Journal of Planar Chromatography 29 (2016) 3 327

Summary

Oxalis corniculata is an important medicinal plant, but chromatog-raphic analysis for the simultaneous quantifi cation of diff erent phenolic compounds has not been performed till date in this plant species. A high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of caff eic acid, vanillic acid, and syringic acid in O. corniculata L. methanolic fraction was developed for the fi rst time. In HPTLC, for achieving good separation, the mobile phase of toluene‒ethyl acetate‒formic acid (7:3:1, v/v) was used. The densitometric determination was car-ried out at 300 nm in refl ection/absorption mode. The calibration curves were linear in the range of 100–700 ng per spot for caff eic acid, vanillic acid, and syringic acid. HPTLC analysis has indicat-ed the presence of optimum amount of caff eic acid (0.025 ± 0.0002), vanillic acid (0.018 ± 0.0005), and syringic acid (0.04 ± 0.0006) in the methanolic fractions. The optimized method was successfully applied for the analysis of three major phenolics in O. corniculata L. The proposed method is simple, precise, specifi c, and accurate. The quantifi cation of these phenolic compounds has not yet been reported in this species which may be utilized for the proper stan-dardization of the drug.

1 Introduction

Oxalis corniculata L., a sub-tropical plant being native to India, is commonly known as creeping wood sorrel [1]. It is a somewhat delicate-appearing, low-growing herbaceous plant abundantly distributed in damp shady places, roadsides, plantations, lawns, and nearly all regions throughout the warmer parts of India, especially in the Himalayas up to 8000 ft-cosmopolitan [2, 3].

A. Gupta, P. Kumar, O. Goshain, and A.K. Rai, Department of Pharmacy, Pran-veer Singh institute of Technology, Kanpur, India; and J. Dwivedi, Pharma-cognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow, India.*E-mail: abhishek.nbri@gmail.com

Phytochemical investigation of O. corniculata has revealed the presence of fl avanoids, tannins, phytosterol, phenol, glycosides, fatty acids, and volatile oil. The leaves contain fl avonoids, iso-vitexine, and vitexine-2-O-- -glucopyrunoside. It is a rich source of essential fatty acids like palmitic, oleic, linoleic, lino-lenic, and stearic acids. They are good source of vitamin C and carotene but contain a high content of oxalates. The leaves and stem contain tartaric and citric acid; the stem contains malic acid [4–6].

Methanolic and ethanolic extracts of this plant show the pres-ence of carbohydrate, glycosides, phytosterols, phenolic com-pounds, fl avonoids, proteins, amino acids, and volatile oil [7]. Leaves contain carbohydrate (1.36 ± 0.3%), fatty acids (13.2 ± 0.7%), palmitic acid (1.8%), and a mixture of oleic, linoleic and lenolenic acid, protein, fi ber, tannin, and moisture [8]. The whole plant extract is suspected to contain phytoestrogens [9]. O. corniculata identifi ed several compounds that were char-acterized by nuclear magnetic resonance, infrared, and mass spectrometry as (1) Oc-1, a mixture of saturated fatty acids C24 to C28; (2) Oc-2, a mixture of long-chain alcohols C18 to C28; and (3) Oc-3, a single compound that was a galacto-glycerolipid [10]. The plant is well known for its medicinal value as a good appetizer and as a remover of Kapha, vata, anemia, dyspepsia, cancer, dementia, convulsion, and piles [1, 11]. The plant boiled with butter milk is a home remedy for indigestion and diar-rhea in children. The leaf paste is applied over forehead to cure headache. The juice of the plant is given to relieve the intoxica-tion produced by Datura [3, 12]. The leaves are well masticated, and the juice is kept in the mouth for some time to get relief from aphthae. The extract of the plant is applied in the case of scorpion sting; the fresh leaves of O. corniculata are crushed and are used to stop bleeding from wounds [13]. The HPTLC quantitation of vitexin in O. corniculata leaves [14] and an esti-mation of fl avonoids has been performed earlier in chamomiles by some authors [15]. We have earlier performed the quantifi ca-tion of phenolic compounds in Bauhinia species [16, 17], but till date, the reported phenolic compounds form a substantial part of plant foods and are used as antioxidants with benefi cial bio-logical activities. These compounds have received considerable attention because of their role in preventing human diseases

High-Performance Thin-Layer Chromatography Analysis for Simultaneous Quantifi cations of Phenolic Biomarkers in Oxalis corniculata L. Methanolic Fraction

Abhishek Gupta*, Prashant Kumar, Omprakash Goshain, A.K. Rai, and Jyotsana Dwivedi

Key Words:

Caffeic acidHigh-performance thin-layer chromatographyOxalis corniculataSyringic acidVanillic acid

Journal of Planar Chromatography 31 (2018) 4, 327–331 DOI: 10.1556/1006.2018.31.4.80933-4173 © Akadémiai Kiadó, Budapest

HPTLC Analysis of Phenolic Compounds in Oxalis corniculata L.

328 Journal of Planar Chromatography 31 (2018) 4

[18]. Caff eic acid is a powerful antioxidant, which increases collagen production and prevents premature aging. It also has antimicrobial activity and may also have an eff ect in the treat-ment of dermal diseases [19]. Vanillic acid is a fl avoring agent found in edible plants and fruits. It is an oxidized form of van-illin. Vanillin has antibacterial, antimicrobial, and chemopre-ventive eff ects [18]. Syringic acid is a phenolic compound that acts pharmacologically as an antioxidant to clear free radicals. Syringic acid may act to inhibit the development of diabetic cataract [20]. The study demonstrates a comparative validated simultaneous quantifi cation of these three phenolic compounds in the whole plant of O. corniculata L. by HPTLC.

2 Experimental

2.1 Chemicals and Reagents

Caff eic acid, vanillic acid, and syringic acid were supplied by Sigma-Aldrich (Taufkirchen, Germany). All the reagents used in the experiment were of HPLC grade and were supplied by E. Merck (Darmstadt, Germany).

2.2 Preparation of Standard Solutions

A stock solution of vanillic, caff eic, and syringic acid stand-ards (1 mg mL−1) was prepared by dissolving 1 mg of accurately weighed standards each in 1 mL HPLC grade methanol, and further working solution of 100 μg mL−1 was prepared by add-ing 900 μL HPLC grade mobile phase into 100 μL of the stock solution and storing it at 4°C for analysis by HPTLC.

2.3 Plant material

The plant specimen, i.e., the whole plant of O. corniculata L., was collected from the National Botanical Research Institute, Lucknow, India, in the month of November, 2015. The plant was identifi ed and authenticated by Dr. A.K.S. Rawat, NBRI. A voucher specimen has been submitted in the institute’s herbarium.

2.4 Sample Preparation

The fresh plant was collected and thoroughly washed with water to remove all debris. The plants were dried in hot-air oven at 30°C. The dried plants were powdered by using an electric grinder at 100 mesh size. Extraction was performed by cold per-colation and soxhlation. First, the powdered plants are defatted under a Soxhlet assembly using 250 mL of 98% petroleum ether for 6 h. This is followed by 9-h soxhlation of the defatted pow-der by using 250 mL of chloroform followed by methanol and water. The fi nal extracts obtained were passed through What-man No. 1 fi lter paper. The fi ltrates obtained were concentrated under vacuum in a rotary evaporator at 40°C and stored at 4°C for further use. The crude extracts were obtained by dissolving a known amount of the dry extract in 98% methanol to obtain a stock solution of 1000 μg mL−1.

2.5 Development of HPTLC Fingerprinting: Instrumentation and Chromatographic Conditions

The following were the instruments and chromatographic conditions used: spotting device, Linomat V automatic sam-ple spotter (CAMAG, Muttenz, Switzerland); syringe, 100 μL Hamilton (Bonaduz, Switzerland); TLC chamber, glass twin-trough chamber (20 × 10 × 4 cm; CAMAG); densitometer, TLC Scanner 3 linked to winCATS software V. 4.06 (CAMAG); HPTLC plates, 10 × 10 cm, 0.2 mm thickness precoated with silica gel 60 F254 (E. Merck); experimental conditions, tempera-ture, 25 ± 2°C; relative humidity, 40%; solvent system, toluene–ethyl acetate–formic acid (7:3:1); detection wavelength, 300 nm for caff eic acid, vanillic acid, and syringic acid; slit dimension, 6.00 × 0.20 mm; scanning speed, 20 mm s–1; and source of radi-ation, deuterium lamp.

2.6 Calibration Curve of Caffeic Acid, Vanillic Acid, and Syringic Acid

A stock solution of caff eic acid, vanillic acid, and syringic acid (100 μg mL–1) was prepared in methanol. Diff erent volumes of stock solution were spotted on the TLC plate to obtain concen-trations of 100–700 ng per band of caff eic acid, vanillic acid, and syringic acid respectively. The data of peak areas plotted against the corresponding concentrations were treated by least square regression analysis method validation. The method was validated according to the International Conference on Harmo-nization (ICH) guidelines [21, 22], and the statistical analysis was done using Excel 2000 (MS Offi ce®).

3 Results and Discussion

3.1 Method Optimization for Separation of Analytes

The HPTLC method was developed and optimized in view of developing a simple and precise assay method. Several easily available solvents in diff erent combinations and concentrations were tried to obtain good resolution, compact spot, and better separation of degradation products. Lastly, the mobile phase consisting of toluene–ethyl acetate–formic acid (7:3:1, v/v), which gives a sharp and well-defi ned peak of caff eic acid, van-illic acid, and syringic acid (Figures 2 and 3), was selected. It was found that the solvent system has a very good resolution for

Figure 1

HPTLC profi le of O. corniculata methanolic fraction with standard caffeic acid, vanillic acid, and syringic acid at 254 and 366 nm.

HPTLC Analysis of Phenolic Compounds in Oxalis corniculata L.

Journal of Planar Chromatography 31 (2018) 4 329

the separation of phenolic compounds and also from the other components of crude drug and its extracts.

3.2 Calibration Curves

The linear regression data obtained for the calibration curves (n = 6) showed a good linear relationship over wide concentra-tion range 100–700 ng per spot with respect to peak area. The calibration equation obtained (Table 1) proves that the standard plot is linear and does not depart from linearity.

3.3 Validation

The method was validated with respect to parameters includ-ing linearity, accuracy, precision, robustness, limit of detection (LOD), limit of quantifi cation (LOQ), and specifi city in accord-ance with the ICH guidelines (Table 1).

3.4 Precision

Precision studies have been performed by analyzing intra- and inter-day variation for the determination of caff eic acid, van-illic acid, and syringic acid, carried out at three diff erent con-centration levels of 200, 400, and 600 ng per band; the mean percentage relative standard deviations were found to be 0.533, 0.396, and 0.163 for caff eic acid, vanillic acid, and syringic acid, respectively, in intra-day analysis, while the inter-day analysis showed mean percentage relative standard deviations of 1.4, 1.653, and 1.05 for caff eic acid, vanillic acid, and syringic acid, respectively, showing good precision (Table 2).

Figure 2

HPTLC chromatogram of caffeic, vanillic, and syringic acid in O. corniculata methanolic fraction and standards.

Figure 3

HPTLC densitogram of caffeic, vanillic, and syringic acid in O. cor-niculata methanolic fraction.

Table 1

Summary of validation parameters by HPTLC.

Parameters Caff eic acid Vanillic acid Syringic acid

R 0.36 ± 0.00 0.44 ± 0.005 0.54 ± 0.00

Linearity range 100–700 ng 100–700 ng 100–700 ng

Regression via area y = 164.551 + 3.504 * x y = 638.777 + 6.375 * x y = 164.551 + 3.504 * x

r 0.998 0.992 0.998

Slope 3.504 6.375 3.504

Intercept 164.551 638.777 164.551

LOD [ng] 40 40 40

LOQ [ng] 100 100 100

HPTLC Analysis of Phenolic Compounds in Oxalis corniculata L.

330 Journal of Planar Chromatography 31 (2018) 4

3.5 Robustness

The robustness of the method was studied, during method development, by determining the eff ects of small variations of mobile phase composition (±2%), duration of plate pre-wash-ing, chamber saturation period, development distance, and scanning time (10% variation of each). When the abovemen-tioned changes were made to the method conditions, there were no marked changes in the chromatographic behavior, indicating the method was robust. However, the developed method was robust at gradient elution regardless of small variations within the same mobile phase and to small changes revels with changes in the pH, temperature, flow rate, and concentration of reagents on mobile phase.

3.6 Limit of Detection and Limit of Quantifi cation

The LOD of the proposed method, with a signal-to-noise ratio of 3:1, was found to be 40 ng per spot, whereas LOQ, with a signal-to-noise ratio of 10:1, was found to be 100 ng per spot. This method was selected for the quantifi cation of all analytes because no peak overlapping of analytes among each other was detected in the plant samples.

3.7 Recovery Studies

The proposed TLC method used for the extraction and subse-quent estimation of caff eic acid, vanillic acid, and syringic acid from O. corniculata after spiking with 50%, 100%, and 150% of additional drug demonstrates good recovery. The experi-mental data are expressed as mean percentages of the recovered analytes and standard deviation, and relative standard deviation is also presented (Table 3).

3.8 Determination of Caffeic Acid, Vanillic Acid, and Syringic Acid in O. corniculata

A well resolved single spot was observed in the chromato-gram of the methanolic fraction of O. corniculata. It was also observed from the comparative HPTLC chromatogram (Figure 1) that there was no interference from the other

components present in the methanolic fraction of O. cornic-ulata. The HPTLC of methanolic fraction of O. corniculata was performed with marker compounds at six-point calibration curve; all markers were observed and quantifi ed in O. cornic-ulata. The total amount of phenolic compounds was quantifi ed in all the accessions (Table 4).

Table 2

Inter-day and intra-day precision data of biomarkers vanillic acid, caffeic acid, syringic acid, and kaempferol on HPTLC.

Standard markers Conc. [ng band−1]Intra-day Inter-day

%RSD Mean RSD %RSD Mean RSD

Caff eic acid

200 0.63

0.533

1.73

1.4400 0.51 1.36

600 0.46 1.11

Vanillic acid

200 0.52

0.396

1.61

1.653400 0.39 1.53

600 0.28 1.82

Syringic acid

200 0.18

0.163

0.92

1.05400 0.15 1.04

600 0.16 1.19

Table 3

Recovery analysis of caffeic, vanillic, and syringic acid from O. cor-niculata methanolic fraction by HPTLC.

Standard Amount added [%]

Amount recovered [%] Mean SD RSD

Caff eic acid

50 97.073 0.545 0.562

100 97.560 1.706 1.748

150 98.770 1.359 1.376

Vanillic acid

50 97.386 1.646 1.690

100 99.606 1.694 1.701

150 98.333 1.950 1.983

Syringic acid

50 99.813 1.938 1.942

100 98.936 1.878 1.898

150 98.883 1.416 1.432

Table 4

Quantifi cation of caffeic, vanillic, and syringic acid in O. corniculata methanolic fraction by HPTLC.

Standards Conc. (%)

Caff eic acid 0.026 ± 0.0002

Vanillic acid 0.018 ± 0.0005

Syringic acid 0.04 ± 0.0006

HPTLC Analysis of Phenolic Compounds in Oxalis corniculata L.

Journal of Planar Chromatography 31 (2018) 4 331

4 Discussion

The research on phenolic compounds is of current interest since they have important biological and pharmacological proper-ties. To our knowledge, our study is the fi rst to simultaneously quantify these phenolic acids present in O. corniculata. The present study confi rmed the presence of phenolic acids in O. corniculata. High concentrations of phenolic acids were found in O. corniculata with signifi cant variations in the amount. This indicates that O. corniculata should be consumed for its anti-oxidant eff ects. Out of the four phenolic compounds, syringic acid was found in the highest concentration compared to others amounting to 0.04%. This indicates that O. corniculata is a rich source of syringic acid. Syringic acid is one of the most impor-tant polyphenols that provide health benefi ts; it is widely known for its strong antioxidant properties. Syringic acid is also cor-related with the inhibition of LDL oxidation. Besides syringic acid, other phenolic compounds found in O. corniculata may also contribute to its medicinal and antioxidant properties. Further studies to isolate individual active principles and the antioxidant activity of the individual extracts of the roots and leaves through radical scavenging assay and their pharmaco-logical validation in terms of modern medicine will be of great pharmacological importance in the future which is under our consideration.

5 Conclusion

In this article, an HPTLC method has been established for the simultaneous determination of four phenolic compounds in O. corniculata methanolic extract without pre-treatment. Three phenolics (caff eic acid, vanillic acid, and syringic acid) have been identifi ed in O. corniculata by HPTLC. Syringic acid was found in high concentrations confi rming the antioxidant poten-tial and health benefi ts of O. corniculata. The HPTLC fi nger-prints of these standard phenolic compounds obtained using the method described above would serve the purpose of established benchmarks for future plant research. The qualitative and quan-titative analysis of the actual phenolic compounds present in any unknown plant sample would be facilitated by means of comparison with such standard chromatograms, enabling iden-tifi cation and confi rmation of the presence of any of these four common phenolic compounds in the research sample. The pro-posed method is simple, precise, specifi c, and accurate. The method can be used to determine the purity of the drug availa-ble from various sources by detecting the related impurities as well as for quality control of herbal formulations containing O. corniculata as an ingredient.

Acknowledgment

The authors are thankful to the director of CSIR-NBRI for pro-viding HPTLC facilities to conduct this research work.

Financial Support and Sponsorship

This work was supported by the Council of Science and Tech-nology, Uttar Pradesh, India.

Confl icts of Interest

There are no confl icts of interest.

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Ms received: April 11, 2018Accepted: May 31, 2018