Extraction solvent affects the antioxidant, antimicrobial, cholinesterase and HepG2 human hepatocellular carcinoma cell inhibitory activities of Zanthoxylum bungeanum pericarps and the major chemical components

Industrial Crops & Products 142 (2019) 111872

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Extraction solvent affects the antioxidant, antimicrobial, cholinesterase and HepG2 human hepatocellular carcinoma cell inhibitory activities of Zanthoxylum bungeanum pericarps and the major chemical components

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Yao Ma, Xuan Li, Li-Xiu Hou, An-Zhi Wei Northwest A&F University, Address: No.3 Taicheng Road, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Zanthoxylum bungeanum Maxim. Chemical composition Antimicrobial Cholinesterase inhibitor HepG2 inhibitor

Chinese prickly ash (Zanthoxylum bungeanum Maxim.) is widely grown in China where it is of high economic importance. Its pericarps are rich in bioactive compounds and are in common use both as food additives, for their distinctive flavor, and as traditional medicines, to treat various diseases. The method of extraction of these compounds is important. This study evaluates the effects of the solvent on the yields of the major components and their biological activities - antioxidant, antimicrobial, cholinesterase and HepG2 human hepatocellular carcinoma cell (HepG2) inhibitory. Seven common solvents were used: water, methanol, acetic acid, chloroform, ethanol, ethyl acetate and benzene. Acetic acid produced the highest extract yield (24.69%), while benzene produced the lowest (7.54%). The ethanol extract had the highest phenolic content (81.19 ± 4.81 g gallic acid equivalents (GAE) /kg extract), the methanol extract had the highest flavonoid content (110.69 ± 8.49 g rutin equivalents (RE)/kg extract) and the benzene extract had the lowest (22.42 ± 2.1 g RE/kg extract). Procyanidin was the most abundant component in the extracts (125.35–350.98 mg/100 g pericarps). The water extract showed strong antioxidant activity for OH scavenging activity (0.45 mg/mL of half-maximal inhibitory concentration (IC50)), reducing power (RP) (RP0.6 mg/mL = 3.93) and ferric reducing ability of plasma (FRAP) (FRAP0.6 mg/mL = 0.48). Methanol and acetic acid were the most effective extracts for inhibition of Staphylococcus aureus, while chloroform was most effective for antimicrobial activity against Candida albicans and HepG2 growth inhibition rate (0.39 mg/mL of IC50). The inhibitory activities of the water and methanol extracts on butyrylcholinesterase (BChE) were stronger than on acetylcholinesterase (AChE) with selectivity indices of 0.74 and 0.28, respectively. Methanol (1.02 mg/mL of IC50 for AChE and 0.28 mg/mL of IC50 for BChE) was most effective for cholinesterase inhibitory activity. The total phenolic content, total flavonoid content, total alkaloid content and warfarin content were significantly correlated with the first two principal component analysis axes, indicating some relationship between these components and their biological activities. Extracts of Z. bungeanum pericarps are a good source of compounds having significant biological activities for the pharmaceutical industry.

1. Introduction Zanthoxylum bungeanum Maxim. is a member of the family Rutaceae. It is an aromatic plant well known for both its medicinal and food properties (J. Li et al., 2015; P. Li et al., 2015; Xiang et al., 2016). It is widely distributed in China and southeast Asia due to its excellent tolerance of adverse conditions (Zhang et al., 2016a, b). The pericarp, the outer part of the fruit, is often used as a food additive because of its distinctive flavor (Bader et al., 2014; Yang, 2008). It is also used as a traditional herbal medicine to treat a range of diseases (Li et al., 2016; Wu et al., 2015). There are a number of reports on the quantitative

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determination of the chemical components of Z. bungeanum pericarp extracts which include essential oils (Lan et al., 2014; Li et al., 2016), polysaccharides (J. Li et al., 2015; P. Li et al., 2015) and alkylamides (Huang et al., 2012). These extracts show antioxidant (J. Li et al., 2015; P. Li et al., 2015), antifeedant (Wang et al., 2015), anti-inflammatory (Zhang et al., 2016a, b) and anticancer activities (Li et al., 2016). Consumers and producers are showing growing interest in the potential nutritional and health-promoting properties of extracts of Z. bungeanum pericarps. These are important primary products already in use in a wide range of products and are of relatively high economic value. Thus, Z. bungeanum pericarps are considered a natural source of food

Corresponding author. E-mail addresses: [email protected] (Y. Ma), [email protected] (X. Li), [email protected] (L.-X. Hou), [email protected] (A.-Z. Wei).

https://doi.org/10.1016/j.indcrop.2019.111872 Received 17 June 2019; Received in revised form 11 October 2019; Accepted 15 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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52AA rotary evaporator, Yarong Biochemical Instrument Factory, Shanghai, China) under reduced pressure (SHB-IIIA multipurpose water circulating vacuum pump, Great Wall Science & Trade Co. Ltd., Zhengzhou, China). Then the mixture was transferred to a beaker and dried to obtain the crude extract. The yield of the extract was calculated as {(W1/W2) × 100}, where W1 is the weight of the crude extract after evaporation and dry, and W2 is the dry weight of the dry pericarp sample. The extracts were then stored in colored vials at 4 °C pending further analysis.

additives, medicines and cosmetics. Extraction is the usual first step in obtaining biological components from a raw plant material. However, the components obtained are affected by the extraction solvent (Abd Hamid et al., 2017), or extraction temperature (Efthymiopoulos et al., 2018) or extraction technique (Rafińska et al., 2019). Among these factors, the nature of the solvent is considered the most critical. Organic solvents such as methanol, ethanol, acetone and ethylene glycol and their aqueous solutions are the most common ones used (Metrouh-Amir et al., 2015; Miao et al., 2018; Pintać et al., 2018). However, due to the varied physical and chemical natures of the components present in a range of plant materials, it is often unclear which solvent is most effective. We are unaware of any information on solvent effects on the compositions and biological activities of extracts from Z. bungeanum pericarps. Most previous studies on Z. bungeanum used a single solvent for extraction and compared the range of extract components available from samples taken from different tree varieties, and sourced from different geographical locations. Here, we undertake a comparative investigation of the yields, compositions (phenols, alkaloids and coumarins) and biological activities (in vitro antioxidant, antimicrobial, cholinesterase inhibitory and anticancer) of extracts of a range of Z. bungeanum pericarp samples. We then determine relationships between the extract components and their biological activities. The results provide new information on the potential utility of Z. bungeanum pericarps as a natural source of foods and medicines.

2.3. Determination of total phenolics, flavonoids and alkaloids The total phenolic content (TPC) of the extracts was estimated using Folin–Ciocalteu reagent (Metrouh-Amir et al., 2015; Miao et al., 2018; Pintać et al., 2018). Briefly, 0.5 mL of Folin–Ciocalteu reagent (diluted two-fold in distilled water) was added to the extract, followed by 1.5 mL of saturated Na2CO3 solution. The volume of the reaction mixture was then brought to 10 mL with distilled water. Absorbance was measured at 765 nm using an ultraviolet–visible (UV–Vis) spectrophotometer (UV-1700, Shimadzu Corp., Kyoto, Japan) after incubation for 60 min at room temperature. The TPC was expressed as gallic acid equivalents (g GAE/kg extract). The total flavonoid content (TFC) of the extracts was measured using an ultraviolet–visible (UV–Vis) spectrophotometer (Peña-Cerda et al., 2017). The reaction mixture was prepared by mixing 1.0 mL of working solution, 4.0 mL of 60% ethanol, 0.3 mL of 5% NaNO2 (stand for 5 min), 0.3 mL of 10% Al(NO3)3•9H2O (stand for 6 min) and 4 mL of 4% NaOH. The volume was brought to 10 mL with distilled water and the absorbance was measured at 510 nm. The TFC in each sample was determined from the standard rutin curve and is presented as rutin equivalents (g RE/kg extract). The total alkaloid content (TAC) of the extracts was measured using the acid dye colorimetric method described previously by Tian et al. (2018a, b) with slight modification. The extracts were dissolved in 2.5 mL of methanol and filtered. This solution was transferred to a separating funnel, 2 mL of bromocresol green solution and 2 mL of phosphate buffer were added. The mixture was shaken with 10 mL of chloroform by vigorous shaking and collected in a 25 mL volumetric flask. A series of reference standard solutions of chelerythrine was prepared in the same manner as described earlier. The absorbances for test and standard solutions were determined against the reagent blank at 470 nm with an UV–vis spectrophotometer. The TAC was expressed as mg of chelerythrine equivalents per gram (mg CE /g extract).

2. Materials and methods 2.1. Materials and chemicals The Z. bungeanum was grown in the field at the Research Centre for Engineering and Technology of Zanthoxylum, State Forestry Administration, Northwest A&F University, Fengxian, Shaanxi Province, China. The pericarps were harvested by hand when the fruits were mature and were dried at 40 °C for 18 h in a ventilated oven (YiHeng, DHG-9140A, Shanghai Yiheng Scientific Instruments Co., Ltd.) before being powdered in a disintegrator/grinder (FW-100 Test Instrument Co., Ltd., Tianjin, China). The powder was passed through a 40-mesh sieve (average particle diameter of 0.45 mm) and stored in a desiccator pending analysis. All reagents used in the extractions were of analytical grade, while solvents used in the HPLC analyses were of HPLC grade. Methanol, ethanol, acetic acid, ethyl acetate, chloroform, benzene, sodium carbonate anhydrous (Na2CO3), aluminum nitrate nonahydrate [Al (NO3)3•9H2O], iron(II) sulfate heptahydrate (FeSO4•7H2O), potassium ferrocyanate [K3Fe(CN)6], and phosphate buffer saline (PBS) were purchased from Guanghua Sci-Tech Co., Ltd. (Guangdong, China). Ascorbic acid, Folin–Ciocalteau reagent, acetylthiocholine iodide (ATCI) and S-butyrylthiocholine iodide (BTCI) were purchased from Solario Sci-Tech Co., Ltd. (Beijing, China). Phenolic acid, flavonoid, alkaloids, and coumarins standards (canadine, rutaecarpine, ethoxychelerythrin, dicoumarolum, warfarin, p-coumaric acid, guajavarin, quercetin, quercetin-3-O-glucuronide, hyperoside, rutin, epicatechin, procyanidin B1, procyanidin B2, chlorogenic acid, chelerythrine, catechin, and chelerythrine), and bacterial strains (Staphylococcus aureus and Candida albicans) were purchased from Beina Chuanglian Biotechnology Institute (Beijing, China).

2.4. Identification and quantification of chemical components Chemical components including phenolics, flavonoids, alkaloids and coumarins in the extracts were determined using an HPLC system (Waters, Milford, MA, USA) equipped with a Waters 2998 photodiode matrix detector, a Waters 1525 high-precision pump, a Waters SunFire C18 column (250 mm × 4.6 mm, 5 μm) and an automatic sample injector (Waters 2707). The HPLC gradient system was operated at 30 °C with a flow rate of 1.0 mL/min. The sample solution (10 μL) was injected into the HPLC system. Two solvents were used to separate the components. The mobile phase was composed of solvent (A) containing 99.99% (w/v) acetonitrile and solvent (B) containing 1% (w/v) formic acid with a flow rate of 1 mL/ min using the following program: linear 0–25.0 min 5%–14% A; linear 25.0–45 min 25.0% A; linear 45–55 min 40% A; linear 55–65 min 60% A; linear 65–66 min 5% A; and isocratic 66–80 min 5% A. The authentic standards were monitored simultaneously at the corresponding wavelengths, and peaks were identified by comparison of retention times with authentic standards. Quantification was carried out using the external standard method with five-point calibration curves for each standard based on peak height. The results were expressed as milligrams of compound per 100 g pericarps (mg/100 g

2.2. Preparation of extracts Samples of dry pericarp powder (5 g) were percolated at room temperature for 24 h with 200 mL of solvent: water, methanol, ethanol, acetic acid, ethyl acetate, chloroform or benzene. This was done in an ultrasound bath for the first hour. This process was repeated three times. The extracts were filtered through quantitative filter papers (202, Xinhua Paper Industry Co. Ltd., Hangzhou, China) and evaporated (RE2

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mixture (S) was composed of 140.0 μL PBS (0.1 M, pH 7.8), 20.0 μL of extract solution, and 20.0 μL of AChE or BChE solution (0.5 units/mL) was incubated at 25 °C for 15 min. Thereafter, the reaction was initiated with the addition of 10.0 μL of DTNB and 10.0 μL of acetylthiocholine iodide ATCI or BTCI (7.5 mM). The absorbance was measured at 412 nm using a Tecan Sunrise SN microplate reader equipped by XFluor4 software after 37 °C for 30 min. Mixture without AChE (BChE) solution was used as a blank and that without extract as a negative control (E). Huperzine was used as a positive control. The percentage of activity inhibition was determined as: Activity inhibition rate (%) = (1 − S/E) × 100

pericarps). 2.5. Evaluation of in vitro antioxidant activity 2.5.1. Reducing power assay (RP) The RP of extracts was determined using the method of Gunathilake and Ranaweera (2016). The extract (50 μL) was added to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of K3Fe(CN)6 (1%). After 20 min of incubation at 50 °C, 2.5 mL of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 5000 r/min for 10 min. The supernatant was collected and mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride (0.1%). The absorbance, which is correlated with RP, was calculated at 700 nm. Ascorbic acid was used as a positive control. To compare the RPs of extracts, the required extract concentration for the reaction system was 0.6 mg/mL.

2.8. Determination of cytotoxicity The 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate cytotoxicity in HepG2 cells (Liang et al., 2019). A volume of 100 μL of HepG2 cells in logarithmic growth phase were seeded on a 96-well plate (7 × 103 cells/well) for 12 h incubation. Subsequently, the cells were cultured in DMEM medium as blank control (B) and pericarp extracts with a series of nine concentrations (0.2–1.4 mg/ mL) for 24 h. After washing with PBS twice, 20 μL of MTT (0.5 mg/mL) was added to each well and the cells then subjected to an additional 4 h incubation. The formazan crystals formed by active cells were dissolved in 150 μL of DMSO and the sample absorbance (A) was read at 450 nm. Cell growth inhibition rate was calculated as:

2.5.2. OH scavenging activity assay The OH scavenging activity was determined according to Ding et al. (2017) with slight modification. Briefly, 0.6 mL of the sample solution at different concentrations was added followed by 0.6 mL of 0.01% H2O2 and 0.6 mL of 5 mM FeSO4. The mixture was incubated at 37 °C in the dark for 15 min and then mixed with 3.0 mL distilled water. The absorbance of the mixture was measured at 510 nm. Ascorbic acid was used as a positive control. 2.5.3. Ferric reducing ability of plasma assay(FRAP) The total antioxidant power of the extracts to reduce the ferric tripyridyltriazine (Fe(III)-TPTZ) complex to ferrous tripyridyltriazine (Fe (II)-TPTZ) at low pH was evaluated according to Alimpić et al. (2017) with slight modification. Freshly prepared FRAP reagent was composed of sodium acetate buffer (3 mol/L, pH 3.6), a solution of TPTZ (0.1 mol/ L) in HCl (0.4 mol/L) and FeCl3·6H2O solution (0.2 mol/L) in the proportions 10:1:1 (v/v/v). The working FRAP solution was heated to 37 °C prior use. Extract (380 μL) was added to 3.62 mL of working FRAP reagent and incubated at room temperature for 5 min. The absorbance of the reaction mixture was then measured spectrophotometrically at 593 nm. Ascorbic acid was used as a positive control. FRAP values were calculated from the standard curve equation and expressed as the concentration of FeSO4·7H2O (g/mL). To compare the FRAPs of extracts, the required extract concentration for the reaction system was 0.6 mg/mL.

Cell growth inhibition rate = (1 − A/B) × 100

2.9. Statistical analyses Data are reported as means ± standard deviations of triplicate measurements. Basic statistical analyses were carried out using Excel 2010. Principal component analysis (PCA) was use to visualize the data structure and to classify samples using the Canoco 5 program. One-way analysis and the test of S-N-K(s) with confidence interval of 0.05 were used to compare means. The means were considered not significantly different for (p > 0.05) and significantly different for (p < 0.05). 3. Results and discussion

2.6. Determination of antimicrobial activity

3.1. Yield, total phenolic, flavonoid and alkaloid content of extracts

The bacterium Staphylococcus aureus (ATCC 25923) and the fungus Candida albican (ATCC 90028) were selected for the antimicrobial activity assay. The cultures of the strains were maintained in their appropriate agar slants at 4 °C throughout the study. All strains were subcultured at 37 °C for 24 h on nutrient agar slants prior to culturing them in nutrient broth overnight. The density of the strains was adjusted with de-ionized water to approximately 1.0 × 106 CFU/mL. The antimicrobial activity assay was carried out according to Moradi et al. (2016) using the disc diffusion method with slight modification. Briefly, uniform microbial lawns were made using 50-μL inocula on a nutrient agar plate. Filter paper discs (5.0 mm) soaked with test extracts and ventilated-oven dried. The dry filter paper discs were placed over seeded plates and incubated at 37 °C for 24 h. Activity was observed in terms of the diameter (mm) of the inhibition. The filter paper discs (without extracts) served as negative controls. All tests were carried out in triplicate.

As expected, the yields, total phenolic, flavonoid and alkaloid contents of Z. bungeanum extracts were affected by the extraction solvent used (Table 1). The highest yield was with acetic acid extract (24.69%), while lowest was with water (7.54%). The total phenolics ranged from 56.46 to 81.19 mg GAE/g of extract indicating Z. bungeanum pericarps are a good source of polyphenols. The methanol extract (110.69 ± 8.49 mg RE/g extract) had the highest flavonoid content, whereas the benzene extract (22.42 ± 2.1 mg RE/g extract) had the lowest. The ethyl acetate extract (57.44 ± 4.57 mg CE/g extract) had the highest alkaloid content and the water extract (11.01 ± 1.43 mg CE /g extract) had the lowest. Due to the presence of complex ingredients in materials, it is difficult to quantify each component separately (Dong et al., 2015). The amounts of phenolic, flavonoid, and alkaloid compounds extracted are affected by the polarity of the solvent and also by the solubility of those components in the solvent (Alimpić et al., 2015). The extracts of phenolic, flavonoid and alkaloid compounds include a range of other substances including proteins, polysaccharides, terpenes, chlorophyll, lipids and inorganic compounds (Drosou et al., 2015). Thus, the relative amounts of total phenolic, flavonoid and alkaloid compounds in the different solvents did not necessarily correlate with the yields. It has been shown that the yields and contents of total phenolic, flavonoid,

2.7. Determination of cholinesterase inhibitory activity Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were selected to evaluate the level of cholinesterase inhibition using 96-well plates according to the method of Liu et al. (2017). The reaction 3

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Table 1 Effect of solvent on yield, total phenolics, total flavonoids and total alkaloids in extracts of Zanthoxylum bungeanum pericarps. Extract

Relative polarity

Yield (%)

Total phenolic (mg GAE/g extract)

Total flavonoid (mg RE/g extract)

Total alkaloid (mg CE/g extract)

Water Methanol Acetic acid Chloroform Ethanol Ethyl acetate Benzene

10.2 6.6 6.2 4.4 4.3 4.3 3.0

12.79 ± 1.56c 20.25 ± 1.17b 24.69 ± 0.94a 10.23 ± 0.15 cd 9.90 ± 0.40 cd 9.84 ± 0.21 cd 7.54 ± 0.64d

56.46 77.16 65.46 62.90 81.19 64.33 81.14

45.82 ± 4.28c 110.69 ± 8.49a 44.49 ± 3.07c 28.24 ± 2.14d 83.29 ± 5.14b 41.01 ± 3.49c 22.42 ± 2.17d

11.01 13.39 29.09 33.05 27.25 57.44 27.39

± ± ± ± ± ± ±

3.91d 5.63b 3.21c 3.93c 4.81a 5.74c 4.85a

± ± ± ± ± ± ±

1.43b 2.21b 3.35ab 2.78ab 2.89ab 4.57a 2.81ab

Results are means ± standard deviations of three independent measurements. Different letters in a column indicate significant difference (p < 0.01); GAE, gallic acid equivalents; RE, rutin equivalents; CE, chelerythrine equivalents.

of the components, followed by benzene (13), ethanol (12), chloroform (11) and methanol (10). Water and ethanol extracts showed certain similarities with regard to the types and contents of components. The efficiency of extraction did not rise with increasing solvent polarity as reported by Akkol et al. (2008) and Lim et al. (2019). Present in all extracts were procyanidin B1 (125.35–350.98 mg/100 g pericarps), quercetin-3-O-glucuronide (0.31–0.47 mg/100 g pericarps), quercetin (0.54–0.69 mg/100 g pericarps), hyperoside (0.04–14.90 mg/100 g pericarps), chlorogenic acid (0.03–0.10 mg/100 g pericarps), warfarin (0.97–15.33 mg/100 g pericarps) and tetrahydroberberine (1.09–2.51 mg/100 g pericarps). Procyanidin B1 was the most abundant component. Rutin was not found in the water, methanol or ethanol extracts, although it has been reported as readily extracted by these solvents in some other research (Shi et al., 2013). Our result was similar that of Sepahpour et al. (2018) who reported that 80% acetone extracts of turmeric contained large quantities of rutin. Although flavonoids with sugar moieties (flavonoid glycosides) have been reported to be more soluble in water than in other solvents, the contents of quercetin3-O-α-L-arabinoside and quercetin-3-O-glucuronide in our water extract were no greater than in the other solvents. The content of dicoumarol (22.10–100.82 mg/100 g pericarps) was quite high in most extracts but was not detectable in acetic acid. Therefore, the high extract yield in acetic acid may be attributed to unknown components that are extracted only in acetic acid. In contrast, the absence of ethoxychelerythrin in the water extract (0.26 mg/100 g pericarps) - a water-soluble alkaloid reported to ameliorate acute cardiac allograft rejection (Zhang et al., 2016a, b) - could be due to the extract polarity

and alkaloid substances in plant extracts increase with increasing polarity of the solvent (Akkol et al., 2008; Lim et al., 2019). However, for our Z. bungeanum pericarp extracts this relationship was not perfect. Thus, water (12.79%) was not a much more efficient extraction solvent than methanol (20.25%) or acetic acid (24.69%). Polar solvents, such as acetic acid, methanol and water showed higher yields than the less polar and non-polar solvents (9.90%, 9.84%, and 7.54% for ethanol, ethyl acetate, and benzene, respectively). In contrast, total phenolic content (56.46 mg GAE/g extract), total flavonoid content (45.82 mg RE/g extract) and total alkaloid content (mg CE/g extract) in extracted in water were lower than in alcohol, while total phenolic content in less or non-polar solvents was higher than in the polar solvents. A mixture of solvents is reported to be more effective for the extraction of these bioactive components than mono-solvent systems (Alcantara et al., 2019; Kim et al., 2013; Sukadeetad et al., 2018). Overall, most components in Z. bungeanum pericarps were not readily soluble in water and were of a less polar or non-polar nature.

3.2. Chemical components of extracts The identification of all peaks appearing in the HPLC outputs was impossible due to limited availability of commercial standards. Our analyses here focused on the four main groups found in Z. bungeanum; namely flavonoids, phenolic acids, coumarins and alkaloids. These results are summarized in Table 2. The types and contents of extracted compounds depended on solvent. Water (14), acetic acid (14) and ethyl acetate (14) extracted most

Table 2 Chemical components in various extracts of Zanthoxylum bungeanum pericarps. (mg/100 g pericarps). Compound name Flavanones Procyanidin B1 Procyanidin B2 Catechin L-Epicatechin Flavonols Quercetin-3-O-glucuronide Quercetin Rutin Hyperoside Quercetin-3 -O-α-L-arabinoside Phenolic acids Chlorogenic acid p- coumaric acid Coumarins Warfarin Dicoumarol Alkaloids Tetrahydroberberine Ethoxychelerythrin Rutecarpine

Water

Methanol

Acetic acid

Chloroform

Ethanol

Ethyl acetate

Benzene

307.33 ± 12.45b 1.88 ± 0.12b 0.52 ± 0.04b 0.28 ± 0.02c

278.58 ± 6.56c – – –

150.40 ± 8.24e 4.80 ± 0.25a 0.69 ± 0.52a 0.23 ± 0.02d

159.00 ± 8.67d – – –

350.98 ± 10.28a 0.38 ± 0.21d 0.31 ± 0.03c 0.21 ± 0.02d

301.41 ± 15.32b 0.62 ± 0.05c 0.70 ± 0.06a 0.55 ± 0.04a

125.35 ± 8.45f 0.31 ± 0.02d – 0.40 ± 0.03b

0.37 ± 0.03c 0.54 ± 0.04e – 0.07 ± 0.01f –

0.32 ± 0.02d 0.55 ± 0.04e – 0.17 ± 0.01e –

0.31 0.69 0.54 0.42 0.23

0.37 0.59 0.29 1.47 0.26

0.25c 0.06c 0.02c 0.15c 0.03c

0.33 ± 0.03d 0.54 ± 0.04e – 0.04 ± 0.00f –

0.47 0.63 – 6.51 0.75

0.40 ± 0.03b 0.57 ± 0.02d 5.38 ± 0.45a 14.90 ± 0.52a 0.35 ± 0.02b

0.03 ± 0.00d 0.75 ± 0.05b

0.03 ± 0.00d 0.79 ± 0.05a

0.10 ± 0.01a 0.75 ± 0.06b

0.05 ± 0.00c –

0.03 ± 0.00d 0.77 ± 0.06b

0.05 ± 0.00c 0.76 ± 0.03b

0.06 ± 0.00b –

2.14 ± 0.10d 74.34 ± 5.48c

4.41 ± 0.32b 85.47 ± 4.32b

0.97 ± 0.08e –

15.33 ± 1.35a 35.24 ± 2.85e

3.45 ± 0.21c 100.82 ± 5.23a

2.10 ± 0.15d 53.89 ± 2.85d

1.11 ± 0.10e 22.10 ± 1.86f

1.98 ± 0.15c 0.26 ± 0.03 5.26 ± 0.04e

1.88 ± 0.09d – 21.52 ± 1.85a

1.09 ± 0.55f – 7.99 ± 0.42c

1.40 ± 0.15e – 3.69 ± 0.25f

2.51 ± 0.18a – –

2.24 ± 0.25b – 15.94 ± 1.35b

1.11 ± 0.10f – 5.53 ± 0.05d

± ± ± ± ±

0.03d 0.05a 0.05b 0.02d 0.02c

± ± ± ± ±

± 0.03a ± 0.05b ± 0.32b ± 0.05a

Results are means ± standard deviations of three independent measurements; - component is below the measurement threshold; Different letters in a row indicate a significant difference (p < 0.01). 4

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Fig. 1. Antioxidant activities of various extracts of Zanthoxylum bungeanum pericarps. OH, OH free radical scavenging activity; FRAP, ferric reducing ability of plasma; RP, reducing power.

3.4. Antimicrobial activity of extracts

and solubility in the solvent.

C. albicans is an important human pathogenic fungus, while S. aureus is a gram-positive bacterium. Both species can cause serious infections and are also often used to test antimicrobial activity (Pereira et al., 2015). The antimicrobial activity assays show the growth of both C. albicans and S. aureus were inhibited by all extracts (Table 3). For inhibition of S. aureus growth was greater with the methanol and acetic acid extracts than with water, ethanol, ethyl acetate, chloroform or benzene. The chloroform extract showed highest antimicrobial activity against C. albicans with the highest inhibition diameter at all concentrations. The antimicrobial activity for C. albicans was similar to that for Mentha piperita (Pereira et al., 2015). The antimicrobial activities of the extracts may be linked to the presence of phenolic acids such as gallic, protocatechuic, 3-hydroxybenzoic and chlorogenic acids and of flavonoids (Pereira et al., 2015; Sepahpour et al. 2018). Variations between our results and those reported by others may be attributable to factors such as the choice of microbial strain or the incubation conditions (time, temperature, oxygen levels etc.) and the methods used to determine the in vitro antibacterial activity (Ben Yakoub et al., 2018; Da Silva Andrade et al., 2018; Radenkovs et al., 2018; Tian et al., 2018a, b).

3.3. Antioxidant activity of extracts The antioxidant activities of Z. bungeanum pericarps extracts varied among the solvents and increased with extract concentration (Fig. 1). Radical scavenging activity can be expressed as the half-maximal inhibitory concentration (IC50). A lower IC50 value of OH indicates a higher antioxidant activity, as do higher RP0.6 mg/mL and FRAP0.6 mg/mL values. The water extract exhibited the highest antioxidant activity with the lowest IC50 for OH (0.45 mg/mL), the highest RP (RP0.6 mg/ mL = 3.93) and FRAP (FRAP0.6 mg/mL = 0.48). The RP assay results correspond in part with solvent polarity. The ethyl acetate extract had the highest IC50 values (5.63 mg/mL for OH), while that with benzene was lowest RP (RP0.6 mg/mL = 0.32) and FRAP (FRAP0.6 mg/mL = 0.06). These levels were markedly lower than those of synthetic antioxidants such as ascorbic acid (used in the present work) and trolox (Peña-Cerda et al., 2017; Perera et al., 2016). The differences in antioxidant activity are likely due to other (unknown) components of the extracts and their interactions (Alimpić et al., 2015; Drosou et al., 2015; Rizhikovs et al., 2015). Some researchers have reported significant relationships between antioxidant activity and total phenolic or flavonoid content in plant extracts (Joshi et al., 2015), whereas others have found no such correlations (Joshi et al., 2015). The antioxidant activities of plant extracts may be affected by the extraction procedures along with the source environment, cultivar, time of year and the physiological age of the source plant (Dong et al., 2015; Pereira et al., 2015).

3.5. Cholinesterase inhibitory activity of extracts We also examined the ability of the various extracts to inhibit AChE and BChE (Table 4). In the AChE inhibition assay, the activity ranking was huperzine-A (0.134 mg/mL) > acetic acid extract (0.98 mg/ mL) > methanol/ ethyl acetate extract (1.02 mg/mL) > benzene 5

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Table 3 Antimicrobial activities of different extracts of Zanthoxylum bungeanum pericarps. 0.5 (mg/mL) Staphylococcus Water Methanol Acetic acid Chloroform Ethanol Ethyl acetate Benzene Candida Water Methanol Acetic acid Chloroform Ethanol Ethyl acetate Benzene

aureus 8.03 ± 8.24 ± 8.71 ± 7.85 ± 7.95 ± 7.88 ± 7.90 ± albicans 7.87 ± 7.95 ± 7.80 ± 9.29 ± 7.53 ± 8.06 ± 8.87 ±

0.8 (mg/mL)

1.1 (mg/mL)

1.4 (mg/mL)

1.7 (mg/mL)

0.21b 0.13b 0.45a 0.35b 0.26b 0.34b 0.33b

8.76 ± 0.36d 11.44 ± 0.15a 11.01 ± 0.29b 8.57 ± 0.06d 9.25 ± 0.20c 8.51 ± 0.27d 8.74 ± 0.32d

10.55 ± 0.43d 13.31 ± 0.26b 14.34 ± 0.56a 10.08 ± 0.28de 11.39 ± 0.35c 9.69 ± 0.33e 10.00 ± 0.38e

11.36 14.57 15.36 12.54 15.21 11.36 11.17

± ± ± ± ± ± ±

0.37d 0.36b 0.34a 0.18c 0.44a 0.25d 0.13d

12.61 17.06 18.13 13.61 16.92 12.22 12.25

± ± ± ± ± ± ±

0.59d 0.27b 0.54a 0.38c 0.39b 0.23d 0.27d

0.23b 0.55b 0.46b 0.37a 0.53b 0.30b 0.42a

8.90 ± 0.18ef 10.99 ± 0.42b 8.41 ± 0.23f 12.09 ± 0.32a 9.61 ± 0.88d 9.22 ± 0.25de 10.24 ± 0.21c

10.55 12.15 10.02 14.60 11.70 10.82 12.56

11.77 14.07 11.49 16.80 13.17 12.08 14.82

± ± ± ± ± ± ±

0.25ef 0.60c 0.04f 0.46a 0.21d 0.13e 0.58b

13.01 16.16 12.63 19.46 15.25 13.48 16.64

± ± ± ± ± ± ±

0.25de 0.53b 0.12d 0.61a 0.42c 0.47e 0.68b

± ± ± ± ± ± ±

0.50de 0.19bc 0.33e 0.44a 0.54c 0.66d 0.56b

Results are means ± standard deviations of three independent measurements; Different letters in a row indicate a significant difference (p < 0.01). Table 4 Inhibitory activity of various extracts of Zanthoxylum bungeanum pericarps on cholinesterase. Extract

Water Methanol Acetic acid Chloroform Ethanol Ethyl acetate Benzene Huperzine-A

IC50(mg/ml)

SI

AChE

BChE

2.94 ± 0.23a 1.02 ± 0.08d 0.98 ± 0.05d 1.28 ± 0.12b 1.17 ± 0.06c 1.02 ± 0.11d 1.14 ± 0.11c 0.134 ± 0.01e

2.17 0.28 1.41 2.08 1.37 2.39 2.21 0.19

± ± ± ± ± ± ± ±

IC50BChE/IC50AChE 0.02b 0.05e 0.18d 0.35c 0.13d 0.14a 0.17b 0.03f

0.74 0.28 1.43 1.62 1.17 2.33 1.93 1.41

Different letters in a column indicate a significant difference (p < 0.01); SI, selectivity index; IC50, half-maximal inhibitory concentration; AChE, acetylcholinesterase; BChE, butyrylcholinesterase. Fig. 2. Inhibition of growth of HepG2 by various extracts of Zanthoxylum bungeanum pericarps. IC50, half-maximal inhibitory concentration.

extract (1.14 mg/mL) > ethanol extract (1.17 mg/mL) > chloroform extract (1.28 mg/mL) > water extract (2.94 mg/mL). This contrasted with the BChE inhibition assay where the ranking was huperzine-A (0.19 mg/mL) > methanol (0.28 mg/mL) > ethanol (1.37 mg/mL) > acetic acid (1.41 mg/mL) > chloroform (2.08 mg/mL) > water (2.17 mg/mL) > benzene (2.21 mg/mL) > ethyl acetate (2.39 mg/mL). Interestingly, the inhibitory activities of extracts using the strong polar solvents, water and methanol, on BChE were stronger than on AChE with SI values less than one (0.74, and 0.28, respectively). Meanwhile, the less polar and non-polar solvent extracts showed weaker inhibitory activities to BChE than to AChE. This indicates strong polar components are optimal for AChE inhibition properties, while the less polar and non-polar solvent extracts were more active stem nucleus for selective inhibition agents on BChE. A linear relationship between AChE and BChE inhibitory activities is noted and has previously been reported in a number of studies (Aghraz et al., 2018; Liu et al., 2017). Many investigations have shown that various Zanthoxylum oils are strong inhibitors of AChE and BChE (Chakira et al., 2017; Hieu et al., 2012), while Murray et al. (2013) showed a strong inhibition of other plant extracts. Clearly, the extracts using a range of solvents are able to inhibit AChE and BChE indicating there are significant active components present in Z. bungeanum pericarps.

benzene extract (0.452 mg/mL) > ethanol extract (0.500 mg/mL) > acetic acid extract (0.527 mg/mL) > ethyl acetate extract (0.549 mg/ mL) > methanol extract (0.696 mg/mL) > water extract (0.699 mg/ mL). This is based on the IC50 of cytotoxicity, and indicates that less polar and non-polar solvents were best for inhibiting HepG2 cell growth. Many investigations have shown that Zanthoxylum pericarp extracts inhibit HepG2 cell growth. The essential oils and sanshool of Zanthoxylum pericarp can induce apoptosis of HepG2 through increased production of reactive oxygen species (Paik et al., 2005; You et al., 2015). The n-butanol extract fraction from Z. bungeanum pericarps most affects cholesterol metabolism in HepG2 cells (Wu et al., 2015). The variation in HepG2 cell growth inhibition rate was affected by chemical composition and component content in different extracts. 3.7. Relationship between phytochemical component and biological activity The largest value (0.9) of lengths of the gradient axes was obtained from the model based on the data for biological activity. So principal component analysis (PCA) was used to analyze the relationship between phytochemical component and biological activity. Interactive forward selection was used to filter phytochemical component because the coordination model was unconstrained (Hou et al., 2019). After filtering, total phenolic content, total flavonoid content, total alkaloid content, quercetin-3-O-glucuronide, hyperoside and warfarin were chosen as variables in the model. As shown in Fig. 3, all phytochemical components can explain

3.6. Cytotoxicity of extracts The cytotoxicity of the various extracts was evaluated by inhibiting HepG2 cell growth. HepG2 growth was inhibited by all extracts (Fig. 2). The cytotoxicity ranking was chloroform extract (0.390 mg/mL) > 6

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HepG2. The inhibitory activities of the water and methanol extracts on BChE were stronger than on AChE, while methanol was the most effective solvent for cholinesterase inhibitory activity. Overall, total phenolic content, total flavonoid content, total alkaloid content and warfarin content were significantly correlated with the first two PCA axes, indicating some relationships between phytochemical component and biological activity. The total phenolic, total alkaloid and warfarin were key factors influencing the biological activity of pericarp extracts. Extracts of Z. bungeanum pericarps are effective sources of natural compounds with significant and useful biological activities for the pharmaceutical industry. Ongoing work seeks to identify further components present in these Z. bungeanum pericarps extracts. Particularly, those components that may interfere with the activities of the useful ones. It also seeks to better understand the sources of natural variation in the levels of these bioactive components with a view to increasing their production. It is also hoped that new chemical components will be discovered that will lead to formulation of new and more potent pharmaceutical products. Declaration of Competing Interest The authors declare no competing interests.

Fig. 3. Ordination plots of the principal component analysis identifying relationships between various phytochemical components and their biological activities. WE, water extract; ME, methanol extract; AAE, acetic acid extract; CE, chloroform extract; EE, ethanol extract; EAE, ethyl acetate extract; BE, benzene extract; TFC, total flavonoid content; TPC, total phenolic content; TAC, total alkaloid content; W, warfarin; H, hyperoside; Q-3-O-g, Quercetin-3-Oglucuronide; S, Staphylococcus aureus; C, Candida albicans; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; OH, OH scavenging activity; RP, reducing power, FRAP, total antioxidant power; HePG2, HepG2 inhibition rate.

Acknowledgments This study received financial support from the National Key Research and Development Program Project Funding (2018YFD1000605) and the Research Centre for Engineering and Technology of Zanthoxylum, State Forestry Administration, Northwest A&F University [grant numbers 45,2016]. The support of all staff in the research center is gratefully acknowledged.

99.63% of the total variance of the various pericarp extracts. The explanation proportions for the first two PCA axes were 99.27% and 0.36%, respectively, representing the main relationship between phytochemical component and biological activity. The lengths of the phytochemical component arrows represent the effects on biological activity. Total phenolic content, total flavonoid content, total alkaloid content and warfarin content with the contributions of 45.9%, 4.3%, 37.6% and 11.3%, respectively, were significantly correlated with the first two PCA axes (p < 0.1). Therefore, our observations confirm that biological activity of pericarp extracts is influenced by total phenolic, total alkaloid and warfarin. The distance between the points can represent the relationship between samples, while the cosine values represent the relationships between two variables (Bagheri Bodaghabadi et al., 2019). Ordination plots show the chloroform extract, ethanol extract, ethyl acetate extract and benzene extract were adjacent. In addition, phytochemical component and biological activity represent positive or negative correlation according to the cosines of the two variables. This indicates total alkaloid content, hyperoxide and warfarin increased antioxidant activity for FRAP and RP, and reduced HepG2 growth. Moreover, warfarin increased AChE inhibitory activity, while total flavonoid increased BChE inhibitory activity.

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