High‑Performance Thin‑Layer Chromatography Analysis of Gallic Acid and Other Phytoconstituents of Methanolic Extracts of Myrica Nagi Fruit Yash Prashar, Nilesh J
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Pharmacogn. Res. ORIGINAL ARTICLE A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products www.phcogres.com | www.phcog.net High‑Performance Thin‑Layer Chromatography Analysis of Gallic Acid and Other Phytoconstituents of Methanolic Extracts of Myrica nagi Fruit Yash Prashar, Nilesh J. Patel1 Ph.D Research Scholar, Ganpat University, Ganpat Vidyanagar, Mehsana-Gozaria Highway, 1Department of Pharmacology, Shree S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar, Mehsana-Gozaria Highway, Kherva, Gujarat, India ABSTRACT Background: Myrica nagi Thunb. (family: Myricaceae) is effective against gastric, metabolic, and hepatic disorders. The therapeutic effect of its fruit, which is consumed in North India, has not been confirmed, and detailed chemical profiling of the fruit is thus required. Objectives: The study objective was to develop and optimize a high‑performance thin‑layer chromatography (HPTLC) method for the characterization of gallic acid, quercetin, myricetin, and caffeic acid in the methanolic extract of M. nagi fruit and the quantification of gallic acid. Materials and Methods: Analyses were performed using HPTLC, and liquid chromatography‑mass spectrometry. HPTLC experiments were carried out using an optimized solvent mixture, which enabled the separation and detection (at 254 and 366 nm) of four flavonoid compounds in the dried M. nagi extract. Gallic acid was quantified using calibration curves. Results: The proposed method enabled the detection of gallic acid, quercetin, myricetin, and caffeic acid. Validation took into account the estimation of linearity, limit of detection, limit of quantification, accuracy, and recovery of gallic acid. Gallic acid was quantified at 12.93 µg/mg of dry plant concentrate. Conclusion: This study describes the development of an HPTLC method for the analysis Abbreviations Used: HPLC: High‑pressure liquid chromatography, and characterization of phytoconstitutents in the methanolic solution of HPTLC: High‑performance thin‑layer chromatography, ESI: Electrospray a dried M. nagi fruit extract. The method was successfully validated for ionization, LC‑MS: Liquid chromatography‑mass spectrometry, LOD: Limit the analysis of gallic acid. of detection, LOQ: Limit of quantification, MS: Mass spectrometry, Rf: Key words: Flavonoid, gallic acid, high‑performance thin‑layer Retention factor, TLC: Thin‑layer chromatography. chromatography, liquid chromatography‑mass spectrometry, Myrica nagi Correspondence: Access this article online SUMMARY Dr. Nilesh J. Patel, Website: www.phcogres.com We performed full chemical profiling of the fruit extract ofMyrica nagi and Department of Pharmacology, Shree S. K. provided insights into the various phytoconstituents present in the fruit Patel College of Pharmaceutical Education Quick Response Code: through LC‑MS. The presence of various phytocompounds was confirmed and Research, Ganpat University, Ganpat through HPTLC. The method for gallic acid analysis was validated in a Vidyanagar, Mehsana‑Gozaria Highway, Kherva, solvent system of ethyl formate/toluene/formic acid/water 20:1:2.6:0.5 Gujarat ‑ 384 012, India. (v/v/v/v). The method allowed excellent separation of the compounds and E‑mail: [email protected]; can be of high importance for further quantification of phytocompounds [email protected] and for the development of herbal formulations. DOI: 10.4103/pr.pr_104_19 INTRODUCTION the first essential step and involves the extraction of analytes of interest. Modern, state‑of‑the‑art sample preparation and separation methods Myrica nagi Thunb. (syn.Myrica esculenta), commonly known as offer substantial benefits over standard methods for the characterization box myrtle, katphala, boxberry, and orkaphal, belongs to the family of medicinal plants, playing an essential role in the commercialization Myricaceae and is a widely used medicinal plant. M. nagi is sourced of high‑quality herbal products.[17] Thin‑layer chromatography (TLC) [1] primarily for its fruits, which are similar to raspberries and are deep is a simple and cost‑effective separation technique enabling the analysis red in color, with a small amount of pulp and a round seed at the center. The bark of this plant contains chemical compounds, such as myricetin, This is an open access journal, and articles are distributed under the terms of the myricitrin, and specific glycosides. It has been reported to possess Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which anti‑allergic,[2] anti‑inflammatory,[3] antioxidant,[4] antihelminthic,[5] allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. antimicrobial,[6] anxiolytic,[7] chemoprotective,[8] hypertensive,[9] [10] [11] [12] [13] mast cell‑stabilizing, analgesic, antiulcerating, antidiabetic, For reprints contact: [email protected] hepatoprotective,[14] and wound‑healing[15] properties. This has led to the [16] development of new therapeutic medicines using M. nagi extract. Cite this article as: Prashar Y, Patel NJ. High-performance thin-layer Analysis of herbal samples typically involves several steps, including chromatography analysis of gallic acid and other phytoconstituents of methanolic sample preparation, separation, and detection. Sample preparation is extracts of Myrica nagi fruit. Phcog Res 2020;12:95-101. © 2020 Pharmacognosy Research | Published by Wolters Kluwer ‑ Medknow 95 YASH PRASHAR and NILESH PATEL: Isolation and Quantitation of Biomarker Compounds from Myrica nagi of several samples simultaneously; hence, it is often considered the first 15 min. The solutions were applied to the plates using an automatic TLC option for diverse medicinal analytical applications. High‑pressure sampler (CAMAG® ATS; CAMAG, Muttenz, Switzerland) equipped liquid chromatography (HPLC) and high‑performance thin‑layer with a 100‑µL syringe at a steady implementation rate of 150 nL/s. The chromatography (HPTLC) are widely employed to characterize and solutions were applied onto prewashed TLC plates in 5‑mm broad bands, analyze numerous bioactive mixtures, enabling the detection of 10 mm from the bottom edge, 10 mm from the side and top edges, and constituents, including secondary metabolites, nutrients, and amino with 6 mm gaps between each spot. The CAMAG® Twin Trough Chamber acids.[18] Hence, HPTLC represents an attractive technique for qualitative was presaturated for 20 min at 25°C ± 2°C and 40% relative humidity, and quantitative analyses of herbal extracts.[19] Mass spectrometry (MS) with the mobile phase composed of a mixture of ethyl formate/toluene/ provides highly accurate structural information about the constituents formic acid/water 20:1:2.6:0.5 (v/v/v/v).[23‑25] The chromatographic run of herbal extracts, allowing the identification of compounds of interest. was approximately 80 mm. After separation, the TLC plates were dried Overall, an effective, high‑throughput screening and separation using an air current. Densitometric visualization of the chromatographic technique combined with MS detection is crucial for the identification spot was performed at 254 nm and 366 nm using deuterium and mercury of potential natural therapeutic candidates.[20] lamps in the CAMAG® HPTLC instrument equipped with visionCATS This study aimed to develop and validate a high‑throughput HPTLC software (CAMAG, Muttenz, Switzerland). method for the characterization of gallic acid, quercetin, myricetin, and Characterization of methanolic extracts using liquid caffeic acid in methanolic extracts of the M. nagi fruit. The developed chromatography–mass spectrometry method was validated for gallic acid. The methanolic extract was analyzed using the 2795 Alliance HPLC MATERIALS AND METHODS system (Waters, Milford, MA, USA) coupled with a Micromass Q‑TOF Micro Mass Spectrometer (Waters, Milford, MA, USA). Chemicals Separation was carried out on an XBridge C18 column (130 Å, 3.5 µm, All solvents, including methanol, butanol, acetic acid, formic acid, 4.6 mm × 150 mm; Waters). The mobile phase was composed of 80% and toluene, were of analytical grade and purchased from SD Fine methanol and 20% water and run in the isocratic mode. The flow rate Chemicals (Mumbai, Maharashtra, India). Flavonoid standards, such was set at 0.7 mL/min. The injection volume was 20 µL. MS acquisition as those for gallic acid, quercetin, myricetin, and caffeic acid, were was performed using the electrospray ionization (ESI)‑positive mode purchased from Natural Remedies (Bengaluru, Karnataka, India). and multiple reaction monitoring with unit resolution. Desolvation gas and cone gas flow rates were set to 550 L/h and 30 L/h, respectively. Collection of Myrica nagi Desolvation gas and source temperatures were set to 300°C and 110°C, Fruit samples of M. nagi were collected from Mandi district, Himachal respectively. The ESI capillary voltage was fixed at 3000 V, and the cone Pradesh, India, in July 2017. The collected plant material was voltage was set at 30 V. Compounds were fragmented using collision authenticated by NISCAIR, Delhi, India (Ref. No. NISCAIR/RHMD/ energy of 4 eV. Nitrogen and argon were used at pressures of 6–7 bars Consult//2017/3102‑51‑4). Healthy fruits were separated and kept for and 5–6 bars, respectively. further analyses. Validation of the developed high-performance Methanolic extraction of Myrica nagi fruits thin-layer chromatography method Freshly collected