Anda WEI, Hua ZHU, Fengfeng XIE, Miao ZHANG, Piaoling HUANG, Wenqi YANG

Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] The differences in secondary metabolites of Herba Polygoni Chinensis from Guangxi were analyzed, in order to find flavonoids which can antagonize liver injury and provide a basis for the quality evaluation of Herba Polygoni Chinensis. [Methods] UPLC-MS was used to analyze the secondary metabolites of Herba Polygoni Chinensis, and emphasis was placed on flavonoids. [Results] Flavonoids were abundant in the secondary metabolites of Herba Polygoni Chinensis. Quercitrin, catechin gallate, epicatechin gallate, hyperoside and so on were screened as the components with high content and antagonistic effect on liver injury. [Conclusions] Quercitrin was the flavonoid compound with the highest content among the three origins. It is stable and has a good protective effect on liver, which is representative. Quercitrin can be considered as the target component to be used as an important index for quality evaluation of Herba Polygoni Chinensis.

Key words Herba Polygoni Chinensis, UPLC-MS, Secondary metabolite, Flavonoid, Quercitrin

1 Introduction

Herba Polygoni Chinensis is the dried whole plant ofPolygonumchinenseL. which belongs to family Polygonaceae and genusPolygonum. It was first recorded inIllustratedClassicsofMateriaMedica, and has the functions of clearing away heat, removing dampness, cooling blood, detoxifying, benefiting liver, improving eyesight, relaxing menstruation and activating blood circulation. Herba Polygoni Chinensis is a common folk medicine, and in particular, it is widely used in the south of the Five Ridges. The curative effect of Herba Polygoni Chinensis has been well received, with development value in health care and clinical treatment.

Previous studies have shown that total flavonoids of Herba Polygoni Chinensis have pharmacological effect of antagonizing liver injury. However, which flavonoids and which components in Herba Polygoni Chinensis play the pharmacological effect still needs to be explored. In this study, UPLC-MS technology was used to analyze the secondary metabolites of Herba Polygoni Chinensis from different areas in Guangxi, focusing on flavonoids, in order to find flavonoids which can antagonize liver injury and provide a basis for the quality evaluation of Herba Polygoni Chinensis.

Metabonomics is the qualitative and quantitative analysis of all metabolites in organisms. It is a technology that integrates holistic and comprehensive analysis, and most of its research objects are small molecular substances with relative molecular weight less than 1 000. Metabonomics has obvious advantages in chemical analysis of traditional Chinese medicine. In the absence of reference substance, the components can be identified by analyzing the mass spectrum information of peaks or comparing with the literature data[1]. This technology plays an important role and advantage in the screening of quality markers of medicinal materials. It can accurately and quickly identify a group of different components in traditional Chinese medicine due to germplasm resources, ecological environment and other factors, including secondary metabolites and primary metabolites, so as to evaluate the quality of medicinal materials[2]. The key technology of metabonomics is sample data acquisition. Due to the advantages of high sensitivity, fast separation speed, strong specificity and accurate qualitative analysis, UPLC-MS is widely used in metabonomics[3]. In this study, UPLC-MS technology was used to identify the chemical components of Herba Polygoni Chinensis, and the secondary metabolites of Herba Polygoni Chinensis from different areas were analyzed, to provide a basis for the quality evaluation of Herba Polygoni Chinensis in Guangxi.

2 Materials and methods

2.1 Materials

2.1.1Instruments and equipment. The instruments and equipment used mainly included ultra performance liquid chromatography (UPLC) (Shim-pack UFLC Shimadzu CBM30A, http://www.shimadzu.com.cn/) and tandem mass spectrometry (MS/MS), (Applied Biosystems 4500 QTRAP, http://www.appliedbiosystems.com.cn/). The drugs used mainly included methanol, acetonitrile and ethanol (chromatographically pure, Merck, Germany).

2.1.2Medicinal materials. The samples were collected from Laibin, Fangcheng and Baise in Guangxi. They were identified as the dried whole plants ofPolygonumchinensisL. by Professor Teng Jianbei from the Department of Traditional Chinese Medicine Identification of Guangxi University of Chinese Medicine.

2.2 Methods

2.2.1Detection conditions. Chromatographic conditions were as follows: column, Waters ACQUITY UPLC HSS T3 C18(1.8 μm, 2.1 mm×100 mm); mobile phase, ultrapure water (added with 0.04% acetic acid)-acetonitrile (added with 0.04% acetic acid); elution gradient, 95∶5 (V/V, water/acetonitrile, 0 min) → 5∶95 (V/V, 11 min) → 5∶95 (V/V, 12 min) → 95∶5 (V/V, 12.1 min) → 95∶5 (V/V, 15 min); flow rate, 0.4 mL/min; column temperature, 40 ℃; sample size, 5 μL.

Mass spectrometry conditions were as follows. Ion source, electrospray ionization (ESI); temperature, 550 ℃; voltage, 5 500 V; curtain gas (CUR), 25 psi.

2.2.2Sample preparation. The Herba Polygoni Chinensis samples were freeze-dried in vacuum and ground (30 Hz, 1.5 min) into powder with grinder (MM 400, Retsch), respectively. A certain amount (100 mg) of the powder of each sample was dissolved in 1.0 mL extract (70% methanol aqueous solution), refrigerated at 4 ℃ overnight (vortexing were performed three times to improve the extraction rate) and centrifuged at 1 000 r/min for 10 min, and the supernatant was collected, filtered through microporous membrane (0.22 μm) and stored in injection bottle for UPLC-MS/MS analysis.

Metabolite quantification was performed by multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry. In MRM mode, after obtaining the metabolite mass spectrometry data of different samples, the peak areas of all the mass spectra were integrated, and the mass spectra peaks of the same metabolite in different samples were integrated and corrected.

3 Results and analysis

3.1 Overall analysis of metabolitesThe metabolites of Herba Polygoni Chinensis mainly included seven types, such as phenolic acids, flavonoids and alkaloids (Table 1). The abundance of metabolites of Herba Polygoni Chinensis is shown in Fig.1.

In Herba Polygoni Chinensis produced in Guangxi, phenolic acids had the highest content, followed by flavonoids and alkaloids. There was no significant difference in phenolic acids among different producing areas, and phenolic acids in Herba Polygoni Chinensis produced in Fangcheng showed the most kinds. There were certain differences in flavonoids among the three producing areas, and the most flavonoids were found in Baise. According to literature, flavonoids, phenolic acids and alkaloids in natural medicines are the material basis for the efficacy. As far as Herba Polygoni Chinensis is concerned, its total flavonoids have the pharmacological effect of antagonizing liver injury. Thus, it is necessary to do further analysis and research on flavonoids.

Table 1 Metabolites of Herba Polygoni Chinensis from different producing areas in Guangxi

3.2 Difference analysis of flavonoidsA total of 78 kinds of flavonoids were identified in Herba Polygoni Chinensis from three different producing areas, including 44 kinds of flavonols, 17 kinds of flavonoids, 8 kinds of flavanols, 4 kinds of dihydroflavones, 3 kinds of isoflavones and 2 kinds of anthocyanins. Flavonoids in Herba Polygoni Chinensis from Baise had the most kinds (78), followed by those from Fangcheng (72), and flavonoids in Herba Polygoni Chinensis from Laibin had the least kinds (68). There were differences in flavonoid types and contents among the three producing areas. The statistical results of differential components are shown in Fig.2.

According to the Venn diagram, there were differences in 10 kinds of flavonoids among the three producing areas. Compared with that of Baise, the count of flavonoid kinds of Linbin was 10 smaller, and that of Fangcheng was 6 smaller (Table 2). Compared with those of Baise, the missing components in Laibin and Fangcheng were repetitive, indicating that the differences in metabolites between the two producing areas were small, while the differences between the two producing areas and Baise were great. The results show that the differences in metabolites of Herba Polygoni Chinensis could be caused by the factor of producing area.

Table 2 Difference analysis of flavonoids in Herba Polygoni Chinensis from different producing areas of Guangxi

3.3 Analysis of flavonoids against liver injuryIn terms of composition of flavonoids, in Herba Polygoni Chinensis from the three different producing areas, quercitrin had the highest content, followed by catechin gallate, epicatechin gallate and hyperoside. Among the three producing areas, the content of quercitrin ranked as Baise > Fangcheng > Laibin, and the quercitrin content of Herba Polygoni Chinensis produced in Baise was much higher than that produced in Laibin. Studies have shown that quercitrin could inhibit the apoptosis of L02 cells treated with H2O2and improve their survival rate, which might be achieved by inhibiting Nrf2/HO-1 pathway and reducing oxidative stress inflammation[6]. It is reported that quercitrin can also improve the fatty liver symptom of experimental mice, and it has a good protective effect on liver[7]. Catechin gallate and epicatechin gallate have antagonistic effect on nonalcoholic fatty liver injury[8]. Hyperoside has protective effect on acute alcoholic liver injury in mice, andinvivoandinvitro, it can antagonize oxidative stress-induced liver injury through PHLPP2-AKT-GSK-3β signal pathway[9]. It is also found that hyperoside has protective effects on CCl4-induced acute liver injury and H2O2-induced L02 cell injury in rats[10-11]. The contents of these four flavonoids were relatively high in each producing area. They have the pharmacological effect of protecting liver from damage, and may be the material basis of antagonizing liver injury. Quercitrin has advantages in content and stability, so it can be considered as an index for quality evaluation of Herba Polygoni Chinensis.

4 Discussion and conclusions

Metabonomics has advantages in quality evaluation of medicinal materials. It can quickly and accurately reflect the related differences between herbs, and is also an important means of screening quality markers of medicinal materials. Flavonoids are abundant and diversified in the secondary metabolites of Herba Polygoni Chinensis. Quercitrin, catechin gallate, epicatechin gallate and hyperoside are screened as the components with high content and antagonistic effect on liver injury. In this experiment, quercitrin was the most abundant flavonoid in the Herba Polygoni Chinensis from the three producing areas of Guangxi. It is stable, and has a good protective effect on liver, which is representative. Quercitrin can be considered as the target component to be used as an important index for quality evaluation of Herba Polygoni Chinensis. If quercitrin was used as the target component, the quality of Herba Polygoni Chinensis from Baise was the best, followed by that from Fancheng. However, quercitrin as an evaluation index needs to be verified in many aspects. This method is stable and accurate, can realize fast analysis, and can be used as a method for quality evaluation of Herba Polygoni Chinensis.