

The optimized HPTLC methods were validated with respect to following parameters.

Linearity: The capacity of an analytical method to produce test findings that are exactly proportionate to the analyte concentration in the sample (within a specified range) is known as linearity. Plotting a graph of peak area vs. standard concentration allowed for the determination of the correlation coefficient (R2) and line equation.

Accuracy: The percentage recovery by assaying a known additional amount of analyte in the sample or the difference between the mean and the acknowledged real value along with the confidence intervals are two ways to report accuracy. A minimum of nine determinations across a minimum of three concentration levels spanning the given range should be used to evaluate accuracy (e.g., three concentrations /3 replicates each of the entire analytical technique). By conducting recovery trials in triplicates at three different concentration levels—80%, 100%, and 120%—and adding a known quantity of a standard mixture of piperine and gallic acid, the percentage recovery was determined. Following analysis of these samples, the outcomes were contrasted with those predicted.

Precision: Precision, which is typically reported as the percent relative standard deviation (%RSD) for a statistically significant number of samples, is a measure of an analytical method's degree of repeatability under typical operating conditions. Precision should be carried out at three separate levels, namely low quality control (LQC), medium quality control (MQC), and high quality control (HQC), in accordance with ICH recommendations. Repeatability is the ability to convey precision over a brief period of time under the same operational conditions. Another name for repeatability is intra-assay precision. It was evaluated using a minimum of nine determinations that fell within the procedure's defined range. While the inter-assay precision was carried out across three separate days, the intra-day assay precision was carried out three times on the same day.

Specificity: The capacity to evaluate the analyte in the presence of elements that would be anticipated to be present in the sample matrix is known as specificity. The Rf value was compared to determine the method's specificity, and the sample and standard spectrums were compared to determine the method's peak purity.

Limit of Detection (LOD) and Limit of Quantification (LOQ): The lowest concentration of an analyte in a sample that, given the specified experimental conditions, may be detected—but not necessarily quantified—is known as the limit of detection, or LOD. The lowest amount of analyte in a sample that can be identified with sufficient precision and accuracy under the specified experimental conditions is known as the limit of quantification, or LOC. The formula for determining LOD and LOQ is k x SD/s, where s is the calibration curve's slope, SD is the analytical signal's standard deviation, and k is a constant (3.3 for LOD and 10 for LOQ).

Robustness: An analytical procedure's resilience to tiny, intentional changes in method parameters is measured by its robustness, which also indicates how reliable it is under typical operating conditions. By making minor adjustments to the saturation duration and composition of the mobile phase, it was investigated in triplicate at 5µg/spot. The final data were analysed using the concentration's percent RSD.

Table 3: Method Validation Study

S.No.	Parameter	Gallic acid	Piperine
1.	Wavelength	254 nm	366 nm
2.	Accuracy	98.91±2.04	99.1± 1.9
3.	Retention factor (Rf)	0.55	0.75
4.	Linearity range (µg/spot)	1-5µl	1-5µl
5.	Correlation coefficient (r2)	0.991	0.987
6.	Intercept	0.0001521	0.0000136
7.	% RSD of slope	0.0003288	0.0000466
8.	LOD (µg/spot)	0.0785650	0.1337842
9.	LOQ (µg/spot)	2.2836769	0.36012

Table 4: Recovery Study

S.No.	Level of % Recovery	Recovery (%)	RSD	Mean Recovery
Gallic acid
1.	80	98.18	1.26	99.15
2.	100	99.17	1.39
3.	120	100.10	0.94
Piperine
1.	80	99.85	1.26	99.01
2.	100	98.03	2.94
3.	120	99.17	1.39

RESULTS AND DISCUSSION:

These days, traditional treatments like ayurvedic medicine are becoming more and more well-liked worldwide. Ayurvedic medicine is a widely used method that uses natural herbs to treat a wide range of illnesses and disorders. One of the best methods now available for the standardisation and measurement of physiologically active ingredients found in herbal and ayurvedic formulations is high performance thin-layer chromatography (HPTLC). Avipattikar Churna (AVC) is a polyherbal ayurvedic remedy. The adulteration and substitution of herbal remedies is the main concern affecting the industry of herbal pharmaceuticals as well as research on commercial natural goods. A wide range of intentional or inadvertent circumstances are referred to as adulteration of an article. Instead of using crude pharmaceuticals, which might or might not have the same therapeutic potential as the original drug, lesser commercial versions are utilised as adulterants. When an article is tampered with, it becomes debased. Intentional adulteration is usually motivated by commercial reasons related to spoilage, mixing, degradation, inferiority, and other unknown issues. The purpose of this study was to ascertain the amount of piperine and gallic acid contained in the polyherbal ayurvedic formulation Avipattikar Churna (AVC). Clear filterate from the 0.45 µ membrane filter was utilised for the HPTLC analysis after the sample was extracted using methanol and sonicated for ten minutes. Better separation was obtained in the solvent system with Toluene: Ethyl acetate: Ethanol: Formic acid (6:2:2:0.3 v/v/v/v). Using visionCat software, the plate was scanned at 254 nm for chromatographic evaluation on a Camag Scanner IV after being photographed at 254 and 366 nm. The existence and quantity of the marker compound in the sample are confirmed by the overlap of all relevant spectra with the marker. A gramme of Avipattikar Churna (AVC) methanolic extract contained 2.51 mg of piperine and 2.70 mg of gallic acid, the marker compounds.

CONCLUSION:

The quality of formulations cannot be guaranteed by the Ayurvedic Pharmacopeial standards. A marker component is required for both qualitative and quantitative formulation evaluation in order to evaluate each formulation's quality. It was manufactured using pharmacopoeial standards at the laboratory scale and put through a number of quality control tests. Using gallic acid and piperine as marker compounds, an HPTLC method has been devised to ensure the quality of Avipattikar Churna (AVC), a polyherbal ayurvedic preparation. Based on the results described above, it can be said that the HPTLC approach is a low-cost, quick, accurate, and precise method that may be used to successfully quantify plant markers. The developed HPTLC method can be applied to regular analysis, quality control, and standardisation.

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Received on 30.10.2023 Accepted on 04.12.2023

Int. J. Tech. 2023; 13(2):108-113.

DOI: 10.52711/2231-3915.2023.00014