UPLC–Q–TOF–MS/MS Analysis of Phenolic Compounds from the Fruit of Cephalostachyum fuchsianum Gamble and Their Antioxidant and Cytoprotective Activities

Bamboo is a widely distributed graminaceous plant in China and is a potential source of bioactive substances. Incidentally, bamboo’s fruit is rich in phytochemicals such as polyphenols and flavonoids, which are significant to human health. In this study, we identified the phenolic compounds of the fruit and investigated the antioxidant activities of Cephalostachyum fuchsianum Gamble (CFG) fruit polyphenols with in vitro and in vivo tests for the first time. UPLC–Q–TOF–MS/MS analysis results showed that the fruit contained 43 phenolic compounds, including 7 hydroxybenzoic acids, 12 flavonoids, 7 coumarins, 10 hydroxycinnamic acids, 1 terpenoid, and 5 lignans. The TPC of SP extracts was higher than that of IBPs extracts in FP and FF. The SP extracts in FP showed better antioxidant activities in vitro compared to those in FF. In addition, polyphenols from CFG fruits protected against H2O2-induced oxidative damage in HepG2 cells, and the protective effect of polyphenols in FP was superior to that in FF. The analysis results showed that CFG fruit has great potential in exploiting natural chemical substances, which can provide valuable pieces of information for the further development and utilization of CFG.


Introduction
Bamboo is a perennial one-time flowering plant and is widely known for its economic value and environmental benefits. In addition, bamboo has been used for centuries to treat diverse maladies, including cough, fever, and leprosy [1].
Cephalostachyum fuchsianum Gamble (CFG) was named by Gamble J. S. in 1896 [2]. CFG, a member of Bambusoideae (Cephalostachyum), is distributed naturally in west and southwest China, Bhutan, Northeast India, and Myanmar. However, similar to other perennial flowering bamboo species, CFG undergoes a vegetative phase for decades [3], followed by massive flowering, bearing fruit, and subsequent death [4]. It is reported that the flowering cycles of CFG generally last for 48 years under natural conditions. Due to their high nutrition and nutraceutical values, CFG fruits have been traditionally used for food and foraging. To date, the fruits of CFG are frequently consumed by local residents as healthy food ingredients, which are stewed in soup with meat, eaten as congee, or consumed like rice [5].
However, to the best of our knowledge, there are few studies on the phytochemicals related to the biological activities of this underutilized fruit. In addition, no one has investigated the cytoprotective activities and antioxidant effects of phenolic extracts from CFG. In order to contribute to facilitating a more comprehensive assessment of the chemical Here, A 0 is the absorbance of the DPPH solution and A 1 is the absorbance of the sample. The results were expressed as µmol of Trolox equivalent (TE) per g of dry sample (DS) (µmol TE/g DS).

Trolox Equivalent Antioxidant Capacity (TEAC)
The TEAC assay was performed according to the procedure of Li [9] and Zhu [10], with minor modifications. ABTS solution was made by mixing 7 mmol/L ABTS working solution and potassium persulfate (2.45 mmol/L) in a volume ratio of 1:1. The sample solution (100 µL) was mixed with 3.8 mL of ABTS working solution. Then, the absorbance of the samples was read at 734 nm. TEAC level was calculated from the calibration curve for Trolox, and the percentage of ABTS radical scavenging was calculated based on the following equation: where A 1 is absorbance of the sample and A 0 is the absorbance of the control sample (ABTS solution). The results were expressed as µmol of TE per g of DS (µmol TE/g DS).

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay of the CFG extract was estimated according to Li's [9] and Zhu's [10] procedures. Methanol was used as blank, and ferrous sulfate was used as a standard reference. The results were expressed as µmol of Fe 2+ equivalents (FE) per gram of DS (µmol FE/g DS).

Hydrogen Peroxide Scavenging Assay (HPSA)
The HPSA was carried out following the method of Zhu [10] and Li [9]. In short, CFG extract (0.6 mL) was mixed with 0.9 mL H 2 O 2 (400 mmol/L) and 1.5 mL of sodium phosphate buffer (45 mmol/L, pH 7.4). The reaction mixture was kept in the dark for 40 min, and then its absorbance was measured at 230 nm. The value of the HPSA was calculated from the standard curve for Trolox, and the percentage of H 2 O 2 radical scavenging was evaluated using the following formula: where A 0 is the absorbance of control and A 1 is absorbance of the sample. The results were expressed as µmol of TE per g of DS (µmol TE/g DS).
2.7. Cell Assays for Antioxidative Activities 2.7.1. Cell Culture HepG2 cells were purchased from the cell bank of Sebachem (Shanghai, China). Cell culture was prepared following Bak [13] with certain modifications. First, HepG2 cells were cultured in DMEM medium supplemented with 1% penicillin-streptomycin (Fisher, Houston, TX, USA) and 10% fetal bovine serum. Then, all the cells were placed at 37 • C in a 5% CO 2 incubator for 24~48 h. When the cell concentration reached 80%, the cells were digested with trypsin.

Cell Viability Assay
Cell viability was performed on the previous method of Tan [14] with some modifications. Briefly, 2 × 10 4 cells/well were seeded into a 96-well plate for 24 h. Then, the cells were treated with the CFG extract for 24 h. MTT solution (10 µL) was added to each well, and the cells were incubated at 37 • C in 5% CO 2 for 24 h. Finally, the medium was removed, 150 µL DMSO was added, and the plate was gently shaken. The absorbance was measured at 517 nm.
Cell viability (%) was calculated with the equation: where A 0 is the absorbance of the sample and A 1 the absorbance of the blank.

Determination of Oxidative Stress Parameters
The cellular reactive oxygen species (ROS) levels were determined following a previous report [15] with some modifications. In brief, cells were seeded at a density of 2 × 10 4 cells/well into 96-well plates, and cultured in a 37 • C, 5% CO 2 incubator for 24 h. Then, 100 µL of no-toxic polyphenol extracts at the concentrations of 5, 10, 25 µg/mL was added into each well. Subsequently, 800 µmol/L H 2 O 2 was added and incubated for 6 h. Finally, the cells were washed with PBS, and 10 µmol/L DCFH-DA (0.0125 mg/mL in medium without FBS) was added. The absorbance was determined at 488 (excitation wavelength) and 525 nm (emission wavelength) using a fluorescence enzyme labeler. (BioTek Synergy H1, Burlington, VT, USA).
The SOD, CAT, MDA activity and GSH levels were measured using the assay kits obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). All the procedures were carried out in accordance with the manufacturer's instructions.

Data and Statistical Analysis
Data were presented as mean ± standard deviation (SD) of three replicates. To analyze the differences between the means of the treatment group and the control group, one-way ANOVA was applied to calculate the statistical significance. All graphs were generated using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). The data were statistically analyzed using SPSS Version 18.0 software. (SPSS Inc., Chicago, IL, USA). p < 0.05 was regarded as the level of significance.

Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)
The total phenolic content (TPC) and total flavonoid content (TFC) of the different extracts from CGF (FF and FP) are presented in Table 1. Phenolic compounds are common secondary metabolites which are widely distributed in plants. Bamboo fruit, such as the fruit of Melocanna baccifera, is generally rich in nutrients and polyphenolic compounds [16]. As shown in Table 1, the TPC of SPs in FF and FP was 8.721 and 17.679 µmol FAE/g DS, respectively, and the TPC of IBPs in FF and FP was 7.544 and 12.903 µmol FAE/g DS, respectively. It can be observed that the TPC values of soluble fraction were higher than those of the insoluble fraction, whether in FF or FP of CFG, which is in agreement with previous studies of mistletoes [9] and red sorghums [17]. In addition, these results suggest that polyphenol molecules were more enriched in the FP than in the FF.
Flavonoids are the largest group of phenolic compounds. Increasing evidence indicates that flavonoids can possess antioxidant and anti-inflammatory effects [18,19]. From Table 1, it can be seen that the TFC of SP extracts was 1.237 and 1.052 CE/g DS in FF and FP, respectively, and the TFC of IBP extracts was 0.622 and 0.837 CE/g DS in FF and FP, respectively. In line with the TPC results, TFC of SPs was significantly higher than those of IBPs in two fractions of CFG fruit. The TFC of IBP extract in FP was higher than that of FF and similar to the TPC values in FP of FF and CFG. However, the TFC of SP extracts in FP was lower than that in FF, which may be due to the presence of different types of polyphenols in FF and FP or because SPs and IBPs in FF and FP can bind to polysaccharides on the cell wall with different affinities [20].
We can infer that the FP of CFG may contain more phenolic compounds due to the difference in polyphenol content between the FP and FF. Therefore, we decided to investigate the polyphenol composition of CFG fruit.

Identification of Phenolic Compositions
The polyphenols of FF and FP from CFG fruit were identified using UPLC-Q-TOF-MS/MS. Representative UPLC-QTOF-MS/MS total ion chromatograms (TIC) in the negative ion mode of polyphenols in CFG are shown in Figure 1. The phenolic compounds were tentatively identified by matching retention times (RT), m/z values, MS/MS fragments with compounds from the reported data in literature and database resources [21]. In addition, the relevant MS/MS spectra are provided in the Supplementary Materials.
A total of 43 compounds were initially identified in the negative mode. Table 2 lists the retention time (RT), molecular formulas, experimental molecular weights, and major fragment ions. Of these, 9 compounds were identified in the FF and 40 compounds in FP, with 6 of them present in both samples.
nutrients and polyphenolic compounds [16]. As shown in Table 1, the TPC and FP was 8.721 and 17.679 μmol FAE/g DS, respectively, and the TPC of I FP was 7.544 and 12.903 μmol FAE/g DS, respectively. It can be observed values of soluble fraction were higher than those of the insoluble fraction, w or FP of CFG, which is in agreement with previous studies of mistletoes [9 ghums [17]. In addition, these results suggest that polyphenol molecules w riched in the FP than in the FF.
Flavonoids are the largest group of phenolic compounds. Increasing e cates that flavonoids can possess antioxidant and anti-inflammatory effects Table 1, it can be seen that the TFC of SP extracts was 1.237 and 1.052 CE/g FP, respectively, and the TFC of IBP extracts was 0.622 and 0.837 CE/g DS respectively. In line with the TPC results, TFC of SPs was significantly high of IBPs in two fractions of CFG fruit. The TFC of IBP extract in FP was highe FF and similar to the TPC values in FP of FF and CFG. However, the TFC of FP was lower than that in FF, which may be due to the presence of different yphenols in FF and FP or because SPs and IBPs in FF and FP can bind to po on the cell wall with different affinities [20].
We can infer that the FP of CFG may contain more phenolic compoun difference in polyphenol content between the FP and FF. Therefore, we dec tigate the polyphenol composition of CFG fruit.

Identification of Phenolic Compositions
The polyphenols of FF and FP from CFG fruit were identified using UP MS/MS. Representative UPLC-QTOF-MS/MS total ion chromatograms (TIC ative ion mode of polyphenols in CFG are shown in Figure 1. The phenoli were tentatively identified by matching retention times (RT), m/z values, ments with compounds from the reported data in literature and database r In addition, the relevant MS/MS spectra are provided in the Supplementary

Analysis of UPLC-QTOF-MS/MS
Generally, we found that the phenolic compounds detected in the present study were also found in bamboo fruits of Melocanna baccifera [61], including cinnamic acid and syringic acid. Cinnamic acid is a phenolic compound naturally occurring in various vegetables, seeds, and also enriched in daily diets [62]. In addition, syringic acid is a phenolic compound that acts as a free radical scavenging antioxidant in pharmacology [63] and is rich in many edible mushrooms and vegetables and food and beverage plants [64]. The presence of polyphenols may be responsible for their antioxidant activities. Hence, the phenolic compounds in CFG fruits have potential for further research.

In Vitro Antioxidant Activities
The results of four in vitro antioxidant capacity evaluation tests are shown in Table 3. DPPH radical scavenging capacity assay was used for assessing the hydrogen atom or electron donor capacity of phenolic compounds. As shown in Table 3, the DPPH radical scavenging of SPs in FF and FP was 1.355 and 4.686 µmol TE/g DS, and that of IBPs was 1.124 and 1.292 µmol TE/g DS. Thus, DPPH scavenging activity of SPs was higher than that of IBPs. Aside from DRSA, the FRAP of the FF and FP extracts of CFG was also determined in this study. The FRAP of SPs was 11.098 µmol FE/g DW in FF and 17.424 µmol FE/g DW in FP, while that for IBPs was 6.433 µmol FE/g DW in FF and 10.597 µ mol FE/g DW in FP. In addition, the TEAC of SPs was 35.328 µmol TE/g DS in FF and 59.847 µmol TE/g DS in FP, while that for IBPs was 17.758 µmol FE/g DW in FF and 56.299 µmol TE/g DS in FP. The HPSA results of SPs in FF and FP were 47.547 and 72.884 µmol TE/g DS, and those of IBPs were 39.281 and 64.843 µmol TE/g DS. In short, like DPPH radical scavenging ability, the polyphenols in FP showed a more robust antioxidant capacity in the TEAC, HPSA and FRAP assays than in FF of CFG. The SP extract in CFG also had significantly higher antioxidant capacities in terms of FRAP, HPSA, DRSA and TEAC than the IBP extracts (p < 0.05). These findings are similar to those reported in previous studies on other plants, such as mistletoe [9] and L. macranthoides [65].
Thus, the in vitro antioxidant test results suggested that CFG fruit had antioxidant properties, and the FP with higher polyphenol content had more vigorous antioxidant activities than the FF.

Cell Viability
Since the fruit of CFG exhibited notable antioxidant activity in vitro, the inner effect on cell levels required further study. Cell viability is often employed as an indicator of cytotoxicity [66], and the cytotoxic effects in FF and FP of CFG fruit on the HepG2 cells were evaluated with the MTT assay. Cytotoxicity was considered when the cell viability was less than 90%.
In this study, H 2 O 2 was used to induce oxidative stress injury in HepG2 cells and to assess the protective effect of polyphenols in CFG.
As shown in Figure 2, the polyphenols in FP showed no cytotoxicity at polyphenol concentrations of 5, 10, or 25 µg/mL, respectively, and the FF showed no cytotoxicity at polyphenol concentrations of 5, 10, 25, or 50 µg/mL. Consequently, to ensure that the polyphenol concentrations remained consistent, the polyphenol concentrations of 5, 10, and 25 µg/mL were employed for subsequent experiments. were evaluated with the MTT assay. Cytotoxicity was considered when the cell viability was less than 90%. In this study, H2O2 was used to induce oxidative stress injury in HepG2 cells and to assess the protective effect of polyphenols in CFG.
As shown in Figure 2, the polyphenols in FP showed no cytotoxicity at polyphenol concentrations of 5, 10, or 25 μg/mL, respectively, and the FF showed no cytotoxicity at polyphenol concentrations of 5, 10, 25, or 50 μg/mL. Consequently, to ensure that the polyphenol concentrations remained consistent, the polyphenol concentrations of 5, 10, and 25 μg/mL were employed for subsequent experiments.

Protective Effects of Polyphenols from CFG on H2O2-Induced Intracellular ROS Production in HepG2 Cells
The effects of the polyphenol on H2O2-induced ROS generation in HepG2 cells are shown in Figure 3. There were 40-60% viable cells in the presence of 800 μmol/L H2O2 compared to control cells. Therefore, in the following experiments, 800 μmol/L of H2O2 treatment for 24 h was used to induce HepG2 cell injury. Compared with the control group, the levels of intracellular ROS in HepG2 cells were prominently increased after H2O2 induction (Figure 3). Those results showed that the increased intracellular ROS levels caused by H2O2-induction were attenuated in the HepG2 cells pretreated with polyphenols. Among them, compared with those in control cells, the intracellular ROS levels were decreased from 215.152% to 87.147% with increasing polyphenol concentrations of FF. Similarly, we observed that the ROS levels were reduced by the treatment of the polyphenol in FP from 193.575% to 69.575%. The same trends of ROS production were observed in Tamarindus indica leaf extract [67] and resveratrol [68]. Our results indicated that the effects of polyphenols were more prominent at the highest concentration (25 μg/mL)

Protective Effects of Polyphenols from CFG on H 2 O 2 -Induced Intracellular ROS Production in HepG2 Cells
The effects of the polyphenol on H 2 O 2 -induced ROS generation in HepG2 cells are shown in Figure 3. There were 40-60% viable cells in the presence of 800 µmol/L H 2 O 2 compared to control cells. Therefore, in the following experiments, 800 µmol/L of H 2 O 2 treatment for 24 h was used to induce HepG2 cell injury. Compared with the control group, the levels of intracellular ROS in HepG2 cells were prominently increased after H 2 O 2 induction (Figure 3). Those results showed that the increased intracellular ROS levels caused by H 2 O 2 -induction were attenuated in the HepG2 cells pretreated with polyphenols. Among them, compared with those in control cells, the intracellular ROS levels were decreased from 215.152% to 87.147% with increasing polyphenol concentrations of FF. Similarly, we observed that the ROS levels were reduced by the treatment of the polyphenol in FP from 193.575% to 69.575%. The same trends of ROS production were observed in Tamarindus indica leaf extract [67] and resveratrol [68]. Our results indicated that the effects of polyphenols were more prominent at the highest concentration (25 µg/mL) than at the lowest concentration (5 µg/mL), both in the FF and FP. Hence, we can conclude that the polyphenols of FF and FP in CFG could protect cells from damage imposed by ROS.
3.6. The Effects of CFG on the Activities of SOD, CAT, GSH and MDA in H 2 O 2 -Induced HepG2 Cells SOD and CAT are critical antioxidant enzymes that can play an essential role in oxygen metabolizing cells. SOD can convert superoxide to H 2 O 2 , which is further converted via CAT into H 2 O and O 2 [69], and the SOD activity levels indirectly reflect the body's ability to scavenge oxygen free radicals [70]. In addition, reduced glutathione (GSH) is an important intracellular antioxidant that can scavenge H 2 O 2 in favor of scavenging oxidants [71]. Malondialdehyde (MDA), a byproduct of lipid peroxidation, is widely used as a crucial indicator for oxidative stress [15]. To investigate the protective effects of polyphenols from CFG on H 2 O 2 -induced cell injury in HepG2 cells, the SOD and CAT activities, GSH and MDA levels were measured using commercial kits.
As shown in Figure 4, compared with the control group, the activities of SOD, CAT and

The Effects of CFG on the Activities of SOD, CAT, GSH and MDA in H 2 O 2 -Induced HepG2 Cells
SOD and CAT are critical antioxidant enzymes that can play an essential role in oxygen metabolizing cells. SOD can convert superoxide to H2O2, which is further converted via CAT into H2O and O2 [69], and the SOD activity levels indirectly reflect the body's ability to scavenge oxygen free radicals [70]. In addition, reduced glutathione (GSH) is an important intracellular antioxidant that can scavenge H2O2 in favor of scavenging oxidants [71]. Malondialdehyde (MDA), a byproduct of lipid peroxidation, is widely used as a crucial indicator for oxidative stress [15]. To investigate the protective effects of polyphenols from CFG on H2O2-induced cell injury in HepG2 cells, the SOD and CAT activities, GSH and MDA levels were measured using commercial kits.
As shown in Figure 4, compared with the control group, the activities of SOD, CAT The results indicated that the polyphenols of the FF and FP from CGF could exert protective action against H 2 O 2 -induced oxidative damage to HepG2 cells, especially at a high concentration.

Conclusions
The phenolic compounds in CFG fruits were identified for the first time using UPLC/Q-TOF-MS/MS. A total of 43 phenolic compounds were identified, including 7 hydroxybenzoic acids, 12 flavonoids, 7 coumarins, 10 hydroxycinnamic acids, 1 terpenoid, and 6 lignans. The antioxidant activities of phenolic compounds in CFG were reported for the first time. Moreover, the SP and IBP extract contents and the in vivo and in vitro antioxidant activity in FF and FP were compared. The results showed that the TPC of SPs and

Conclusions
The phenolic compounds in CFG fruits were identified for the first time using UPLC/Q-TOF-MS/MS. A total of 43 phenolic compounds were identified, including 7 hydroxybenzoic acids, 12 flavonoids, 7 coumarins, 10 hydroxycinnamic acids, 1 terpenoid, and 6 lignans. The antioxidant activities of phenolic compounds in CFG were reported for the first time. Moreover, the SP and IBP extract contents and the in vivo and in vitro antioxidant activity in FF and FP were compared. The results showed that the TPC of SPs and

Conclusions
The phenolic compounds in CFG fruits were identified for the first time using UPLC/Q -TOF-MS/MS. A total of 43 phenolic compounds were identified, including 7 hydroxybenzoic acids, 12 flavonoids, 7 coumarins, 10 hydroxycinnamic acids, 1 terpenoid, and 6 lignans. The antioxidant activities of phenolic compounds in CFG were reported for the first time. Moreover, the SP and IBP extract contents and the in vivo and in vitro antioxidant activity in FF and FP were compared. The results showed that the TPC of SPs and IBPs in FP was significantly higher than that in FF. The SP extracts in FP showed higher antioxidant activity compared to those in FF. In addition, the polyphenol extracts of FF and FP from CFG protected against H 2 O 2 -induced oxidative stress in HepG2 cells. Therefore, this study provides a basis for further research on CFG fruits and a scientific basis for the exploitation of CFG.