Characterization, antioxidant and antitumor activities of phenolic compounds from Amomum villosum Lour.

Amomum villosum Lour. (A. villosum), known as Sharen in China, is widely used for culinary and medicinal purposes due to containing a diverse set of bioactive compounds. In this study, the optimum ethanol extraction process was optimized and the composition and biological activities (antioxidant and antitumor) of five different fractions (dichloromethane, petroleum ether, ethyl acetate, n-butanol and H2O) extracted from the ethanol extract of A. villosum were investigated. The results showed that the optimal extraction conditions were extraction temperature 80°C, extraction time 120 min, ethanol concentration 40% and solid–liquid ratio 1:25 g/mL. Moreover, 35 bioactive compounds were successfully identified by UPLC-ESI-QTOF-MS/MS from five factions for the first time, including 12 phenolic acids and derivatives, 2 organic acids, 12 flavonoids and derivatives, 2 oxylipins and 7 proanthocyanidins. Among them, ethyl acetate fraction (Fr-EtOAc) exhibited the highest content of total phenolic (374.01 mg GAE/g DW) and flavonoid (93.11 mg RE/g DW), where vanillic acid, catechin, epicatechin and protocatechuic acid were the predominant phenolic compounds that accounting for 81.65% of the quantified bioactive compounds. In addition, Fr-EtOAc demonstrated excellent total antioxidant activity (IC50 of DPPH and ABTS assays were 0.23, 0.08 mg/mL, respectively, and FRAP assay was 322.91 mg VCE/100 g DW) and antitumor activity (1,000 μg/mL, 79.04% inhibition rate). The results could provide guidance for the industrial production and application of A. villosum.


Introduction
Amomum villosum Lour.(A. villosum), usually called sharen in China, is a member of Zingiberaceae family and is mainly cultivated in Southern China and Southeast Asian countries.The fruit of A. villosum was used for medicine purposes could be traced back to the seventh century, and together with Areca catechu L., Morinda officinalis How. and Alpinia oxyphylla Miq. were called "four southern medicines" (1).Nowadays, the fruit of A. villosum is also widely used as a spice in culinary due to its non-toxic, aromatic smell and biological activities (2).There is no doubt that the medicinal and edible values of A. villosum were related to the chemical ingredients, including volatile oils, non-volatile compounds (phenolics, flavonoids, polysaccharides, et al.) and so on (3).Currently, lots of research had focused on the volatile oils and polysaccharides from A. villosum and their pharmacological effects.As previously reported, Tang et al. (4) found that A. villosum volatile oil exhibited antibacterial activity by interfering with the metabolism of methicillin-resistant Staphylococcus aureus, and the report described by Liu et al. (5) revealed that A. villosum polysaccharides could reduce gastric mucosal injury by promoting the level of reactive oxygen species and inflammatory factors.Although there were several studies on the biological activities of the water extract of A. villosum, including absorption characteristics (6), weight loss (7), mitigate hyperlipidemia (8), the composition of phenolic compounds from A. villosum was still unclear, which restricted the further utilization of A. villosum.
Phenolic compounds have a wide range of bioactive properties, including antioxidant (9,10), antitumor (11) and other properties.Meanwhile, an increasing number of reports had proved that biological activities were related to the composition of phenolic compounds (12, 13).Therefore, it was very important to take suitable methods for identifying and quantifying the phenolic compounds in plants.Notably, isolation was an essential procedure to enrich the phenolic compounds.And, fractional extraction, as an efficient isolation method, had been utilized to enrich and isolate the target natural compounds by many researchers (13-15).However, there was no report on the systematic study of phenolic compounds from A. villosum, such as phenolic compounds composition, antioxidant activities and their correlation.
Based on the aforementioned, this study focused on optimizing the extraction conditions of phenolic compounds in the seeds of A. villosum and obtaining five fractions from the ethanol extract by fractional extraction method with five different polarities solvents, and then identifying and quantifying the phenolic compounds composition by UHPLC-ESI-QTOF-MS/MS.Moreover, the antioxidant and antitumor activities were determined and the correlation with phenolic compounds was evaluated by Person correlation analysis.These results could provide guidance for the development and application of A. villosum.

Preparation and extraction of phenolic compounds from Amomum villosum
The seeds of A. villosum were washed with distilled water and then placed into a hot air dryer (101-A1, Wuxi marit Co., Ltd., Jiangsu, China) at 50°C until constant weight.The dried seeds were ground to powder (particle size less than 50 μm) using a high-speed grinder (HR-10, Zhejiang harui Co. Ltd., Zhejiang, China).The A. villosum powder was taken into extraction vessel and mixed with a defined solid-to-solvent ratio of ethanol solution, and then extracted at the set temperature and time according to the experimental design (Table 1).After extraction, the extract was separated from the solid by centrifuging at 4500 rpm for 15 min, and the supernatant was collected and concentrated to approximate 50 mL at 50°C under vacuum by a rotary evaporator (RE-2000A, Shanghai yarong Co. Ltd., Shanghai).The concentrated solution was diluted to a total volume of 100 mL with distilled water, and successively extracted using petroleum ether, dichloromethane, ethyl acetate and n-butanol at a ratio of 1:1 (v/v), 3 times.And the extract solutions were condensed to 5 mL, and the organic phase and H 2 O phase extracts were dried by nitrogen blowing concentrator and lyophilizer, respectively.Finally, there were five fractions were prepared from the ethanol extract of A. villosum, including petroleum ether fraction (Fr-PE, 0.47%), dichloromethane fraction (Fr-CH 2 Cl 2 , 0.95%), ethyl acetate fraction (Fr-EtOAc, 0.46%), n-butanol fraction (Fr-nBuOH, 1.95%) and H 2 O fraction (Fr-H 2 O, 10.30%), and the yields were shown in Table 2.The dried samples were collected and stored at-18°C (in a dark condition) for further investigations.A schematic representation of the experiments carried out in this study was shown in Figure 1.

Determination of total phenolic content and total flavonoid content
The TPC and TFC of five fractions from A. villosum were evaluated by the Folin-Ciocalteu method (16) and aluminum chloride colorimetric method (17), respectively.The absorbance values of TPC and TFC assays were read at 750 nm and 510 nm by a UV-Vis spectrophotometer (U-T1810, Beijing puxi Co., Ltd., Beijing, China), respectively.Finally, the results of TPC were quantified as mg gallic acid equivalent (GAE) per gram of dried weight (DW) of five fractions (mg GAE/g DW), while the results of TFC were expressed as mg rutin equivalent (RE) per gram of DW of five fractions (mg RE/g DW).

DPPH• and ABTS + free radical scavenging capacities
The DPPH• and ABTS + free radical scavenging assays were performed according to the previous reports by Zhou et al. (18) and    The schematic diagram of the detailed trial protocol of this study.

( ) (
) DPPH• or ABTS free radical scavenging rate % 1 A / A 100 where A 0 and A 1 refer to the absorbance values of the sample and control, respectively.Finally, the DPPH• and ABTS + free radical scavenging capacities denoted as IC 50 , represented the concentration of the sample when the DPPH• or ABTS + free radical scavenging rate was 50% which was calculated by the fitting curves.

The ferric ion reducing antioxidant power
The FRAP assay was measured according to the method reported by Pourshoaib et al. (20).The absorbance value was measured at 593 nm and the results of FRAP were expressed as mg vitamin C equivalent (VCE) per 100 grams of DW of five fractions (mg VCE/100 g DW).

Cytotoxicity and antitumor activity
To determine the cytotoxicity and antitumor activity of five fractions of A. villosum, the human umbilical vein endothelial (HUVE) cells (Shanghai Meixuan Biotechnology Co., Ltd., Shanghai, China) and HeLa cell lines (Beyotime Institute of Biotechnology, Shanghai, China) were used, respectively.The cells were cultured in DMEM basic medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin at 37°C in an atmosphere with 5% CO 2 (21).When the cell confluence reached ~70%, the cells were then further used for the cytotoxicity and antitumor activity assays.In this study, the cytotoxicity and antitumor activity were analyzed by CCK8 assays based on the method described by Pu et al. (22) with slightly modifications.Briefly, the cells were seeded into 96-well plates at a density of 5 × 10 4 cells/well and allowed to grow at 37°C for 12 h.For determination of the cytotoxicity of five fractions, 10 μL of 100, 500, 1,000 μg/mL of five fractions were added into the wells, respectively, and incubated at 37°C for 48 h followed by 10 μL of CCK8 reagent for 2 h.Subsequently, the absorbance value was measured at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, United States).For determination of the antitumor activity of five fractions, 100, 200, 400, 600, 800, and 1,000 μg/mL of five fractions were evaluated according to the procedure of cytotoxicity assay.Finally, the results of the cytotoxicity and antitumor activity of five fractions were expressed by cell viability (%) and inhibition rate (%), respectively.

UHPLC-ESI-QTOF-MS/MS analysis of phenolic compounds
The phenolic compounds in five fractions were identified using a Ultra-High Performance Liquid Chromatography (UHPLC; 1,290 Infinity, Agilent, United States) equipped with ZORBAX Eclipse Plus C18 (100 mm × 2.1 mm, 1.8 μm, Agilent Technologies, Santa Clara, CA) column and Triple TOF™ 5600 + electrospray time-of-flight high resolution mass (AB Sciex, Foster City, CA, United States).The gradient elution of the mobile phase was conducted using solvent A (water) and B (acetonitrile), respectively, and the process was as follows: 0 min, 99% B; 4 min, 95% B; 23 min, 45% B, 26 min, 10% B, 27 min, 10% B, 28 min, 99% B and hold it for 4 min, flow rate = 0.3 mL/ min, injection volume = 10 μL, column temperature = 40°C.The UHPLC system was coupled to a quadrupole-time-of-flight orthogonal accelerated Q-TOF mass spectrometer equipped with an electrospray ionization source (ESI) and operating parameters as described by Wang et al. (23).Finally, the composition of phenolic compounds from A. villosum was analyzed by matching the database.The standard compounds, calibration lines and method validations for quantification of phenolic compounds from A. villosum were presented in Table 3.The content of phenolic compounds was expressed as mg of phenolic compounds per gram of DW of five fractions (mg/g DW).

Statistical analyses
All experiments were conducted in triplicate and all results were shown as mean value ± standard deviation.The analysis of significant differences was performed at p = 0.05 by Duncan's tests through SPSS Statistic 26.0 (IBM software, United States).The nonlinear polynomial fit of antioxidant activities results and graph drawing were conducted by Origin 9.0 (Origin Lab Co., Northampton, MA, United States).

The extraction of phenolic compounds from Amomum villosum
The yield of phenolic compounds was affected by many extraction factors, such as extraction time, solvent type and temperature, etc.The single-factor experiments in this study were performed at various solid-liquid ratio (g/mL), ethanol concentration (%), temperature (°C) and time (min).Figure 2 presented the results of four singlefactor experiments, and it showed that the TPC value of ethanol concentration, temperature and time experiments exhibited a tendency of increasing first and then decreasing, while the solidliquid ratio experiment showed an increasing trend first and then stabilizing.Considering the cost, energy consumption and other factors, the optimal extraction conditions for single-factor were obtained from the response of TPC to extraction parameters: extraction temperature of 70°C, extraction time of 120 min, ethanol concentration of 50% and solid-liquid ratio of 1:30 g/mL, respectively.
Based on the above results, an orthogonal test (L 9 (3 4 )) was employed to optimize the extraction conditions of phenolic compounds in the seeds of A. villosum, and the results were listed in Table 1.It was shown that the TPC values were in a range of 13.75 ± 0.67 mg GAE/g DW to 19.18 ± 0.08 GAE/g DW.Meanwhile, the influence order of four factors was obtained from the range of Table 1 and the F ratio of

TPC and TFC
Phenolic compounds, as the plant secondary metabolites, their contents are commonly considered to be responsible for biological activities, including antioxidant, antitumor and antibacterial (24,25).As presented in Figure 3, TPC and TFC values ranged from 2.29 to 374.01 mg GAE/g DW and 0.38 to 93.11 mg RE/g DW, respectively, and significant differences were observed in TPC and TFC values of five fractions (p < 0.05).Among five fractions, the highest TPC and TFC values were found in Fr-EtOAc, while Fr-H 2 O displayed the lowest, which were consistent with the studies conducted by Fan et al. (13) and Bhardwaj et al. (26) that solvents with too high or too low polarity showed low response for TPC and TFC.Meanwhile, the results were also in line with the fact that water was not effective for the extraction of phenolic compounds (15).Therefore, the higher TPC

Identification of phenolic compounds from Amomum villosum
In this study, the phenolic compounds in five fractions of A. villosum were analyzed by UHPLC-ESI-QTOF-MS/MS according to the method illustrated in Section 2.6.As shown in Table 5 and Figure 4, a total of 35 bioactive compounds in five fractions of A. villosum were successfully verified based on the retention time (RT), fragmentation pattern of MS and MS 2 mass spectra, including 12 phenolic acids and derivatives, 2 organic acids, 12 flavonoids and derivatives, 2 oxylipins and 7 proanthocyanidins.

Organic acids
As shown in Figure 4A, peaks 1 and 2 presented characteristic MS 2 fragment ions at m/z 115 and 111, respectively, resulting from the loss of a water molecule from malic acid and the absence of a water molecule as well as a CO 2 group from citric acid, which had been verified by Oldoni et al. (28).These compounds had also been reported in apple (29), grape (30) and Elaeagnus angustifolia L. flower (31).It is worth noting that malic acid and citric acid are the important  (33), gallic acid (28) and 4-hydroxybenzoic acid (34).In addition, peak 13 also presented characteristic MS 2 fragment ions at m/z 152 [M-H-CH 3 ] − and 108 [M-H-CO 2 -CH 3 ] − , suggesting peak 13 was vanillic acid according to a previous report in ribes stenocarpum by Jiang et al. (33).The MS 2 fragment ions of peak 18 further showed characteristic MS 2 fragment ions at m/z 182 and 166, resulting from the loss of-CH 3 (15) and -OCH 3 (31) in negative ion mode, respectively, in the structures, which presented a characteristic fragmentation behavior of syringic acid (35).

Hydroxycinnamic acids
Peak 22 (RT = 12.095 min) showed a deprotonated molecule ion at m/z 163.0403 [M-H] − , which produced a MS 2 fragment ion The effects of temperature, time, ethanol concentration and solid-liquid ratio on total phenolic content (TPC).GAE: gallic acid equivalent, DW: dried weight.Superscript letters in each point indicated significant differences between different letters (p < 0.05).(36).Furthermore, it could be observed that peak 15 presented a characteristic MS 2 fragment ion at m/z 135 that indicated the addition of a oxygen atom compared with peak 22, suggesting peak 15 was caffeic acid, which was agreed with the characteristic fragment ions of caffeic acid in adlay bran (34).Moreover, it was inferred that peak 25 was formed by the introduction of -OCH 3 into the benzene ring of peak 15 compared with the molecular weight.And peak 25 was further identified based on the MS 2 fragment ions at m/z 178 and 149, suggesting the loss of -CH 3 and CO 2 from the precursor ion at m/z 193.0403 [M-H] − (36), respectively.Thus, peak 25 was identified as ferulic acid.

Other phenolic acids derivatives
Peaks 5 (RT = 7.609 min) and 11 (RT = 9.941 min) exhibited the same precursor ion [M-H] − at m/z 329.0898 with the same MS 2 fragment ions at m/z 269, 209, 167, 123, suggesting that peaks 5 and 11 were structural isomers.It also could be observed a characteristic MS 2 fragment ion at m/z 167 originated from the loss of glucose (162 Da), which corresponded to a glucose linked to a vanillic acid moiety (33).Therefore, peaks 5 and 11 were identified as vanillic acid hexoside isomer.Peak 7 was tentatively identified as protocatechualdehyde via its MS 2 fragment ion at m/z 108, resulting from the loss of -CHO from parent ion 137.0250 [M-H] − (37).Peak 30 was characterized as syringaldehyde due to two characteristic MS 2 fragment ions at m/z 166 and 151, implying the loss of one or two methyl groups (38).

Flavanols
Peaks 10 and 19 presented the same parent ion at m/z 289.0745 [M-H] − with different retention times, which gave MS 2 fragment ions at m/z 245 [M-H-CO 2 ] − and 109 [M-H-C 9 H 8 O 4 ] − , indicating that both peaks were isomers, corresponding to catechin and epicatechin as report by Liu et al. (43) who investigated the phenolic compounds in the internal fruit septum of walnuts.

Other flavonoid derivatives
Peaks 9 and 12 exhibited the same deprotonated molecule ion at m/z 451.1250 [M-H] − and the entire same MS 2 fragment ions at m/z 361, 331, 289 and 245, leading to the fact that peaks 9 and 12 were isomers.Furthermore, the characteristic MS 2 fragment ions at m/z 245 and 109, suggesting the loss of CO 2 and C 9 H 8 O 4 , respectively from parent ion, indicating peaks 9 and 12 were catechin and epicatechin (43).Peak 21 was characterized as a catechin-pentoside isomer, owing to the precursor ion at m/z 435.1315 [M-H] − (C 21 H 23 O 10 ), and MS 2 fragment ions at m/z 331 and 289, indicating the addition of a pentose to catechin (44).Peak 31, with parent ion at m/z 465.1242 [M-H] − , was identified as catechin glucuronide, and it was further confirmed by the MS 2 fragment ion at m/z 421 [M-H-CO 2 ] − and 289 (catechin) (44).

Procyanidins
Procyanidins are formed by the polymerization of different amounts of catechin or epicatechin.Obviously, the fragment ions of procyanidins contain the parent ions of catechin or epicatechin.In this study, peak 23 was speculated to be procyanidin A, owing to the precursor ion at m/z 575.1252 [M-H] − (C 30 H 24 O 12 ), and MS 2 fragment ion at m/z 289 assigned to catechin (45).Moreover, peaks 8, 16 and 26 exhibited the same parent ion at m/z 577.1349  demonstrated the existence of monomer dimeric and trimeric of catechin and its multiple, respectively.Thus, peak 17 was identified as procyanidin D.

Quantification of phenolic compounds in five Amomum villosum fractions
In the present work, phenolic compounds in five A. villosum fractions were quantified using an UHPLC-ESI-QTOF-MS/MS.As shown in Figure 4B, a total of 17 major phenolic compounds from A. villosum were successfully quantified, including quercitrin, vanillic acid, gallic acid, syringic acid, protocatechuic acid, catechin, epicatechin, isorhamnetin, rutin, ferulic acid, caffeic acid, hyperoside, isoquercitrin, protocatechualdehyde, quercetin, p-coumaric acid and 4-hydroxybenzoic acid, which was very essential to identify the phenolic compounds composition for the further utilization of A. villosum.
As depicted in Table 3, the highest phenolic compound content was found in Fr-EtOAc (337.03 mg/g DW), followed by Fr-nBuOH (14.85 mg/g DW), Fr-CH 2 Cl 2 (3.18 mg/g DW), Fr-H 2 O (0.31 mg/g DW) and Fr-PE, which were consistent with the results of TPC and TFC (Figure 3).Interestingly, Fr-EtOAc contained all of the 17 quantified phenolic compounds, while only 9, 3 and 2 kinds of the quantified phenolic compounds were found in Fr-nBuOH, Fr-CH 2 Cl 2 and Fr-H 2 O, respectively.Interestingly, Fr-EtOAc contained all of the 17 quantified phenolic compounds, while only 9, 3 and 2 kinds of the quantified phenolic compounds were found in Fr-nBuOH, Fr-CH 2 Cl 2 and Fr-H 2 O, respectively.Strangely, among the 17 quantified phenolic compounds, no phenolic compounds were determined in Fr-PE.On one hand, low TPC and TFC of Fr-PE (Figure 3) were responsible for the results.On the other hand, it might be due to the polarity difference between quantified phenolic compounds and the mobile phase of UHPLC, resulting in the poor solubility of target phenolic compounds in the mobile phase.
For individual phenolic compounds, mostly individual phenolic compound contents (except rutin) of Fr-EtOAc were higher than the other four fractions.Meanwhile, the predominant phenolic compounds in Fr-EtOAc were vanillic acid (93.70 mg/g DW), catechin (72.67 mg/g DW), epicatechin (69.87 mg/g DW) and protocatechuic acid (38.93 mg/g DW), accounting for 81.65% of 17 phenolic compounds.In other words, Fr-EtOAc was rich in phenolic compounds with hydroxybenzoic acids and flavonols as major phenolic compounds.Similar results were also found in the report by Fan et al. (13) who found that catechin and epicatechin were the predominant phenolic compounds in Fr-EtOAc of A. tsaoko.Although some reports confirmed that the water extract of A. villosum had many biological activities (7, 8), there was not much research on the composition of phenolic compounds from A. villosum.And this is the first time to report the composition of phenolic compounds from the seeds of A. villosum.Additionally, the quantitative results confirmed that Fr-EtOAc was the best fraction for the further utilization of phenolic compounds from A. villosum.

Antioxidant activities
It was well known that each evaluation method of antioxidant activity has its particularities.Therefore, the ABTS, DPPH and FRAP assays (48,49) were used to evaluate the antioxidant activities of phenolic compounds in five fractions of A. villosum.It was noted that the results of ABTS and DPPH assays were expressed by IC 50 values in this study, which represented the concentration of phenolic compounds when the scavenging rate of ABTS + or DPPH• free radicals was 50%.Thus, the lower IC 50 value indicates stronger free radicals scavenging capacities and antioxidant activities.Meanwhile, the VCE was used to express the FRAP value, which was positively correlated with the antioxidant activities.
As shown in Figure 5, the antioxidant activities of five fractions were 0.23-7.67mg/mL, 0.08-2.11mg/mL and 0.84-322.91 mg VCE/100 g DW for DPPH, ABTS and FRAP assays, respectively.Thus, five fractions exhibited a significant difference in antioxidant activities, and the Fr-EtOAc displayed the highest scavenging capacities of ABTS + and DPPH• free radicals and reducing ability of Fe 3+ , followed by Fr-nBuOH and Fr-CH 2 Cl 2 , which agreed with the results of TPC and TFC (Figure 3).Similar findings had also been reported by Clodoveo et al. (50) who evaluated the antioxidant activities of phenolic compounds in sweet cherry pulp.Meanwhile, the ABTS + and DPPH• free radicals scavenging capacities of five fractions were lower than vitamin C, but Fr-EtOAc and Fr-nBuOH exhibited excellent antioxidant activities with low IC 50 values of DPPH (0.23, 0.38 mg/ mL) and ABTS (0.08, 0.16 mg/mL) assays.Additionally, the IC 50 value (DPPH) of Fr-EtOAc was lower than cascara (0.43 mg/mL) (51) and Piper chaba stem methanolic extract (0.31 mg/mL) (52).Previously, phenolic compounds could act through different antioxidant mechanisms to scavenge free radicals and reduce the high valence ions to lower valence ions (53).Furthermore, the extract of medicinal plants was also reported to remove oxidant precursors to reduce or prevent oxidative damage (54).However, the extract of the seeds of A. villosum was a complex mixture, and it was necessary to further explore the specific phenolic compounds that played an important role in the antioxidant activities by correlation analysis.

Cytotoxicity and antitumor activity
Amomum villosum, as a medicinal and edible plant in China, can display various biological activities, including antitumor activity.Firstly, the cytotoxicity of five fractions was evaluated by measuring the cell viability of HUVE cells using a CCK8 assay.This assay is based on an analysis of the mitochondrial activity of the cells.The CCK8 assay is more sensitive than the MTT assay, which is also often used to ascertain cell viability (55).As depicted in Figure 6A, the cell viability of the HUVE cells was greater than 90% when treated with 100, 500 and 1,000 μg/mL of five fractions, respectively, indicating that these concentrations would not be considered to be cytotoxic (56).As a result, 100-1,000 μg/mL of five fractions were used in subsequent experiments because they did not promote cytotoxicity.
Subsequently, CCK8 method was further employed to evaluate the effect of five fractions on HeLa cells proliferation at concentrations of 100-1,000 μg/mL, and the results were shown in Figure 6B.The results showed that the inhibition of five fractions on the proliferation of HeLa cells was in a dosedependent manner (100-1,000 μg/mL).Meanwhile, it was also observed that Fr-EtOAc exhibited the highest inhibition rate, followed by Fr-nBuOH, Fr-CH 2 Cl 2 , Fr-PE and Fr-H 2 O, which fitted well the trends of TPC and TFC values (Figure 3) and antioxidant activities (Figure 5).Moreover, the Fr-H 2 O had no inhibitory effect on the proliferation of HeLa cells at the test concentration (100-1,000 μg/mL).
Excitingly, in the present work, Figure 6B depicted that the inhibition rates of Fr-EtOAc and Fr-nBuOH on HeLa cells increased sharply from 6.02 to 79.04% and 5.86 to 63.45%, respectively, when the concentration increased from 100 μg/mL to 1,000 μg/mL.However, Fr-CH 2 Cl 2 and Fr-PE displayed a slightly inhibitory effect on HeLa cells proliferation.Additionally, a previous study had also shown that ethanol extract of Euphorbia lathyris exhibited good antitumor activity at low concentrations (57).Therefore, the phenolic constituents of the extract may be responsible for the antitumor activity.Meanwhile, Pu et al. (58) demonstrated that ferulic acid, chlorogenic acid, caffeic acid and feruloysinapic acid in jackfruit pulp provided a significant positive contribution to the antitumor effect.Overall, the results provided foundation support for the further development of novel antitumor drugs.

Correlation analysis
Correlation analysis has been proved to be a useful method to evaluate the correlation between individual compounds and bioactivities, which is very important to the further utilization of biological components.In this study, the correlation analysis between individual phenolic compounds and antioxidant activities (ABTS, DPPH, FRAP) was depicted in Figure 7. Intuitively, DPPH and ABTS presented a negative correlation with TPC, TFC and 17 individual phenolic compounds, while FRAP had a positive correlation with them, resulting from the results of DPPH and ABTS assays were expressed by IC 50 values (Section 3.5).Specifically, TPC and TFC were extremely significantly (p < 0.01) positive correlated with FRAP and showed a negative correlation with DPPH (r = −0.67,−0.67, respectively) and ABTS (r = −0.63,−0.62, respectively).And these results also confirmed the fact that Fr-EtOAc had the highest antioxidant activities due to the highest TPC and TFC.Furthermore, it was observed that FRAP depicted extremely significant (p < 0.01) positive correlated with 16 individual compounds (except rutin), while DPPH was significantly (p < 0.05) negative correlated with rutin.Similar results were also reported by Clodoveo et al. (50) who evaluated the correlation between phenolic compounds from sweet cherry pulp and antioxidant activities (DPPH and ABTS assays).And the results of the present work demonstrated and confirmed the speculations of several recent researches on some unique phenolic compounds that contributed to higher antioxidant activities (59,60).

Conclusion
In this work, five fractions were obtained from ethanol extract of the seeds of A. villosum by fractional extraction method, and the identification, quantification and biological activities (antioxidant and antitumor) of bioactive compounds in five fractions were evaluated.This study was the first report on the composition of phenolic compounds from the seeds of A. villosum.And the results revealed that the seeds of A. villosum were rich in phenolic compounds, mainly including vanillic acid, catechin, epicatechin, protocatechuic acid, quercetin and quercitrin.Meanwhile, the extract of the seeds of A. villosum represented excellent antioxidant and antitumor activities, which confirmed the fact that A. villosum had the potential to be an antitumor drug or antioxidant.However, further research is required to be conducted to better understand the antioxidant and antitumor mechanisms.

FIGURE 1
FIGURE 1 and TFC values of Fr-EtOAc indicated that extraction solvents with medium polarity were more effective for the extraction of phenolic compounds from A. villosum.However, EtOAc presented a low response for TPC in the study of Dias et al. (27) who evaluated the extraction ability of different solvents for bioactive compounds from C. baccatum fruit, which confirmed the assumption of Herrera-Pool et al. (14) that different species of plants and extraction conditions of phenolic compounds related to the different responses for TPC.

FIGURE 2
FIGURE 2 According to the report by Zhong et al. (41), peak 28 was characterized as hyperoside due to the same deprotonated ion with peak 27 at m/z 463.0903 [M-H] − and a characteristic MS 2 fragment ion at m/z 255.Zhou et al. (42) suggested deprotonated ion at m/z 315.0539 [M-H] − and MS 2 fragment ion at m/z 151 as characteristic fragment ions in the identification of isorhamnetin in ginkgo biloba fallen leaves, providing the tentative analysis of peak 33.

[
M-H] − (C 30 H 26 O 12 ) and base peak fragment ions at m/z 425, 407, 289 and 245, suggesting that the three peaks should be isomers of procyanidin B (9), respectively.Peaks 14 and 20 were characterized as procyanidin C due to the parent ion at m/z 865.2103 [M-H] − presented a characteristic fragmentation behavior of procyanidin C at m/z 695, 577, 425, 407 and 289(43).Piątczak et al.(46) suggested that parent ion at m/z 1153.2767[M-H] − and the characteristic MS 2 fragment ions at m/z 865, 575 and 287

FIGURE 6 (
FIGURE 6 (A) Represented the effects of five fractions of A. villosum on the human umbilical vein endothelial cells viability.(B) Denoted the antitumor activities of five fractions of A. villosum.Superscript letters in each column indicated significant differences between different letters (p < 0.05).Fr-PE, petroleum ether fraction; Fr-CH 2 Cl 2 , dichloromethane fraction; Fr-EtOAc, ethyl acetate fraction; Fr-nBuOH, n-butanol fraction; Fr-H 2 O, H 2 O fraction.Capital letters represented the significant differences between different samples at the same concentration (p < 0.05).Lowercase letters denoted the significant differences at different concentrations of the same sample (p < 0.05).

TABLE 1
The design of orthogonal test (L 9(3 4)) and range analysis.

TABLE 2
The yields of different fractions.

Table 4 :
extraction temperature > ethanol concentration > solid-liquid ratio > extraction time.In general, the extraction temperature was related to the mass transfer rate and the TPC increased within a certain temperature range, and the extraction time showed a positive response of TPC within a certain time range.While the excessive solid-liquid rate and ethanol concentration led to more energy consumption.Therefore, considering the k i (i = 1, 2, 3) values from Table1and the effect of factors, the optimum extraction conditions of phenolic compounds from A. villosum were as follows: extraction temperature of 80°C, extraction time of 120 min, ethanol concentration of 40% and solid-liquid ratio of 1:25 g/mL.Meanwhile, the TPC value was 19.65 ± 0.13 mg GAE/g DW under the aboveoptimized extraction conditions, which was 0.47 mg GAE/g DW higher than the highest TPC value (19.18 mg GAE/g DW) of orthogonal test, suggesting that the extraction conditions optimized by orthogonal test were credible.

TABLE 4
The variance analysis of orthogonal test.
at m/z 119 [M-H-CO 2 ] − , corresponding to the p-coumaric acid as the report by Ali et al.

TABLE 5
The chemical composition identified in A. villosum extracts based on the UPLC-ESI-QTOF-MS/MS analysis.