Biomed Res- India 2015 Volume 26 Issue 1 99
Biomedical Research 2015; 26 (1): 99-102 ISSN 0970-938X www.biomedres.info
Determination of hydrolyzed Gallic Acid content in Potentilla chinensis Ser by HPLC. Wenfeng Huang#, Xiaoling Zhang#, Xiaomei Wang, Canhui Ouyang, Tao Shu, Zhijun Xie, Zixiang Zhang* The First Affiliated Hospital of Gannan Medical University, Ganzhou City, Jiangxi Province 341000, China
Abstract
Hydrolyzed gallic acid in Potentilla chinensis Ser. was determined by HPLC using Potentilla chinensis Ser. as a model drug with chromatographic conditions as follows: Hypersil C18 column (250 mm × 4.6 mm, 5 μm); mobile phase methanol-0.05% aqueous solution of phosphoric acid (10:90); volume flow 1.0 mL/min; detection wavelength 271 nm; column temperature 25°C; and injection volume 20 μL. Injection amount of gallic acid was in a good linearity with peak area at a range of 36.080~230.912 ng, average recovery was 99.2%, with a RSD of 1.89%. Free gallic acid could hardly be detected in Potentilla chinensis Ser., while gallic acid amount was relatively high after hydrolysis, suggesting that Potentilla chinensis Ser. contained a certain amount of hy-drolysable tannins. In conclusion, the indirect control of tannin residual amount in Potentilla chinensis Ser. can be achieved by determining hydrolyzed gallic acid in Potentilla chinensis Ser.
Keywords: Potentilla chinensis Ser., tannin, hydrolysis product, gallic acid, HPLC.
Accepted October 15 2014 Introduction
Potentilla chinensis Ser. is a perennial herb in the genus Potentilla of the family Rosaceae. The herb is distrib-uted in most parts of China, which is mainly grown in Anhui, Shandong and Liaoning [1]. Potentilla chinensis Ser. has a light smell and is slightly bitter in taste. The herb possesses astringent, spasmolytic, heat-clearing, detoxifying, blood-cooling and dysentery-checking properties. Due to its vulnerary properties it is used to treat hemorrhoidal bleeding, carbuncles and boils [2]. In folk medicine, the dried whole plant of Potentilla chinensis Ser. is used to treat Type-2 diabetes. Accord-ing to reports, Potentilla chinensis Ser. mainly contains triterpenoids, flavonoids, phenols, and tannins [3-5], which can prevent muscle spasm, and has hepatoprotec-tive function [6-8]. After preliminary experiments, it was found that Potentilla chinensis Ser. contained rela-tively high amount of tannins. The estimation of tannin content in Potentilla chinensis Ser. had some signifi-cance to the quality inspection of crude drugs, and de-velopment of quality standards for medicinal materials, as well as relevant formulations such as injections.
With increasing application of Traditional Chinese Medicine (TCM) injections, reports of adverse reactions associated with such dosage form are increasing, and their quality and safety are causing widespread concern in the society [9-10]. It has been reported that tannins
are a possible cause of allergy. The presence of tannins affect the stability of particle system in TCM injections, leading to the occurrence of adverse reactions [11-12]; moreover, tannins can also bind with tissue proteins to form induration, causing pain and necrosis at injection sites; once released into the bloodstream, they can also bind with amino groups of plasma proteins as a hapten to become a strong allergen, inducing serious adverse reactions.
In this study, a method for HPLC determination of gal-lic acid after hydrolysis was established making use of a property of tannins, i.e. ability to be hydrolyzed to gal-lic acid under certain conditions, which achieved the purpose of indirect control of residual tannin in Poten-tilla chinensis Ser. preparations, providing a new way of thinking for the establishment of highly sensitive, specific method for detection of residual tannin. Materials Instruments Agilent 1200 HPLC, Agilent, USA; GYH210 constant temperature water bath (Chengdu Experimental Instru-ment Co., Ltd.); HD-A-2 low-speed table-top centrifuge (Shanghai Climaveneta Co., Ltd.); 1876-6H pH meter (Nanjing Precision Scientific instrument Co., Ltd.). Gal-lic acid reference (batch No. 20140513-200451) was purchased from the National Institute for the Control of
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Pharmaceutical and Biological Products; methanol (HPLC grade), phosphoric acid, hydrochloric acid and sodium hydroxide were all of analytical grade, and dou-ble distilled water was used for all preparations.
Crude drug Potentilla chinensis Ser. crude drug was purchased from Tianshiqi TCM Decoction Pieces Plant, which was identi-fied by Tian Guangqi, a researcher of Chengdu Institute of Phytochemistry, as Potentilla chinensis Ser. in the genus Potentilla of the family Rosaceae. Its roots were dried to a constant weight at 70°C, pulverized, and then passed through a 60 mesh sieve.
Methods and Results
Selection of wavelength UV-Vis full wavelength scanning of gallic acid reference solution revealed a maximum absorption at 271 nm, so the measuring wavelength was selected to be 271 nm.
Chromatographic conditions Hypersil C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.05% aqueous solution of phosphoric acid (10:90); volume flow 1.0 mL/min; detection wave-length 271 nm; column temperature 25°C and injection volume 20 μL. Under the above conditions, the resolu-tion of gallic acid from two adjacent peaks was greater than 1.5. Number of theoretical plates was calculated to be not less than 4,000 based on the gallic acid peak. Chromatogram is shown in Figure 1. Investigation of linearity Appropriate amount of gallic acid was accurately weighed, placed in a 10 mL volumetric flask, dissolved in mobile phase and diluted to the mark to prepare the reference stock solution. Gallic acid reference stock solution was accurately aspirated, diluted separately with mobile phase to concentrations of 115.456, 57.728, 28.864, 14.432, 7.216, 3.608 and 1.804 μg/mL, and injected into the HPLC, followed by measurement of peak area. Standard curve was plotted with sample amount as the abscissa and peak area as the ordinate, and linear equation was obtained as y＝60091 x-3.153 (r=0.9999), the results showed that the injection amount of gallic acid was in a good linearity with the peak area at a range of 36.080 - 230.912 ng.
Preparation of test solution 5 g of dried Potentilla chinensis Ser. powder was taken, placed in a 100 mL volumetric flask, added with 5 mL of 4 mol/L HCl solution, and hydrolyzed at 100°C for 4 h; after cooling to room temperature, and centrifuging at 3000 r/min, the supernatant was collected, its pH ad-justed between 2-3 with 0.5 mol/L NaOH solution, and total volume was determined; 1 mL of the solution was accurately drawn, placed in a 25 mL volumetric flask,
diluted with distilled water to the mark, mixed well, then filtered through a 0.45 μm microporous membrane to give the test solution. Accuracy test Three mass concentrations, high, medium and low (57.728, 14.432 and 3.608 μg/mL), of reference solu-tions were taken, and injected continuously for HPLC determination, gallic acid peak areas were recorded, and their RSDs were calculated to be 0.23%, 0.47% and 0.41% (n=5), respectively. Three mass concentrations, high, medium and low, of reference solutions were taken, and injected repeatedly for HPLC determination within 5 d, gallic acid peak areas were recorded, and their RSDs were calculated to be 0.32%, 0.41% and 0.42% (n=5), respectively. Stability test Sample with batch number 20140312 was prepared into test solution, and injected for HPLC determination at 0, 1, 2, 4, 6, 8, 12 and 24 h, respectively, at an injection volume of 20 μL, gallic acid peak area was recorded, and its RSD was calculated to be 1.23%.
Reproducibility test Five aliquots of dried Potentilla chinensis Ser. powders with batch number 20140312 were sampled in parallel, prepared into test solutions, and injected at 20 μL each time, gallic acid peak area were recorded, and RSD of mass fraction was calculated to be 1.31%. Table 1. Results of determination of gallic acid in three batches of dried Potentilla chinensis Ser. Powders
Batch No. Gallic acid (mg/g) RSD% 20140312 10.23 1.24 20140313 10.12 0.98 20140314 11.09 2.41
Recovery test Nine aliquots of 2.5 g of dried Potentilla chinensis Ser. powder samples with batch number 20140312 were taken, hydrolyzed, then added with gallic acid reference 80%, 100% and 120% the amount of sample, respec-tively, with each level having three parallels, to prepare into test solutions, and determined, followed by calcula-tion of recovery. The results revealed an average recov-ery of 99.2%, with a RSD of 1.89%. Sample determination Dried Potentilla chinensis Ser. powders with batch numbers 20140312, 20140313 and 20140314 were taken, prepared into test solutions, and injected for HPLC determination at an injection volume of 20 μL, the results are shown in Table 1.
Determination Of Potentilla chinensis Ser.
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Figure 1. Reference substance (A), Sample after hydrolysis (B), Sample before hydrolysis (C) Discussion Quality and safety of TCM injections is one hot topic in pharmaceutical research in recent years. Existing TCM injections minimize the occurrence of pyrogenic and
hemolytic reactions by prescription screening and stan-dardized preparation methods [13-14]. However, there are many other factors which are associated with the safety of TCM injections, for instance, tannin residue is also closely linked to the safety of TCM injections. TCM in-
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jections have complex composition, while the existing tannin detection and determination methods have short-comings of low sensitivity, poor reliability and propensity to false-negative results. Tannins are also known as tannic acid, which can be di-vided into hydrolysable, condensed and complex tannins in terms of chemical structure and properties. Hydroly-sable tannins can produce gallic acid after hydrolysis. In this study, such property of tannins is made use of to in-vestigate the hydrolysis conditions of hydrolysable tan-nins in Potentilla chinensis Ser. taking crude drug of Po-tentilla chinensis Ser. as the study object, the amount of gallic acid produced after hydrolysis of tannins in Poten-tilla chinensis Ser. is determined by HPLC, which achieved the purpose of indirect control of tannin residual amount in Potentilla chinensis Ser. preparations, provid-ing a new way of thinking for the establishment of highly sensitive, specific method for detection of tannins. Free gallic acid can hardly be detected in crude drug of Potentilla chinensis Ser., while the amount of hydrolysed gallic acid is relatively high, suggesting that Potentilla chinensis Ser. crude drug contains a certain amount of combined hydrolysable tannins. After hydrolysis, gallic acid content in Potentilla chinensis Ser. crude drug is de-termined, which is of great significance to its quality con-trol, providing an important basis for the establishment of its quality standards, and an inspection basis for safety and stability of relevant future injections and other dosage forms. References 1. Mesicek J, Sojak J. Annotated Chromosome-numbers
of Selected Asiatic Potentilla Species. Folia Geobotanica & Phytotaxonomica 1993; 28: 437-446.
2. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. Vol. I. Beijing: Chemical Industry Press 2010; 199-199.
3. Shen Y, Wang QH, Lin HW, Shu W, Zhou JB, Li ZY. Study on chemical constituents of Potentilla chinensis Ser. Zhong Yao Cai 2006; 29: 237-239.
4. Xue HJ, Yang XK. Common volatiles are major attrac-tants for neonate larvae of the specialist flea beetle Al-tica koreana (Coleoptera: Chrysomelidae). Naturwis-senschaften 2008; 95: 639-645.
5. Wang QH, Li ZY, Shen Y, Lin HW, Shu W, Zhou JB. Studies on triterpenoids from Potentilla chinensis. Zhongguo Zhong Yao Za Zhi 2006; 31: 1434-1436.
6. Jung CH, Choi JK, Yang Y, Koh HJ, Heo P, Yoon KJ, Kim S, Park WS, Shing HJ, Kweon DH. A botulinum neurotoxin-like function of Potentilla chinensis extract that inhibits neuronal SNARE complex formation, membrane fusion, neuroexocytosis, and muscle con-traction. Pharmaceutical Biology (Formerly Interna-tional Journal of Pharmacognosy) 2012; 50: 1157-1167.
7. Lin X, Zhang S, Huang R, Tan S, Liang S, Wu X, Zhuo L, Huang Q. Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice. Int Immunopharmacol 2014; 19: 365-372.
8. Wei J, Huang Q, Huang R, Chen Y, Lv S, Wei L, Liang C, Liang S, Zhuo L, Lin X. Asiatic acid from Potentilla chinensis attenuate ethanol-induced hepatic injury via suppression of oxidative stress and Kupffer cell activa-tion. Biol Pharm Bull 2013; 36: 1980-1989.
9. Sun SG, Li ZF, Xie YM, Liu J, Lu Y, Song YF, Han YH, Liu LD, Peng TT. Analysis of rational clinical uses of traditional Chinese medicine injections and fac-tors influencing adverse drug reactions. Zhongguo Zhong Yao Za Zhi 2013; 38: 2969-2973.
10. Zhang XL, Li M, Rong P, Ma R. Analysis and coun-termeasures of adverse drug reactions of traditional Chinese medicine injections in children. Zhongguo Zhong Yao Za Zhi 2012; 37: 2807-2809.
11. Abed M, Herrmann T, Alzoubi K, Pakladok T, Lang F. Tannic acid induced suicidal erythrocyte death. Cell Physiol Biochem 2013; 32:1106-1116.
12. Karal'nik BV, Tsarevskiĭ IZ. Role of cell-surface pro-tein screening and the determinant of the other sensitin in polyvalent sensitization of tannin-treated erythro-cytes. Mikrobiol Epidemiol Immunobiol 1982; 7: 46-50.
13. Guo Q, Wu XY, Shi QS, Huang Q. Research ideas, methods and suggestions on quality evaluation of TCM injections. Drug Evaluation Research 2010; 33: 351-360.
14. Li LX, Li SF. Causes of adverse reactions with TCM injections and preventive measures. Chinese Tradi-tional and Herbal Drugs 2009; 40: Attachments 4-6.
#Authors contributed equally to this work. *Correspondence to: Zixiang Zhang The First Affiliated Hospital of Gannan Medical University Ganzhou City Jiangxi Province 341000 China
