Phytochemical Analysis, Antioxidant, Antibacterial, Cytotoxic, and Enzyme Inhibitory Activities of Hedychium flavum Rhizome

Hedychium flavum Roxb., a medicinal, edible, and ornamental plant, is widely cultivated throughout China, India, and Southeast Asia. The rhizome from this plant has been used for food flavoring and in traditional Chinese medicine to treat diverse diseases, but the detailed constituents and bioactivities are still limited known. Therefore, phytochemical analysis by GC-MS and UHPLC-Q-Orbitrap-MS, and antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities tests have been conducted in the current study. Based on the GC-MS results, the essential oil (EO) of rhizome was mainly composed of coronarin E (20.3%), β-pinene (16.8%), E-nerolidol (11.8%), and linalool (8.5%). Among them, coronarin E was reported in H. flavum EO firstly. Furthermore, the spectrophotometric indicated rhizome had high total phenolic content (TPC, 50.08–57.42 mg GAEs/g extract) and total flavonoid content (TFC, 12.45–21.83 mg REs/g extract), no matter in water extract (WE) or in 70% ethanol extract (EE). UHPLC-Q-Orbitrap-MS was applied to further characterize composition, and 86 compounds were putatively identified from WE and EE, including 13 phenolic components. For the bioactivities, both WE and EE showed remarkable antioxidant activity by DPPH and ABTS tests, being superior to the positive control (butylated hydroxytoluene, BTH). EO revealed significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Proteus vulgaris with DIZ (10.34–24.43 mm), MIC (78.13–312.50 μg/mL), and MBC (156.25–625.00 μg/mL). Moreover, EO exhibited a considerable selectivity to human tumor cell K562 (IC50 = 27.16 μg/mL), and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 cell (IC50 = 95.96 μg/mL) and L929 cell (IC50 = 129.91 μg/mL). A series of apoptosis analysis demonstrated that EO induced apoptosis against K562 cells in a dose-dependent manner. In enzyme inhibitory effect assays, WE and EE showed strong α-glucosidase inhibition activity, being superior to the positive control (acarbose). Besides, the EO, WE, and EE didn’t show a promising inhibition on tyrosinase (19.30–32.51 mg KAEs/g sample) and exhibited a weak inhibitory effect on cholinesterase. Based on the current results, H. flavum could be considered as a source of bioactive compounds and has high exploitation potential in the cosmetics, food, and pharmaceutical industries.


INTRODUCTION
The Hedychium genus (Zingiberaceae), commonly known as ginger lily, includes approximately 87 species, which are mainly distributed in China, India, and Southeast Asia (The Plant List, 2010;Prakash et al., 2016). Hedychium species are extensively cultivated for their various uses in fragrance, paper, ornamental, cosmetics, medicine, and food industries (Sakhanokho and Rajasekaran, 2019). Hedychium plants are rich in essential oil and widely used in traditional medicine for the treatment of flu, gastric diseases, diarrhea, bronchitis, asthma, nausea, snake bites, and leishmaniasis (Tushar et al., 2010;Hartati et al., 2014;Sakhanokho and Rajasekaran, 2019). The extracts and essential oils of Hedychium plants have been demonstrated to have multiple bioactivities, such as antioxidant, antimicrobial, cytotoxicity, antiacetylcholinesterase, anti-inflammatory, antidiabetic, analgesic, hepatoprotective, and insecticidal activities (Hartati et al., 2014;Ray et al., 2018a;Tavares et al., 2020).
H. flavum has been developed as an industrial crop with medicinal, ornamental, and spice values. However, in addition to few reports on the chemical components, antifungal and insecticidal activities of EO, there is no detailed study on the chemical composition and bioactivities of H. flavum, which could impede its exploitation in industry. Therefore, phytochemical analysis by GC-MS and UHPLC-Q-Orbitrap-MS, and antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities tests of the rhizome from H. flavum were conducted in this work. To our knowledge, the antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities of EO, as well as the chemical composition and bioactivities of extracts from H. flavum rhizome were reported for the first time.
WE and EE were prepared by reflux extraction method. The fresh, finely chopped rhizomes (0.5 kg) were placed into a round bottom flask (2 L), mixed with distilled water or 70% ethanol, boiled under reflux for 2 h, and then filtered. The reflux extraction was repeated twice. The combined filtrate was concentrated with a rotary evaporator and dried in a freezedrier. Then, WE and EE were weighed and stored at 4°C.

GC-FID/MS for Composition Analysis of EO
The EO was analyzed by an Agilent 6890 gas chromatograph equipped with a flame ionization detector (FID). Capillary column: HP-5MS (60 m × 0.25 mm, 0.25 mm film thickness). The flow rate of carrier gas helium was set at 1 mL/min and the split ratio was 1:20. The following GC oven temperature was used with the injection volume of 1 mL: held at 70°C (2 min), 2°C per min to 180°C (55 min), 10°C per min to 310°C (13 min), and kept at 310°C (12 min). The GC-MS analysis was performed using an Agilent 6890 gas chromatograph equipped with an Agilent 5975C mass selective detector (Agilent Technologies Inc., CA, USA). GC column and parameters were the same as in GC-FID. The mass spectra operated in the mass range (m/z 29 to 500) and EI mode (70 eV). The interface temperature and ion source temperature were 280°C and 230°C, respectively. The relative percentage of chemical constituents was determined by the peak area. The retention index (RI) is determined by referring to a series of nalkanes (C 8 -C 22 ). The identification of chemical constituents was based on comparison of their retention index and mass spectrum with those in Wiley 275 and NIST 2017 databases.

Total Phenolic and Flavonoid Contents
Total Phenolic Content (TPC) The TPC was evaluated according to Folin-Ciocalteu method (Slinkard and Singleton, 1977) with marginal modification. The sample solution (0.5 mL) was mixed with Folin-Ciocalteu reagent (2.5 mL) and incubated for 4 min. Subsequently, Na 2 CO 3 solution (4 mL, 7.5%) was added and incubated at room temperature for 60 min. The sample absorbance was read using a UV-5200 spectrophotometer (Shanghai Metash Instruments Co., Ltd, China) at 760 nm. TPC was expressed as equivalents of gallic acid (mg GAEs/g extract).

Total Flavonoid Content (TFC)
The TFC was assayed by NaNO 2 -Al(NO 3 ) 3 -NaOH colorimetry (China Pharmacopoeia Commission, 2015) with minor modification. The sample solution (5 mL) was mixed with 5% NaNO 2 (0.4 mL) and incubated for 6 min. Subsequently, Al (NO 3 ) 3 solution (0.4 mL, 10%) was added and incubated for 6 min. Then, NaOH solution (4 mL, 4%) was added up to a total volume of 10 mL with distilled water. The absorbance was read at 510 nm after 15 min incubation at room temperature. TFC was expressed as rutin equivalents (mg REs/g extract).
The Thermo Scientific Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometry (Q-Orbitrap-MS) equipped with heated electrospray ionization (HESI-II) source was used for MS data collection. HESI-II source was operated in negative and positive ionization modes with spray voltages of 2.5 and 3.0 kV, respectively. HESI-II source parameters were set as follows: capillary temperature 320°C, vaporizer temperature 350°C, sweep gas pressure 0 arb, auxiliary gas pressure 10 arb, sheath gas pressure 35 arb, RF lens amplitude (S-lens) 60. The mass spectrometer analysis was executed in the mode of full mass/dd-MS 2 , scanned from m/z 100 to 1,500 with a resolution of 70,000 in full scan MS 1 , followed by data-dependent MS 2 acquisition at 17,500 resolution with stepped normalized collision energy (NCE) (20, 40, and 60 eV). Maximum injection time (IT) for MS 1 and MS 2 was separately set at 100 and 50 ms and the target values of automatic gain control (AGC) were 1e 6 and 2e 5 , respectively. Loop count was 3, dynamic exclusion time was 5 s, and quadrupole isolation window (isolation width) was 1.5 m/z. The mass spectrum data of the chemical composition was processed with CD3.0 (Thermo Fisher Scientific) and identified by comparing mzVault, mzCloud, and ChemSpider databases. The allowable calculation error was set as 5 ppm.

DPPH Assay
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was evaluated according to the method with slight modification (Blois, 1958). Two milliliter of sample solution and DPPH solution (2 mL, 0.1 mM) were mixed and incubated for 30 min at room temperature in the dark. The absorbance was recorded at 517 nm. Butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls. The results were expressed using IC 50 values and as equivalents of ascorbic acid (mg AEs/g sample).

ABTS Assay
The 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging capacity was assayed according to the method with marginal modification (Re et al., 1999). The ABTS• + solution was generated by reacting 2.45 mM K 2 S 2 O 8 solution with and 7 mM ABTS solution incubated in the dark at room temperature for 12 h. Before experiments, to obtain an absorbance of 0.70 ± 0.02 at 734 nm, the ABTS• + solution was diluted with methanol. The diluted ABTS• + solution (4 mL) and sample solution (0.4 mL) were mixed and the absorbance was read at 734 nm after 10 min incubation in the dark at room temperature. Ascorbic acid and BHT were used as positive controls. The results were expressed using IC 50 values and as equivalents of ascorbic acid (mg AEs/g sample).

Agar Well Diffusion Assay
The inhibition zone diameter was assayed according to the agar well diffusion method with slight modification (Zhang et al., 2017). Briefly, The WE and EE were dissolved with distilled water (100 mg/mL). Streptomycin distilled water solution (1 mg/ mL) was used as the positive control. The bacterial suspension (100 mL, 10 6 CFU/mL) was evenly spread on the Mueller-Hinton agar medium. Filter paper discs (diameter 6 mm) containing pure essential oil, WE, EE, and streptomycin solution (20 mL) were added and the diameter of inhibition zone (DIZ) was recorded after 24 h incubation at 37°C.

Determination of MIC and MBC
The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were determined by the broth microdilution method with marginal modification (Eloff, 1998). Briefly, the two-fold serially diluted sample solution (100 mL) and the bacterial suspensions (100 mL) were added to each well at a final density of 5 × 10 5 CFU/mL. The 96well plates were incubated for 24 h at 37°C. Subsequently, the resazurin aqueous solution (10 mL, 0.01%) was added to each well and incubated for 2 h at 37°C in the dark. The MIC was determined as the minimum sample concentration without color change. For the determination of MBC value, the sample (10 mL) from the wells without color change was subcultured in Mueller Hinton agar plate for 24 h at 37°C. The MBC was defined as the minimum sample concentration showing no bacterial growth (Mzid et al., 2017).

Cytotoxic Activity
The cytotoxic effect was assayed according to our previously published MTT method (Tian et al., 2020) against murine fibroblast cell line (L929) and five human cell lines: fetal lung fibroblasts (MRC-5), lung adenocarcinoma (A549), and non-small cell lung cancer (NCI-H1299), prostatic carcinoma (PC-3), and leukemic (K562) cell lines. EO solution (0.1% DMSO), WE, and EE distilled water solution were twofold serially diluted with medium. The cell suspensions (80 mL) were added to each well at a density of 5 × 10 3 cells per well and incubated for 24 h. Subsequently, 20 mL of the diluted sample solution was added and cultured for 72 h. After that, the MTT solution (10 mL, 5 mg/mL in PBS) was added and incubated for 4 h. Then, 150 mL of DMSO was added to each well to dissolve formazan crystal after removing the medium. The absorbance was recorded using an iMark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm. The results were expressed using IC 50 values.

Morphology Assay
The K562 cell suspension was added to a 6-well plate (1×10 6 cells per well) and incubated for 24 h. Subsequently, the cells were treated with varying concentrations of EO (0, 30, and 60 mg/mL) for 48 h. Morphological changes of the treated cells were recorded using an inverted microscope (Leica DMi8, Leica Microsystems, Germany).
For Acridine orange/ethidium bromide (AO/EB) staining assay, the cells were treated with EO as described above. The treated cell suspensions (1 mL) were collected, centrifuged at 500 g for 10 min, and resuspended in 50 mL PBS, then stained with AO (10 µg/mL) and EB (10 µg/mL) for 10 min. After that, the stained cell suspension (10 mL) was placed onto a glass slide and photographed using an inverted fluorescence microscope (Leica DMi8, Leica Microsystems, Germany).
For Hoechst 33258 staining assay, the cells were treated with EO as described above. The treated cell suspensions (1 mL) were collected, centrifuged, and resuspended in 1 mL PBS. After removing the PBS, the cells were fixed in 4% paraformaldehyde (10 mL) for 10 min. After two PBS washes, the cells were stained with 10 mL of Hoechst 33258 (Beyotime, Haimen, China) for 10 min. After that, the stained cell suspension (10 mL) was placed onto a glass slide and analyzed by an inverted fluorescence microscope.

Annexin V-FITC/PI Assay
Annexin V-FITC and propidium iodide (PI) staining was performed using flow cytometry to quantify EO-induced apoptosis in K562 cell. The K562 cells were seeded in 6-well plates at 2×10 5 /well, and treated with different concentrations of EO (0, 30, and 60 mg/mL) for 48 h. Subsequently, the cells were collected and stained with Annexin V-FITC/PI as described by the manufacturer using Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, Haimen, China). The labeled cells were analyzed using a BD FACSCalibur flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).

Enzyme Inhibitory Activities a-Glucosidase Inhibitory Activity
The a-glucosidase inhibitory activity was evaluated according to the method with slight modification (Silva et al., 2019). Sample solution (30 mL), phosphate buffer (60 mL, pH 6.8), and aglucosidase solution (10 mL, 0.8 U/mL) were mixed and added to each well. After 15 min incubation at 37°C, p-Nitrophenyl-a-Dglucopyranoside (p-NPG) solution (10 mL, 1 mM) was added and incubated at 37°C for 15 min. Subsequently, the reaction was stopped with the addition of Na 2 CO 3 solution (80 mL, 0.2 M), and the absorbance was recorded at 405 nm. Acarbose was used as positive control. The a-glucosidase inhibitory effect was expressed using IC 50 values and as equivalents of acarbose (mmoL ACEs/g sample).

Tyrosinase Inhibitory Activity
The inhibition of tyrosinase was assayed according to the method with L-tyrosine as substrate (Zardi-Bergaoui et al., 2018). Sample solution (70 mL) and tyrosinase solution (10 mL, 100 U/mL) were mixed in a 96-well plate and incubated at 37°C for 5 min. Then, L-tyrosine solution (80 mL, 5.5mM) was added and incubated at 37°C for 30 min. Kojic acid was used as positive reference, and the absorbance was recorded at 492 nm. The tyrosinase inhibitory activity was expressed using IC 50 values and as equivalents of kojic acid (mg KAEs/g sample).

Statistical Analysis
All data were repeated at least three times and expressed as mean ± standard deviation (SD). SPSS software (version 19.0) was used for statistical analysis. The significant difference between the two groups was compared using the two-tailed unpaired t-test (P < 0.05).
The coronarin E has previously been reported in other Hedychium species EO, such as H. coronarium (Rodrigues et al., 2013;Ray et al., 2018b;Ray et al., 2019), H. acumnatum (Weyerstahl et al., 1995) and H. gardnerianum (Weyerstahl et al., 1998). To our knowledge, the coronarin E, as the most predominant constituent, was identified for the first time in H. flavum EO.

Total Phenolic and Flavonoid Contents
The WE presented a higher yield at 2.04% (w/w) on fresh weight basis than the EE (1.37%). The TPC and TFC of H. flavum rhizome WE and EE were shown in Figure 2. The TPC of WE and EE were 50.08 ± 0.71 and 57.42 ± 0.07 gallic acid equivalents (mg GAEs/g) of dry weight of extract, respectively. The TFC in WE and EE were 12.45 ± 1.01 and 21.83 ± 0.33 rutin equivalents (mg REs/g) of dry weight of extract, respectively. Statistical analysis indicated that TPC and TFC of EE were significantly higher (p< 0.01) than that of WE. Plant polyphenols and flavonoids have been widely used in various medicines and food products due to their wide range of pharmacological activities, such as antioxidants, antimicrobial, anticancer, antiinflammatory, hepatoprotective, antiviral, antiatherosclerotic, antispasmodic, and enzyme inhibitory activities (Fraga, 2010;Agrawal, 2011;Murti and Sharma, 2017). The H. flavum rhizome WE and EE exhibited high TPC and TFC values, which can be used as a rich source of phenolics and flavonoids.

Antioxidant Activity
The antioxidant activity of EO, WE, and EE was evaluated using DPPH and ABTS methods, and BHT and ascorbic acid were used as positive controls ( Table 2). The EO showed low antioxidant activity in both methods. The WE (IC 50 : 15.36 ± 0.15 mg/mL) and EE (IC 50 : 16.24 ± 0.61 mg/mL) exhibited significantly stronger DPPH free radical scavenging capacity than BHT (IC 50 : 109.40 ± 9.68 mg/mL), but lower than ascorbic acid (IC 50 : 9.39 ± 0.79 mg/mL) (p < 0.05). Base on  (Shin et al., 2015;Baloglu et al., 2019). Thus, the antioxidant capacity of WE and EE may be due to the high levels of TPC and TFC. The free radical scavenging agents can be used as a preventive medicine for age-related diseases and cancer (Khansari et al., 2009). The BHT, a synthetic antioxidant, is used as preservative in foods. However, as a food additive, the BHT is rejected by consumers due to its adverse reactions (Imaida et al., 1983;Simon, 2003). Interestingly, the WE and EE showed higher antioxidant activity compared with the BHT. These results indicate that WE and EE can be used as potential substitutes for synthetic antioxidants in the pharmaceutical and food industries. The authors limited the results of the DPPH analysis.

Antibacterial Activity
The antibacterial properties of EO, WE, and EE were qualitatively determined by the diameter of inhibition zones (DIZ) and quantitatively evaluated by MIC and MBC values using streptomycin as positive control (  (Sokovic et al., 2008;Park et al., 2012;Hartati et al., 2015;Silva et al., 2015;Kiryu et al., 2018). Hence, H. flavum EO possessed significant antibacterial ability, probably due to these major constituents, and it can provide natural antibacterial agents for the pharmaceutical industry.

Cytotoxic Activity
The cytotoxic activities of EO, WE, and EE were evaluated against human tumor cell lines (A549, NCI-H1299, PC-3, and K562), human normal cell line , and murine fibroblast cell line (L929) using the MTT method with cisplatin as positive control (  (Reddy et al., 2009;Kumrit et al., 2010). The cytotoxic properties of other main components in EO, such as 1,8cineole, b-pinene, a-pinene, E-nerolidol, linalool, and acurcumene, have been demonstrated in previous reports (Hassan et al., 2010;Shin and Lee, 2013;Dall'Acqua et al., 2017;Salehi et al., 2019). Therefore, the presence of these main components could explain the significant cytotoxic activities of the H. flavum rhizome EO. These results indicate that the H. flavum EO can be used as a new source of natural antitumor active small molecules in the pharmaceutical industry.

EO Induces Apoptosis in K562 Cells
Morphology assay indicated that EO triggered obvious morphological changes with typical characteristics of apoptosis, such as cell shrinkage and fragmentation in K562 cells ( Figure  4A). AO/EB staining assay showed that the number of apoptotic cells with red fluorescence increased, while the number of viable cells with bright green fluorescence decreased when the cells were treated with increasing concentrations of EO ( Figure 4B). Hoechst 33258 staining revealed that the nucleus of EO-treated cells presented more and brighter blue-fluorescence compared with the control group, which indicated typical apoptotic morphology ( Figure 4C). Furthermore, to quantitatively determine the apoptotic effect of the EO, Annexin V-FITC/PI staining assay was performed using a flow-cytometer. As shown in Figure 5, the percentage of apoptotic cells (Q2+Q3) was enhanced to 13.72 ± 0.42% (at 30 mg/mL) and 15.48 ± 0.22% (at 60 mg/mL) in comparison to the control (6.14 ± 0.94%), which suggested that EO triggered apoptosis against K562 cells in a dose-dependent manner.

Enzyme Inhibitory Activities
The inhibitory effect of H. flavum rhizome EO, WE, and EE on a-glucosidase, tyrosinase, and two cholinesterases (AChE and BChE) were investigated and the results are shown in Table 5.
It is well known that a-glucosidase inhibitors reduce post-meal blood glucose and insulin levels by delaying the absorption of carbohydrates in the small intestine. Therefore, a-glucosidase inhibitors are used to treat type 2 diabetes (van de Laar et al., 2005). In this study, the inhibitory effect of a-glucosidase could be summarized with the following order: WE (IC 50 = 0.064 ± 0.004 mg/mL) > EE (IC 50 = 0.19 ± 0.01 mg/mL) > acarbose (IC 50 = 0.25 ± 0.02 mg/mL) > EO (IC 50 = 4.15 ± 0.26 mg/mL) (p < 0.05), which were presented in Table 5 and Figure 6. H. flavum rhizome WE (6.02 ± 0.79 mmoL ACEs/g sample) and EE (2.04 ± 0.20 mmoL ACEs/g sample) showed higher a-glucosidase inhibitory capacity than EO (0.094 ± 0.002 ACEs/g sample), expressed as acarbose equivalents per gram of extract. Interesting findings in this study are that WE and EE had higher a-glucosidase inhibitory activity than acarbose. Hence, H. flavum rhizome WE and EE could be a promising a-glucosidase inhibitor, which could be developed as a therapeutic product for type 2 diabetes.
Tyrosinase is a key enzyme involved in the catalysis of mammalian melanogenesis and the enzymatic browning reaction in fruits and vegetables. Therefore, tyrosinase inhibitors are used to control enzymatic browning in foods and for treatment of hyperpigmentation in human skin (Chang, 2009). As shown in Table 5, the best inhibitory effect on tyrosinase was provided by the EO (IC 50 = 6.12 ± 0.16 mg/mL, 32.51 ± 0.82 mg KAEs/g sample) followed by the EE (IC 50 = 9.62 ± 0.94 mg/mL, 20.85 ± 1.12 mg KAEs/g sample) and WE (IC 50 = 10.49 ± 1.48 mg/mL, 19.30 ± 2.72 mg KAEs/g sample). However, compared to positive control kojic acid (IC 50 = 0.20 ± 0.01 mg/mL), all of EO, WE, and EE didn't show a promising inhibition on tyrosinase.  Cholinesterase inhibitors have become the most effective treatment strategy for Alzheimer's disease by inhibiting the breakdown of acetylcholine to enhance cholinergic neurotransmission (Grutzendler and Morris, 2001). As shown in Table 5, the WE was the most effective AChE inhibitor (IC 50 = 2.27 ± 0.13 mg/mL, 0.108 ± 0.011 mg GALAEs/g sample) and BChE (IC 50 = 0.65 ± 0.04 mg/mL, 7.24 ± 0.26 mg GALAEs/g sample). Compared with the positive control drug galanthamine,  H. flavum rhizome EO, WE, and EE showed weak cholinesterase inhibitory activity. According to previous studies, the IC 50 values of aglucosidase inhibitory effect of 1,8-cineole and a-pinene were 1.118 and 1.420 mg/mL, respectively (Basak and Candan, 2013). a-terpineol, a-pinene, and 1,8-cineole showed significant tyrosinase inhibitory effect (Chao et al., 2017). The significant inhibited AChE activity of 1,8-cineole, b-pinene, a-pinene, and linalool has been demonstrated in previous studies (Abdelgaleil et al., 2009;Kim et al., 2013;Seo et al., 2015). Additionally, the linalool-rich EO was reported to exhibit strong a-glucosidase, tyrosinase, and cholinesterases (AChE and BChE) inhibitory activity (Sarikurkcu et al., 2015). Therefore, the enzyme inhibitory activities of EO may be due to these main components.
Previous studies have indicated that phenolic compounds play an important role in enzyme inhibitory activity (Goncalves and Romano, 2017). In this study, the a-glucosidase, and cholinesterases (AChE and BChE) inhibitory activities of WE were significantly higher than that of EE (p<0.05), except for not significant difference in tyrosinase inhibitory effect. However, WE revealed the significantly lower TPC and TFC compared with EE. This finding could be explained by the presence of nonphenolic and non-flavonoid enzyme inhibitors in the extract. Furthermore, UHPLC-Q-Orbitrap-MS analysis showed that many bioactive compounds may contribute to the enzyme inhibitory effect of the extract. For instance, the tyrosinase inhibitory activity of p-Coumaric acid (12) (IC 50 = 0.66 mg/ mL) was much higher than positive control arbutin (IC 50 > 100 mg/mL), and p-Coumaric acid showed a moderate a-glucosidase inhibitory activity (Pei et al., 2016). According to Ma et al. (2020), carnosol (44) displayed a competitive inhibition (Ki =5.57 mg/mL) on a-glucosidase with IC 50 value of 12.0 ± 0.8 mg/mL, and showed much stronger inhibitory effect than acarbose (IC 50 = 412 ± 26 mg/mL). Ginkgolic acid (C13:0) (62) revealed higher a-glucosidase inhibitory activity (IC 50 = 3.5 ± 0.13 mg/mL) than the positive control quercetin (IC 50 = 3.8 ± 0.12 mg/mL) (Sukito and Tachibana, 2014).

CONCLUSIONS
To our knowledge, the antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities of essential oil (EO), as well as the chemical composition and bioactivities of water extract (WE) and 70% ethanol extract (EE) from H. flavum rhizome were reported for the first time. The coronarin E was the most main constituent in EO and was first detected in H. flavum. The WE and EE exhibited high TPC and TFC values. Moreover, 86 compounds were putatively identified from WE and EE using UHPLC-Q-Orbitrap-MS, and all the compounds were reported in H. flavum for the first time. In addition to the low antioxidant   The cells were treated with EO (0, 30, 60 mg/mL) and the morphological changes of the cells were observed using an inverted microscope (100×). The cells were treated as described above and then stained with AO/EB (B) and Hoechst 33258 (C). The morphological changes were observed using a fluorescence microscope (100×). effect of EO and the weak inhibitory activity of EO and extracts on tyrosinase and cholinesterase, EO demonstrated significant antibacterial activity against P. vulgaris, P. aeruginosa, B. subtilis, and S. aureus, and showed significant a-glucosidase inhibitory effect. Besides, EO exhibited a considerable selectivity to human tumor cell K562, and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 and L929 cells. A series of apoptosis analysis suggested that EO triggered apoptosis against K562 cells in a dose-dependent manner. The very low inhibition against the tumor and non-cancerous cell lines suggested that WE and EE were less-toxic. Interesting findings in this study are that WE and EE displayed remarkable antioxidant and aglucosidase inhibition activities, being superior to their respective positive control drugs BHT and acarbose. Hence, H. flavum could be considered as a source of bioactive compounds and has high exploitation potential in the cosmetics, food, and pharmaceutical industries.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
YZ and MT conceived and designed the experiments. MT and XW performed the experiments. TL, XZ, and FW analyzed the data.  MT and GD drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.