INTRODUCTION:

Indian blackberry S. cumini is also known as Jambolan, Jamun, Jaman, Black plum and Malabar plum¹. The jamun tree is native to the Indian subcontinent and is distributed widely in Bangladesh, Sri Lanka, Pakistan, and India. Due to its economic significance, particularly in fruit, it has been introduced to other regions like America and France. Various parts of the Jamun tree are traditionally used to cure mouth blisters, diarrhoea, flux, acne, stomach pains, piles and even cancer².

The juice from the Jamun fruit is administered three times a day to manage diabetes in human beings³. In Unani medicine, Jamun is utilised as a liver tonic and also to fortify teeth and gums, enhance blood quality, and act as a deworming agent against ringworm infections of the head. The pulp of Jamun fruit is employed to treat gingivitis⁴ and it can also be beneficial in addressing haemorrhoids by applying Jamun for 2-4 months⁵. The leaves have a considerable amount of phenolic compounds^5,6which have been reported for antidiabetic and anti-inflammatory activities. S. cumini L. has total flavonoid content (53.5mg/gm), phenolic content (322.35mg/gm)_ and terpenoid content (4 mg/gm). Various phytoconstituents, including glycosides, alkaloids (jambosine and jamboline), phenolic acids, tannins (like ellagic acid and gallic acid), steroids, flavonoids (anthocyanins, epicatechin, and quercetin), triterpenes and phytosterols, are thought to be biologically active compounds^7,8. Quercetin, caffeic acid, and gallic acid are important constituents present in Syzygium cumini L. leaves in free and bound forms. It has many other pharmacological actions, like antioxidant^9,10,11anti-inflammatory^12,13, anti-diabetic^14,15,16, anti-cancer, antibacterial^17,18, hepatoprotective¹⁹, cardioprotective²⁰, anti-diarrhoeal²¹, antipyretic¹³ and anti-allergic²².

HPTLC serves as an efficient, economical and vital approach for both quantitative and qualitative pharmacological studies of compounds in medicinal plants and herbal formulations²³. Ajam C. Shaikh et.al., identified a single terpenoid peak at Rf 0.96, utilising ethyl acetate and n-hexane in the ratio of 28:72 as the mobile phase at 417nm²⁴. Dalavi NB et al. understood the importance of S. cumini L. extract through HPTLC, detecting ellagic acid and gallic acid peaks at Rf 0.47± 0.02 and 0.57±0.02, respectively, with a mobile phase consisting of formic acid: toluene: ethyl acetate: (1:7:2) under storage conditions of 40ºC±2ºC and 75% RH± 5% RH²⁵. Vivek Joshi et al. generated HPTLC fingerprinting for methanolic extract of S. cumini L. leaves, using a mobile phase of ethyl acetate: chloroform: methanol (6:4:6), resulting in 14 peaks at 254nm and 10 peaks at 366nm²⁶. Jasmeen Kaur et al. developed a distinct fingerprint for S. cumini L. leaves via the HPLC method, identifying gallic acid (1.45% w/w) and ellagic acid (3.97% w/w)²⁷.

Till date, no method has been documented in the literature for the simultaneous determination of caffeic acid, quercetin, and gallic acid in S. cumini L. leaves using HPTLC. Hence, the present study is designed to identify and quantitate the three different polyphenolic biomarkers in the water and ethanolic fractions of S. cumini L. leaves. The International Conference on Harmonisation (ICH, 2002) guideline was followed while validating the suggested approach²⁸.

MATERIALS AND METHODS:

Materials:

The reference standards quercetin, caffeic acid and gallic acid were obtained from Sigma-Aldrich, India. All standards and solvents used in the investigation were purchased from Sigma-Aldrich, India. All the solvents and standards used in the study were greater than 98% purity (HPLC grade). The leaves of Syzygium cumini L. were collected from the National Botanical Research Institute, Lucknow’s herbal garden in September 2024. Botanical Survey of India, Prayagraj authenticated the plant specimen. The herbarium voucher (756/2024-2025) of the plant specimen was submitted and maintained in the institute.

Sample Preparation:

After collection the leaves were washed to clean the dust, silica and foreign organic matter. The leaves were dried in shade for 15 days. After the leaves lost moisture and seemed to be brittle, they were subjected to grinding in a mortar and pestle. The leaves were deliberately powdered as coarse powder. The grinder was avoided to prevent the artefact formation by the heat generated during the process. About 250gm of the powder was defatted using Soxhlet apparatus using 350ml of petroleum ether. The soxhlation was done for a period of 12hours. The sequential extraction was continued further using the same parameters with solvents ethanol and water. The extracts obtained were dried in a rotary evaporator at a temperature less than 40°C. A stock solution of 1000µg/ml was obtained by dissolving 10 mg of extract in 10 ml of pure methanol (>99% purity). Further, analysis was performed using this stock solution.²⁹

Column chromatography:

Silica gel column chromatography:

Silica gel 60-120 was used to run a column of the extracts (aqueous and ethanolic). 4gm of the extract was mixed with 8grams of the silica 60-120 and loaded onto the column. Ethyl acetate: Hexane: Formic acid in the ratio 2:7:1 was utilised as eluent for column chromatography of both ethanol and water extracts. The column was monitored using thin-layer chromatography (TLC) at regular intervals. The TLCs were observed under visible light, ultraviolet 254nm and 366nm.

TLC Results:


Figure 1: TLC of Ethyl acetate, Ethanol and Water extract at 254nm	Figure 2: TLC of Ethyl acetate, Ethanol and Water extract at 366nm

HPTLC Analysis:

Development of HPTLC Fingerprinting:

Instrumentation and Chromatographic Conditions:

The chromatographic conditions and instruments utilised were as given here: CAMAG Linomat 5 spotting device, 100μl syringe, glass identical twin trough chambers (20.0 x 10.0; CAMAG), densitometer, CAMAG TLC Scanner 3.0 combined with winCATS software V, and HPTLC³⁰ (High-Performance Thin Layer Chromatography) plates manufactured of TLC Silica Gel 60F254 (20.00 x 10.00cm). The experimental conditions included a TLC Silica Gel 60 F254 stationary phase, multi-level calibration, peak area evaluation mode, methanol as a solvent system, detection wavelengths of 254nm and 366nm, 4.0 x 0.30mm slit dimension, 20mm/s scanning speed, a deuterium lamp (D2) and a tungsten lamp (W) as radiation sources.

Standard Preparation:

A stock solution of reference standard (RS) caffeic acid, quercetin, and gallic acid was prepared by dissolving 5 mg of the RS in 5ml of methanol (HPLC grade) to obtain a solution of 1mg/ml. To prepare a working solution with a concentration of 100μg ml⁻¹, 900μL of HPLC-grade methanol was combined with 100μL of the stock solution. The working solution was then kept at a set temperature of 4°C for HPTLC examination.

Caffeic acid, Quercetin and Gallic acid Calibration Curve:

A 100μg/mL stock solution of quercetin, caffeic acid and gallic acid was produced in methanol. Several volumes of the stock solution were applied to a TLC plate, resulting in a range from 100 to 900ng per band for each compound. The peak area was integrated with respect to the increasing concentration levels, and the data was analysed by regression analysis (least squares method). The procedure complied with ICH guidelines, with statistical analysis carried out using Microsoft Excel 2000 (MS Office®).

RESULTS AND DISCUSSION:

Method Optimization for the Separation of Analytes:

The HPTLC method was designed and fine-tuned to create a straightforward and precise assay technique^31,32. Different combinations and concentrations of commonly available solvents were tested to enhance resolution, achieve compact spots, and improve the separation of degradation products. Ultimately, a mobile phase consisting of ethyl acetate, toluene, and formic acid (2:7:1, v/v) was selected for its ability to produce sharp, well-defined peaks for caffeic acid, gallic acid, and quercetin. This solvent proved highly effective in resolving phenolic compounds and other constituents present in the traditional medicine and their extracts.

Calibration Curves:

A calibration curve (n = 6) demonstrated a strong linear correlation across a broad concentration range based on the peak area, the concentration ranging between 100–900ng per spot. The derived calibration (Table 1) confirms that the standard plot maintains linearity without any deviation.

Validation:

The method was evaluated for various parameters, including simplicity, precision, accuracy, efficacy, robustness, LOD, LOQ, and specificity, in compliance with ICH guidelines (Table 1)

Table 1: Summary of Validation parameters by HPTLC

Parameters	Quercetin	Caffeic acid	Gallic acid
Rf	0.40	0.04	0.95
Linearity Range	100-900ng	100-900ng	100-900ng
Regression via area	-11.47 + 3.354 * X	2717 + 8.072* X	150.3 + 8.396* X
Slope	5	5	5
r	0.99135	0.97372	0.99048
sdv	8.10	9.55	8.00

Precision:

The reference standards Caffeic acid, gallic acid, and quercetin were subjected to precision studies. Repeatability assays for the sample preparation and peak area estimation were carried out using 9 determinants (3 concentrations with 3 separate experiments each) within the prescribed ranges of 400, 800, and 1200ng per band of caffeic acid. The results are defined as the relative standard deviation. Inter-day and intra-day fluctuations were assessed at three concentration levels – 400, 800, and 1200ng per band. The acceptance specifications for repeatability or intermediate precision were aligned with the intended purpose of the analytical method.

Robustness:

The methodology of robustness was assessed by integrating minor variations in mobile phase content such as volume, saturation time, and the stimulation of TLC plates by using methanol. The impact of these changes was analysed with robustness tests. RS Caffeic acid, gallic acid and quercetin were tested (in triplicate) at a concentration of 200ng per band. Relative standard deviation (RSD) as well as standard deviation (SD) of the peak areas were calculated.

Limit of Quantification (LOQ) and Limit of Quantification (LOD):

For calculating the LOD and LOQ, diluted methanol was administered 6 times, and the signal-to-noise ratio was computed. Limit of Detection was defined in a ratio of 3:1, while LOQ was set at 10:1. These values were experimentally validated by neutralising a known amount of quercetin, gallic acid, and caffeic acid until the usual responses were approximately three or ten times those of 6 separate determinations.

Recovery Studies:

The TLC method proposed for extracting and estimating gallic acid, quercetin, and caffeic acid from S.cumini L. leaves, despite requiring 50 percent, 100 percent, and 150 percent additional drug, shows excellent recovery. Experimental results were reported as the mean proportion of the restored analyte, along with their SD and RSD.

Determination of Quercetin, Gallic Acid, and Caffeic acid in S. cumini L. Leaves:

A distinct, single spot was detected in the chromatogram of the water and ethanolic fractions of S. cumini L. leaves. Additionally, the comparative HPTLC chromatogram indicated no interference from other components in the water and ethanolic fractions of S. cumini L. leaves. Water and ethanolic fractions were subjected to HPTLC to obtain respective chromatograms using marker compounds with a 6-point calibration curve; all the indicators were identified and quantified in S. cumini L. leaves. The total phenolic compound content was quantified in all the accessions.

RESULTS OF CAFFEIC ACID

Figure 3: Chromatogram showing peaks of standard Caffeic acid

Figure 4: HPTLC profile of S. cumini L. ethanolic and water fraction with standard caffeic acid at 254nm and 366nm

Table 2 Calibration Curve table of Caffeic acid

S. No	R_f	Amount	Area (AUC)
1	0.05	228.80ng	3793.63
2	0.05	457.60ng	6842.76
3	0.05	686.40ng	9118.80
4	0.05	915.20ng	10163.68
5	0.05	1144.0ng	11367.50

Figure 5: Calibration curve of Standard Caffeic acid

RESULTS OF GALLIC ACID:

Figure 6: Chromatogram showing peaks of Standard Gallic acid

Figure 7: HPTLC profile of S. cumini L. ethanolic and water fraction with standard Gallic acid at 254nm and 366nm

Table 3: Calibration Curve table of Gallic acid

S. No	R_f	Amount	Area (AUC)
1	0.95	105.30ng	827.08
2	0.94	210.60ng	2032.12
3	0.94	315.90ng	2983.06
4	0.94	421.20ng	3814.94
5	0.94	526.50ng	4356.26

Figure 8: Calibration curve of Standard Gallic acid

RESULTS OF QUERCETIN:

Figure 9: Chromatogram showing peaks of Standard Quercetin

Figure 10: HPTLC profile of S. cumini L. ethanolic and water fraction with standard Quercetin at 254nm and 366nm

Table 4: Calibration Curve table of Quercetin

S. no	R_f	Amount	Area (AUC)
1	0.44	210.80 ng	533.40
2	0.41	421.60 ng	1599.02
3	0.41	632.40 ng	2200.28
4	0.40	843.20 ng	2696.08
5	0.40	1054.0 ng	3520.36

Figure 11: Calibration curve of Standard Quercetin

Table 5: Quantification of Syzygium cumini L. leaves in Ethanolic and Water fraction

S. No.	Biomarkers	Ethanolic fraction	Water fraction
1.	Quercetin	0.129	0.703
2.	Caffeic Acid	2.776	1.087
3.	Gallic Acid	0.208	0.710

DISCUSSION:

Flavonoids and phenolic compounds are currently being studied due to their crucial biological and pharmacological effects. This study represents the first simultaneous quantification of these compounds, phenolics, and flavonoids in S.cumini L. leaves. The present study confirmed the presence of gallic and caffeic acid, and quercetin, suggesting that S. cumini L. leaves could offer anti-diabetic benefits. Among these compounds, caffeic acid was detected in the highest concentration, measuring 2.776 in ethanolic extract and 1.087 in water extract, establishing S. cumini L. leaves as a rich source of caffeic acid. Known for its health-promoting properties, caffeic acid exhibits strong antioxidant, anti-inflammatory, and antimicrobial activities and is linked to the inhibition of LDL oxidation. Additionally, the phenolic compounds and flavonoids in S. cumini L. leaves likely contribute to their pharmacological and antioxidant effects.

CONCLUSION:

Caffeic acid, gallic acid, and quercetin were all quantified simultaneously in plant extracts. Although previous studies have quantified gallic acid^33,34, myricetin, and quercetin³⁵ in Syzygium cumini L. leaf extracts and formulations using HPLC, this is the first report demonstrating the simultaneous quantification of caffeic acid, gallic acid, and quercetin in S. cumini L. leaf extracts via HPTLC. The validated TLC method was employed to accurately quantify and resolve these compounds. A mobile phase of toluene–ethyl acetate–formic acid (7:2:1) exhibited optimal selectivity for resolution, resulting in well-separated bands in the S. cumini L. leaf extracts. The proposed method is simple, precise, specific, and accurate, making it suitable for evaluating the purity of pharmaceutical drugs from various sources, detecting impurities, and ensuring the effectiveness of herbal formulations, including S. cumini L. leaves as a major ingredient.

ACKNOWLEDGMENT:

The authors acknowledged the director of HIPER for performing this research work.

CONFLICT OF INTEREST:

The authors acknowledge that there are no conflicts of interest related to this investigational study.

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Received on 26.04.2025 Revised on 13.08.2025

Accepted on 23.10.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2111-2116.

DOI: 10.52711/0974-360X.2026.00303

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