Activity Guided Isolation Of Phenolic Compositions From Anneslea Fragrans Wall. And Their Cytoprotective Effect Against Hydrogen Peroxide Induced Oxidative Stress in HepG2 Cells Part 1

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Abstract: Anneslea fragrans Wall., commonly known as"Pangpo Tea", is traditionally used as folk medicine and healthy tea for the treatment of liver and intestine diseases. The aim of this study was to purify the antioxidative and cytoprotective polyphenols from A.fragrans leaves. After fractionation with polar and nonpolar organic solvents, the fractions of aqueous ethanol extract were evaluated for their total phenolic (TPC) and flavonoid contents(TFC) and antioxidant activities(DPPH, ABTS, and FRAPassays). The n-butanol fraction (BF)showed the highest TPC and TFC with the strongest antioxidant activity. The bio-guided chromatography of BF led to the purification of six flavonoids (1-6) and one benzoquinone lanthanoid (7). The structures of these compounds were determined by NMR and MS techniques. Compound 6 had the strongest antioxidant capacity, which was followed by 5 and 2. The protective effect of the isolated compounds on hydrogen peroxide (H2O2)-induced oxidative stress in HepG2 cells revealed that the compounds 5 and 6exhibited better protective effects by inhibiting ROS productions, having no significant difference with vitamin C(p>0.05), whereas 6 showed the best anti-apoptosis activity. The results suggest that A.fragrans could serve as a valuable antioxidant phytochemical source for developing functional food and health nutraceutical products.

Keywords: Anneslea fragrans; antioxidant; guided isolation; oxidative stress; flavonoid glycosides

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1. Introduction

Reactive oxygen species (ROS) are produced as by-products through oxidative respiration in normal physiological and biochemical processes [1]. As endogenous free radicals, ROS plays an important role in cell signaling and the maintenance of body constancy in a normal range [2]. If the ROS could not be effectively scavenged, excessive ROS could lead to the occurrence of oxidative stress, which may influence cell proliferation and apoptosis [3]. Oxidative stress is closely related to a variety of diseases including diabetes, hyperlipemia, obesity, cancer, and cardiovascular and neurodegenerative diseases [4]. Recently, many studies have evidenced that a diet enriched with antioxidants possesses a series of beneficial effects owing to their scavenging ability on excessive ROS [5,6].

Natural antioxidants contain a variety of molecules such as polyphenols[], carotenoids [8], vitamins [9], nitrogen-containing compounds [10], and coumarins [9]. Polyphenols are distributed widely throughout the plant kingdom and promote health benefits owing to their antioxidant properties [11]. Many of them, such as resveratrol and some derivatives [12], anthocyanidins [13, isoflavones [14l, catechin [15l, and quercetin [16] are well known for their protective effect by scavenging ROS [1]. For the past few years, polyphenols have been attracting attention in the prevention of cancer[17], cardiovascular dysfunc-tion[18], neurodegenerative diseases [19], and aging [20]. Therefore, searching for effective antioxidants is an urgent need to promote human health.

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Anneslea fragrans Wall. is an evergreen plant, which is mainly distributed in the south of China [21]. In addition to its ornamental purpose, the leaves of A.fragrans are also used as a folk medicine to treat fever, liver protection, invigorating stomach and intestines China and Cambodia [22], which had been recorded in"Yunnan Simao Chinese Herbal Medicine".In addition, the leaves have also been processed as a tea beverage, which is known as"Pangpo Tea".In previous reports [23], the extract of A.fragrans has shown antioxidant and antimalarial activities. However, to date, its antioxidant phenolic compounds from A. fragrances have not been reported.

Thus, the purpose of this research was to isolate and identify the antioxidant compounds from A. fragrans leaves that are responsible for their traditional use for the treatment of liver diseases. The four fractions, dichloromethane fraction (DF), ethyl acetate fraction (EAF), an n-butanol fraction(BF), and residual water fraction (RWF)of aqueous ethanol extract were assessed for their total phenolic(TPC)and total flavonoid contents (TFC) and for their antioxidant capacity. The n-butanol fraction(BF) had the highest TPC and TFC with the strongest antioxidant activity. The bio-guided fractionation of BF allowed the purification of compounds. Furthermore, the cytoprotective effect of the isolated compounds was performed on hydrogen peroxide (H, O,)-induced oxidative stress in human liver cancer HepG2 cells. The intracellular ROS production and cell apoptosis were determined using flow cytometry. Thus, this research afforded a valuable antioxidant phytochemical ingredient for the development and utilization of A.fragrans leaves as a functional supplement (healthy tea) in the food and health industry.

2. Results and Discussion

2.1.Yield Efficiency of Fractions and Subfractions

The liquid-liquid partitioning by organic solvents and column fractionation are important techniques for enriching the bioactive compounds from crude extract [24]. Using liquid-liquid partitioning, ethanol extract(CE) from A. fragrans leaves was successively fractionated with dichloromethane, ethyl acetate, and n-butanol to give four fractions (DF, EAF, BF, and RWF), respectively. Yield percentages of fractions were found to vary from 11 to 27%. The residual water fraction (RWF, 27%)had the highest yield, and the yields of other fractions were in the order as follows: n-butanol fraction (BF, 24%), ethyl acetate fraction (EAF, 21%), dichloromethane fraction (DF, 11%).

The yield percentage of subfractions (BF-A to E)was found to vary from 3.82 to 48.4%. The BF-E presented the highest yield of 48.4%followed by BF-D (24.96%), which has significantly higher yields than the other fractions.

2.2.HPLC Analysis

High-performance liquid chromatography (HPLC) detection has been demonstrated to be a powerful technique for quantitative determination. HPLC analysis revealed that BF had the most compounds with the strongest antioxidant activity. Under the guidance of antioxidant assays and HPLC analysis, the antioxidative fraction was further chromatographed for the isolation of subfractions. The BF was subjected to a hydrated resin D101 column to yield five subfractions(BF-A to E). HPLC analysis revealed that BF-Cto E had the most antioxidant compounds. Bio-guided fractionation of these fractions (BF-C to E) allowed the purification of seven pure compounds.

As shown in Figure 1, the BF and seven pure compounds were profiled by HPLC analysis. Based on comparison with the retention times and UV absorption curves, these seven compounds were confirmed through retention times at 7.51 min (corrosive, 7),7.92 min((epi)-catechin, 6), 9.38 min (confusoside, 1),9.57 min((S)-naringenin-7-O-β-D-glucopyranoside, 4), 10.32 min(vaccinii folin, 2), 12.64 min (2',3,4,4'-tetrahydroxydi dihydrochalcone, 5), and 13.95 min (1-[4-(β-D-glucopyranosyloxy)-2-hydroxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)-1-propanone,3)(Figure 1A).Among them, compounds 1-6 belong to flavonoids,and compound 7 is benzoquinolethanoid.

2.3.Total Phenolic Contents (TPC) and Total Flavonoid Contents (TFC)

The A.fragrans leaves are traditionally used as processed health tea and have been proven to be a good resource of phenolics and flavonoids [25]. According to spectrophotometric assays, the TPC and TFC were tested in different fractions and subfractions from A. fragrans leaves. As shown in Table 1, the BF had the highest TPC value with 238.12±12.05 mg GAE/g extract followed by EAF. The DF showed the lowest TPC value as 66.52±0.57 mg GAE/g extract. Similarly, the highest TFC concentrations were also found in BF with TFCvalue of 165.19±5.21mg RE/extract. With regard to the RWF and EAF (66.92±1.38 and 116.12±2.89mg GAE/extract, respectively), they also had lower TFC concentrations than the BF(165.19±5.21 mg RE/g extract), while DF had the lowest TFC concentration as 47.94±2.84 mg RE/g extract. The contents of TPC and TFCin BF were approximately four times higher than those in DF.

The data depicted the presence of highest TPC and TFC in BF-E with values as 262.03±1.72mg GAE/g extract; 180.52± 6.30 mg RE/g extract, respectively, followed by BF-D(219.84±4.01 and 157.01±2.50 mg RE/g extract, respectively), BF-C (210.69±6.31 and 151.01±4.33 mg RE/g extract, respectively), while BF-B and BF-A had the lowest TPC and TFC values (Table 1).

2.4.Antioxidant Actioity

Most of the polyphenols, especially flavonoids and phenolic acids, are exploited into popular antioxidant foods (nutraceuticals) and present a series of human benefits [17]. In our previous study, the A.fragrans leaves have been proven to be a good resource of polyphenols[26]. However, the antioxidant activity of the extract from A. fragrans leaves and its phytochemicals have not been investigated yet. Due to different antioxidative reaction mechanisms, three assays of ABTS, DPPH, and FRAP were combined to evaluate the antioxidant activity of the fractions and subfractions from A. fragrans leaves.

Among the fractions, BF showed the most potent antioxidant activity in ABTS, DPPH, and FRAP radical-scavenging activities with 1808.46±96.52,951.42±87.75, and 1822.96± 29.24 μmol TE/g extract （Table 1）.PDF showed the lowest antioxidative activity in ABTS and FRAP assays（139.23±8.62 μmol TE/g extract and 180.05± 9.46 μmol TE/g extract, respectively)(Table 1). Whereas the radical scavenging activities of four fractions (DF, EAE, BF, and RWF) in the DPPH radical-scavenging assay were found to vary from 613.38± 45.35 to 951.42±87.75 μmol TE/g extract， respectively， having no significant difference (p>0.05). These results suggested that the phytochemicals from A. fragrans leaves might be insensitive to DPPH. To further obtain the active metabolites, BF was selected for further fractionation. The BF was subjected to D101 macroporous adsorbing resin column chromatography eluting by a gradient of the methanol-aqueous system to yield five subfractions(BF-A to E). Hence, the ABTS radical-scavenging activity of these subfractions can be ranked as BF-E> BF-C>BF-D. The reducing activity in the FRAP assay revealed that BF-Dhad the strongest antioxidative activity, which was followed by BF-C and BF-E.It is noteworthy that three subfractions(BF-C to E) presented higher antioxidative activity than the mother fraction (BF). Taken together, BF-C to E was submitted to column chromatography for isolating and identifying the antioxidative phytochemicals.

2.5. Antioxidant Actioity of the Isolated Compounds

All the isolated compounds (compounds 1-7) were evaluated for antioxidant capacity by ABTS, DPPH radical scavenging activity, and FRAP antioxidant activity. All the data are described in Table 2. Compounds6 and 2 showed the highest antioxidant activity, followed by 5. Compound 4 displayed moderate antioxidant activity and compounds 1, 3, and 7 were considered inactive with ABTS and DPPH radical scavenging activities less than 200 μmol TE/g extract. Using Vc（the radical scavenging activities with 2932.91 ±93.63 and 1873.56±121.68 umol TE/g extract, respectively)as the positive control, compound6 had the strongest antioxidant activity in ABTS and DPPH radical scavenging assays with 1580.37±89.32 and 1953.31±109.93 μmol TE/g extract （p<0.05）， respectively. Furthermore, compound 6 had the best antioxidant capacity in the FRAP assay as 5027.43±620.75 μmol TE/extract， which was much higher than that of Vc as 3291.28±241.02 μmol TE/extract. Moreover， the compound 2and6showed a significant antioxidant activity, which was equivalent to a positive control (Vc).

Additionally, the antioxidant structure-activity relationship of the flavonoids (1-6)is discussed by varying degrees of inhibitory effects. Compound 2 presented more an-tioxidant activity than compounds 1, 3, and 5, which suggested that the 3,4-dihydroxy groups in dihydrochalcones in 2 may play a critical role against ABTS, DPPH radical scavenging activity, and FRAP antioxidant activity, and the glucose moiety substituted at C-4'in 2 may play an important role on its antioxidant capacity [4]. Furthermore, compound 6 exhibited the highest antioxidant activity compared to other compounds, which suggested that flavan-3-ols (catechin) probably exhibit a better radical scavenging activity than dihydrochalcones and flavanones [27]. In summary, compounds 2, 5, and 6 with good antioxidant activity were selected further for cytoprotective effects against oxidative stress by H2O2 in the next study.

2.6. Relationship between Antioxidant Activity and TPC/TFC Contents

Concentrations of the TPC and TFC highly correlated with antioxidant activity from the values of FRAP (r=0.982 and 0.977) and ABTS (r=0.959 and 0.965), respectively. The correlation matrix also showed strong correlation between the ABTS and FRAP values (r =0.992). Furthermore, a multivariate analysis (PCA), which was extracted from the data of Table 1, was carried out. As shown in Figure 2, PCA explained 94.44% of total variation, in which PC1 accounted for 84.73% of the variance and PC2 accounted for 9.71%. The FRAP, ABTS, and DPPH assays with EAF and RWF are placed at the upper right quadrants, and TPC and TFC concentrations with BF and BF-Cto E are located at the lower right quadrants in the PC1 positive scores, respectively. Meanwhile, the DF and BF-A to B with low TPC and TFC concentrations are located along the axis of PC1 negative scores. These findings showed that the TPC and/or TFCconcentrations are closely associated with antioxidant capacity, and the greater the TPCor TFC concentrations in the fractions and subfractions, the higher their antioxidant capacity values. These results revealed that the high phenolic and flavonoid contents in different fractions and subfractions from A. fragrans leaves might contribute to antioxidant activity.

Figure 2. PCA analysis on total phenolics, flavonoids, and antioxidant activity. Means with different letters indicate significant differences (p<0.05).DF, EAF, BF, RWF mean dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and residual water fraction. BF-A to E means BF was subjected to D101 column chromatography to yield five subfractions.

This article is extracted from Molecules 2021, 26, 3690. https://doi.org/10.3390/molecules26123690 https://www.mdpi.com/journal/molecules