INTRODUCTION:

An ancient branch of the Indian traditional healthcare system with origins in Dravidian culture is traditional Siddha medicine (500BCE-500CE). Traditional Siddha medicine uses a comprehensive approach to healing and is predicated on the philosophy of the three doshas and the five fundamental elements. Siddha Polyherbal preparations may be taken alone or in conjunction with other medications.¹ The development of polyherbal formulations with high efficacy, potency, and safety for their future characteristics is a focus of traditional Siddha medicine. More than 80% of people worldwide still use traditional herbal medicine to treat their fundamental medical requirements in today's society. With its various forms, ayurvedic medicine uses a combination of more than 45,000 plant species in India 7500 of these species are used in practice.²

Ayurveda is a holistic healing system that focuses on possible healing by the use of medicinal herbs and is well known for both preserving health and treating diseases and ailments.³ The basic goal of Siddha principles is to restore proper balance by treating the underlying causes of illness with a combination of natural products and preventing the return of imbalance by adopting a healthy lifestyle. Ayurvedic texts like the Sushruta Samhita and the Charaka Samhita emphasise the significant combination of multiple herbs in a particular ratio to decrease toxic principles and increase therapeutic potential.⁴ Due to its inherent receptivity and lack of harmful side effects, traditional medicine has been increasingly popular around the world.

Due to the high cost and scarcity of standard, authentic drugs, the marketing of herbal remedies has resulted in use of inexpensive substitutes and adulterants. As a result, it is now necessary to certify polyherbal formulations in accordance with current research standards in order to assess and standardise their quality and purity.⁵ Traditional medicines must include quality control and evaluation to guarantee the administration of the required dosage of standard drugs.⁶ To verify high-quality pharmaceuticals that are free of substitutes and adulterants, physicochemical and phytochemical evaluation of crude drugs obtained from medicinal plants and their comparison with standard stated values are helpful.⁷ Chromatographic methods such as TLC, HPTLC and HPLC with its effect to evaluate phytochemical constituents of natural products can serve as a useful remedy to intensify batch to batch reliability. ⁸ HPLC is a sophisticated instrument used for the estimation and quantification of marker compounds present in phytoconstituents.⁹ Physicochemical parameters, phytochemical profiling, microbiological evaluation, biochemical analysis and HPTLC profile can be used as quality parameter for the assessment and standardization of phytochemical markers.¹⁰

In this study three phytochemicals ellagic acid, gallic acid and zingerone were used which are commonly present in polyherbal formulations and phytoconstituents in medicinal plants. Ellagic acid (EA) is a polyphenolic compound that many plant species naturally produce as a secondary metabolite.¹¹ Trihydroxybenzoic acid, or gallic acid, has the formula C₆H₂(OH)₃CO₂H. It is categorised under the phenolic acid. Gallnuts, oak bark, sumac, tea leaves, witch hazel and other plants also contain it.¹² Zingiber officinale rhizome contains zingerone, a main flavouring agent in ginger, zingerone, also known as vanillylacetone, gives cooked ginger its sweet flavour.¹³ Fig 1 Indicates the Chemical structures of Ellagic acid, Gallic acid and Zingerone.

Fig. 1: The Chemical structures of Ellagic acid, Gallic acid and Zingerone

HPLC-UVD was used for the synchronous estimation of the indicator compounds quercetin-3-O-glucuronide and ellagic acid, and a technique was designed and validated employing these compounds.¹⁴ Ellagic acid (EA) was estimated by examining HPLC and UV techniques.¹⁵ The quantification of ellagic acid in plasma and tissue samples was examined using a rapid, sensitive, simple, and specific LC-MS approach.¹⁶ An efficient simple approach (LC-ESI-MS/MS) was designed to estimate urolithin C in rat plasma.¹⁷ To estimate (EA), ethanolic fractions of Eugenia uniflora L. leaves (Myrtaceae) family were subjected to a basic HPLC-UV technique study.¹⁸ A robust, sensitive, accurate, and fast technique was determined, developed, assessed, and standardized for Dengue Vati (DNV) by HPTLC and UHPLC analytical techniques.¹⁹

Chromatographic analysis was used to analyze a study that was designed to synchronously quantify three phytomarkers in Syzygium aromaticum (SA): GA, EA, and eugenol.²⁰ A technique to analyze phenolic acids in cereals using HPLC-DAD was developed and assessed. ²¹ Eight phytocompounds found in date palms were quantified using a method that was found to be accurate, precise, simple, and selective.²² Certain phytochemicals in various local wine and fruit wine samples were determined using an HPLC technique.²³

An approach to identify and validate the phytoconstituents found in ginger, such as 6-shogaol, 6-, 8-, and 10-gingerols plasma of human was studied.²⁴ The analysis of 6-gingerol in polyherbal formulations was studied, and a stability-indicating RP-HPLC method was validated.²⁵ A HPLC-UV/VIS approach was established to efficiently standardize and analyze all major constituents gingerols, 6-Paradol, shogaols and zingerone.²⁶

MATERIALS AND METHOD:

Chemicals and Reagents:

The chemicals and reagents required for the study were procured from standard suppliers. All the Phytochemicals (Standards) such as Ellagic acid, Gallic acid and Zingerone were procured from Yucca Enterprises Mumbai, India. Acetonitrile—HPLC grade for liquid chromatography, Water—HPLC grade, (Merck, India).

Instruments and Equipments:

HPLC— Shimadzu Lab Solution HPLC

Pump—Quaternary gradient LC 20 AD

Injector—Auto sampler SIL 20 AC

Column oven—CTO 10 AS

Column—C₁₈, 100A°, 5μm, 4.6 × 150mm

Detector—UV detector SPD M 20 A (Shimadzu, Japan)

Preparation of reference standard solution Ellagic acid:

Ellagic acid standard phytomarker 1mg was precisely measured into a 10ml VF, and 1ml methanol was used for preparing the standard solution of phytomarker. The solution was then adjusted with methanol up to 10ml, and after sonication for 5mins to get the necessary amount of 100µg/ml.

Preparation of reference standard solution Gallic acid:

A 10ml volumetric flask was filled with precisely measured 1mg of Gallic acid standard, then 1ml methanol was used for dissolving the phytomarker. The solution was then adjusted to a final 10ml with methanol and subjected to 5mins sonication to achieve 100µg/ml.

Preparation of reference standard solution Zingerone:

After precisely weighing 1mg of the standard marker zingerone, the solution was transferred into a 10ml VF, 1ml of HPLC water was used for dissolving the contents. The solution was then adjusted with more HPLC- water up to 10ml and sonication was done for 5 mins, to achieve the desired range of 100µg/ml.

Isolation of phytoconstituents from Kabasura Kudineer:

About 10gm of dry pulverized drug was taken and extracted for 24hours with methanol (cold maceration process). The extract was filtered, concentrated by evaporation on the water bath and dried. The concentrated drug was extracted with the 10ml methanol. About 6gm of crude drug was treated 96ml water and heated until the volume was reduced to 12ml. Strained through the clean cotton cloth (4 layered). From that 10ml of decoction was taken and kept on a water bath for 2hours by maintaining the temperature at 80°C until the residue was obtained. The residue was dissolved in 10ml of Methanol by stirring with the glass rod for 20minutes, the dissolved portion was passed through the Whatman filter paper then the clear solution was made up to 10ml in volumetric flask. About 5gm of crude drug (dried) was weighed accurately and added to the 100ml of methanol and macerated for 24 hr and then filtered through Whatman filter paper. The extract was concentrated by Rotary evaporator and dissolved in methanol and solution was used as test solution. ²⁶

Preparation of sample solution:

To achieve the concentration range of 100µg/ml, the sample solution was prepared by precisely taking 1 mg residue in a 10ml VF and using 1ml of HPLC water for dissolving. HPLC water was used for adjusting the volume up to 10ml and subjected for 5 mins sonication.

Analytical method development:

Numerous techniques were developed, validated and optimized for the simple and sensitive separation of specific phytocompounds. To determine the most optimal conditions for selecting a column, mobile phase and detecting wavelength, a number of factors were taken into consideration. Initially several trials were performed to achieve refined chromatographic procedure that achieved optimal peak resolution and high symmetry for the concurrent determination of 3 phytoconstituents.

Validation of developed method:

The accuracy, LOD, LOQ, regression line, precision, robustness, and system the suitability were all evaluated in accordance with the ICH guidelines for method validation.

Linearity:

The procedure for executing a stock solution (100 µg/ml) of all 3 phytoconstituents involved precisely weighing and dissolving 1mg each of ellagic acid, gallic acid, and eugenol in 100mL of methanol, which was then placed into a 100ml VF. This allowed for the establishment of linearity. For each of the three marker compounds, dilution was used to get the desired final range of 25–200µg/ml. The regression line was obtained by plotting the peak area versus the standards.

Detection limit and quantitation limit:

The linearity of the standards was taken to accomplish the LOD and LOQ and the procedure was done thrice to get the standard deviation (SD) and regression equation (S). The formula used to construct and calculate the standard deviation for LOD and LOQ: Precision limits are 3.3×SD/S for detection and 10×SD/S for quantitation.

Precision:

By measuring the sample response six times a day, the method's accuracy was validated. The method was consecutively utilized in the preparation of sample solution. % RSD was utilized to interpret the outcomes of all method and intermediate precision.

Accuracy:

The recovery, was measured in the sample at three distinct concentrations and three spiked levels (50%, 100%, and 150%), was used to compute the methods recovery.

Robustness:

The robustness of the procedure was confirmed by consciously changing the experimental parameters, such as the column temperature and flow rate. To evaluate the method's robustness, the flow rate was adjusted to 0.9, 1 and 1.1 mL/min, while the column temperature was modified to 39, 40, and 41°C.

RESULTS AND DISCUSSION:

Optimization of chromatographic conditions:

Trials were accomplished by using several mobile phase combinations containing methanol: water and ACN: water in various ratios but Rt value was observed to be high. Finally, ortho phosphoric acid was tried in different ratio with acetonitrile and methanol for better resolution of phytocompounds.

Quantification of phytomarkers:

In order to measure the amount of sample solutions and identify the phytoconstituents present, as well as a combination of separate standards, optimized conditions were carried out. Combination of gallic acid, zingerone, and ellagic acid standards (Fig. 3). Kabasura Kudineer sample chromatogram is shown in (Fig. 4). The calibration curve was used as the basis for calculating each individual standard result. Figures 2 shows the NMR spectra of ellagic acid, gallic acid, and zingerone.

Fig. 5: Calibration curve of Ellagic acid

Fig. 6: Calibration curve of Gallic acid

Fig. 7: Calibration curve of Zingerone

Precision:

The precisions were measured six times a day and % relative standard deviation (RSD) ranged from 0.1 to 1.0%. Results obtained of precision study are shown in Table 3.

Table 3:Precision table of Gallic acid, Ellagic acid and Zingerone

S No	Gallic acid	Ellagic acid	Zingerone
1	1778037	1808639	1699207
2	1778922	1790908	1665826
3	1776894	1792284	1662944
4	1779887	1786177	1667692
5	1784473	1782217	1667186
6	1788882	1776121	1667631
Mean	1781183	1789391	1671748
Std Dev	4586.573	11126.95	13570.48
% RSD	0.2	0.6	0.8

Accuracy:

The accuracy of the assay method, measured as relative recovery at three concentration levels, was 100.5– 101.39%, with all RSD values ≤ 2%. Recovery study results are shown in Table 4.

Table 4: Accuracy Data for Gallic acid, Ellagic acid and Zingerone

Sample	Concentration (µg/ml)	Amount Recovered	Mean % Recovery
Gallic acid	50	49.73	99.08%
	100	100.05
	150	150.05
Ellagic acid	50	49	98.9%
	100	99.07
	150	149.03
Zingerone	50	49.05	99.5%
	100	100.02
	150	149.50

Detection limit and quantitation limit:

The LOD and LOQ values were determined and results obtained are shown in Table 5.

Table 5: LOD and LOQ data for Gallic acid, Ellagic acid and Zingerone

S No	Sample	LOD	LOQ
1	Gallic acid	0.39 %	1.18%
2	Ellagic acid	0.21%	0.65%
3	Zingerone	0.34%	1.14%

Robustness:

The robustness of the procedure was confirmed by consciously changing the experimental parameters, such as the column temperature and flow rate. To evaluate the method's robustness, the flow rate was adjusted to 0.9, 1 and 1.1mL/min, while the column temperature was modified to 39, 40, and 41°C. Results obtained for robustness study are shown in Table 6.

Table 6: Robustness data for Gallic acid, Ellagic acid and Zingerone

S No.	Condition	%RSD for Gallic acid	%RSD for Ellagic acid	%RSD for Zingerone
1	Flow rate minus (0.9ml/min)	0.2	0.6	0.8
2	Flow rate plus (1.1 ml/min)	0.5	0.8	1.2
3	Temperature minus (39°C)	0.3	1.1	0.4
4	Temperature plus (41°C)	0.9	1.4	0.9

DISCUSSION:

The process of developing and validating the approach involved establishing a multitude of solvent combinations with different mobile phases. To accurately determine all three phytomarkers, a 20:10:70 isocratic mobile phase containing ACN, CH₃OH, and 0.1% OPA was eventually designed. To quantify the standards and their independent phytoconstituents, a suitable method was identified. The phytoconstituent estimation findings showed the accuracy of the process for routine quality control, as they were in accordance with the ICH parameters established from the quantities of standards used. This developed method was validated by quantifying factors like accuracy, precision, linearity, LOD, quantification robustness, and system suitability according to ICH requirements.^{27, 28}

The system the suitability analysis was established using several factors, such as the tailing factor (Tf), resolution (Rs), and number of theoretical plates (N). It was found that every parameter was within the permitted range. It was discovered that zingerone, gallic acid, and ellagic acid could all be analyzed in the system using the method employed here. The r2 values obtained in the linearity experiment for all standards being lower than 0.999 imply that the developed technique is linear for all three phytocompounds in the prescribed range. LOD and LOQ calculations were also performed to determine the lowest concentration required to quantify and identify the markers in the sample solution. The intra-day precision study was used to determine the precision study.^29,30

According to ICH guidelines, the investigation's findings remained within permissible limits, or % RSD 2%. The accuracy of the phytoconstituents was tested at three different percentage levels. 100% phytomarker recovery for the compounds of interest indicates that the procedure may be able to recover samples entirely. Its slight modification time was the main reason for the recovery and low sample recovery time. The results of the robustness study showed that by modifying the temperature and flow rate, the method was validated as robust for concurrent analysis. These results demonstrate the accuracy, linearity, and precision of the established approach. Moreover, the technique for phytoconstituent estimation makes use of a relatively small sample volume and simple sample preparation procedure.^31,32

These phytocompounds can be employed in Ayurveda, Siddha, Unani and Homeopathy medicines for identification and authentication of standard marker compounds to identify and quantify phytochemical standards and samples and estimate the amount of marker compounds present in natural products and herbal remedies. This method can be used in siddha polyherbal formulations, Ayurvedic, Unani and homeopathy preparations containing these phytoconstituents. These substances can be employed as a standardization method to assess and standardize herbal compositions. This research can be an invaluable resource for discovering new techniques and approving them for use in the herbal industry.^33-35

CONCLUSION:

The multicomponent nature of phytoconstituents found in polyherbal formulations is significant because it guarantees the quality and purity of the final products based on reliable scientific data that was not reported. The goal of the proposed study is to develop and validate a novel analytical technique that will be utilized to assess the phytochemical components in Kabasura Kudineer. Utilizing biologically active compounds, the RP-HPLC method's development and validation will aid in the assessment and quality control of phytoconstituents. For the concurrent estimation of zingerone, gallic acid, and ellagic acid, an HPLC method was developed. It is sensitive, accurate, robust, and simple to use. As there is a need and use of herbal medicine has increased dramatically, so the need for an authentic standardization guidelines and evaluation tool to maintain the quality of these widely used polyherbal medicines.

ABBREVIATIONS:

KK: Kabasura Kudineer; HPLC: High Performance Liquid Chromatography; TLC: Thin Layer Chromatography; HPTLC: High Performance Thin Layer Chromatography; LOD: Limit of Detection; LOQ: Limit of Quantitation; ICH: International Council for Harmonization; RSD: Relative Standard Deviation; LC-MS: Liquid Chromatography Mass Spectrometry; UPLC: Ultra Performance Liquid Chromatography

DECLARATIONS:

Competing interests:

Authors have declared that no competing interests exist.

Availability of data and materials:

Data and material are available upon request.

Funding:

This Research work was funded by AICTE (MODROBS) F. NO. 9-35/IDC/MODROB-REG Policy-1/2021/22

Authors’ Contributions:

All the authors have equally contributed to the article.

ACKNOWLEDGEMENTS:

Authors express their sincere gratitude to Seven Hills College of Pharmacy, Tirupati, for continuous motivation, support, and guidance for research activity and for providing all required facilities to accomplish the entitled work

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Received on 30.01.2024 Revised on 27.06.2024

Accepted on 03.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1688-1695.

DOI: 10.52711/0974-360X.2025.00242

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameter	Gallic acid	Ellagic acid	Zingerone
Retention time(min)	2.418	5.006	8.166
Theoretical plates (N)	6441	4067	11850
Tailing factor (T)	1.123	1.698	1.047

S. No	Concentration (µg/ml)	Area of Ellagic acid	Area of Gallic acid	Area of Zingerone
1	0	0	0	0
2	50	10363518	1497513	2539074
3	75	15496011	2338919	3801134
4	100	20365778	3277123	4914201
5	150	30672785	5099012	6921317
6	175	35594792	6063907	7952420
7	200	40568643	7129973	9042510

Ellagic acid standard phytomarker 1mg was precisely measured into a 10ml VF, and 1ml methanol was used for preparing the standard solution of phytomarker. The solution was then adjusted with methanol up to 10ml, and after sonication for 5mins to get the necessary amount of 100µg/ml.

Preparation of reference standard solution Gallic acid:

A 10ml volumetric flask was filled with precisely measured 1mg of Gallic acid standard, then 1ml methanol was used for dissolving the phytomarker. The solution was then adjusted to a final 10ml with methanol and subjected to 5mins sonication to achieve 100µg/ml.

Preparation of reference standard solution Zingerone:

To achieve the concentration range of 100µg/ml, the sample solution was prepared by precisely taking 1 mg residue in a 10ml VF and using 1ml of HPLC water for dissolving. HPLC water was used for adjusting the volume up to 10ml and subjected for 5 mins sonication.

Analytical method development:

Validation of developed method:

The accuracy, LOD, LOQ, regression line, precision, robustness, and system the suitability were all evaluated in accordance with the ICH guidelines for method validation.

Linearity:

Detection limit and quantitation limit:

Precision:

Accuracy:

The recovery, was measured in the sample at three distinct concentrations and three spiked levels (50%, 100%, and 150%), was used to compute the methods recovery.

Robustness:

Optimization of chromatographic conditions:

Quantification of phytomarkers:

S. No

Concentration (µg/ml)

Area of Ellagic acid

Area of Gallic acid

Area of Zingerone

1

0

0

0

2

50

10363518

1497513

2539074

3

75

15496011

2338919

3801134

4

100

20365778

3277123

4914201

5

150

30672785

5099012

6921317

6

175

35594792

6063907

7952420

7

200

40568643

7129973

9042510