Phytochemical analysis, antioxidant, antimicrobial, and anti-enzymatic properties of Alpinia coriandriodora (sweet ginger) rhizome

Alpinia coriandriodora, also known as sweet ginger, is a medicinal and edible plant. A. coriandriodora rhizome is popularly utilized in traditional Chinese medicine and as flavouring spices, but there are few reports on its constituents and bioactivities. This study analyzed the phytochemical components of A. coriandriodora rhizome by GC-MS and UHPLC-Q-Orbitrap-MS and evaluated its antioxidant, antimicrobial, and anti-enzymatic properties. According to the GC-FID/MS data, its rhizome essential oil (EO) consisted mainly of (E)-2-decenal (53.8%), (E)-2-decenyl acetate (24.4%), (Z)-3-dodecenyl acetate (3.5%), and (E)-2-octenal (3.5%). Its water extract (WE) and 70% ethanol extract (EE) showed high total phenolic content (TPC, 52.99–60.49 mg GAEs/g extract) and total flavonoid content (TFC, 260.69–286.42 mg REs/g extract). In addition, the phytochemicals of WE and EE were further characterized using UHPLC-Q-Orbitrap-MS, and a total of sixty-three compounds were identified, including fourteen phenolic components and twenty-three flavonoid compounds. In the antioxidant assay, WE and EE revealed a potent scavenging effect on DPPH (IC50: 6.59 ± 0.88 mg/mL and 17.70 ± 1.15 mg/mL, respectively), surpassing the BHT (IC50: 21.83 ± 0.89 mg/mL). For the antimicrobial activities, EO displayed excellent antibacterial capabilities against Proteus vulgaris, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus with DIZ (12.60–22.17 mm), MIC (0.78–1.56 mg/mL), and MBC (3.13 mg/mL) and significantly inhibited Aspergillus flavus growth (MIC = 0.313 mg/mL, MFC = 0.625 mg/mL, respectively). In addition to weak tyrosinase and cholinesterase inhibition, EE and WE had a prominent inhibitory effect against α-glucosidase (IC50: 0.013 ± 0.001 mg/mL and 0.017 ± 0.002 mg/mL), which was significantly higher than acarbose (IC50: 0.22 ± 0.01 mg/mL). Hence, the rhizome of A. coriandriodora has excellent potential for utilization in the pharmaceutical and food fields as a source of bioactive substances.

Alpinia coriandriodora D. Fang., also known as sweet ginger, is a perennial plant with a distinctive coriander fragrance that is extensively cultivated in China due to its medicinal and edible value (Wu and Larsen, 2000;Cheng et al., 2021).A. coriandriodora rhizome is used as traditional Chinese medicine for treating fever, asthma, cold, stomachache, and indigestion (CHMC-Chinese Herbal Medicine Company, 1994;Zhang and Li, 2015;Wu et al., 2016).Moreover, its rhizome was utilized as spices in the preparation of soups and stews.Interestingly, wrapping zongzi with its leaves is a specialty of residents in Guangxi, China.In previous studies on A. coriandriodora rhizome, several diarylheptanoids and flavonoids were isolated, and these compounds had intracellular antioxidant and anti-inflammatory actions by inhibiting NO release (Cheng et al., 2021).A. coriandriodora is rich in essential oil, and its EO has been used in traditional medicine (Tran et al., 2022).Previous research indicated that A. coriandriodora rhizome EO was mainly composed of (E)-2decenal (60.4-56.3%)and (E)-2-decenyl acetate (19.6-4.3%) and possessed potent anticancer and anti-malassezia effects (Hong et al., 2022;Tran et al., 2022).
Despite its high edible and medicinal value, there are few reports on the phytochemistry and biological activities of A. coriandriodora rhizome, which may hinder its utilization.Thus, this study analyzed the phytochemical constitution of A. coriandriodora rhizome using GC-FID/MS and UHPLC-Q-Orbitrap-MS and evaluated its antioxidant, antimicrobial, and anti-enzymatic properties.

Plant material
Alpinia coriandriodora D. Fang was cultivated at Guigang City, Guangxi Province, China (latitude: 23°80′89.74′′Nand longitude: 110°18′64.68″E).The plant samples were harvested in August 2020 and authenticated by Professor Guoxiong Hu, a plant taxonomist.A voucher specimen has been deposited at the Herbarium of the College of Life Sciences, Guizhou University, China (Voucher No: AC2020803).

Extraction of EO, WE, and EE
Hydro-distillation was employed to prepare the EO of A. coriandriodora rhizome.In brief, the fresh rhizome (2 kg) was washed using distilled water, crushed, and hydrodistilled (4 h) in a Clevenger's apparatus.After drying with anhydrous Na 2 SO 4 , EO was stored at 4°C until further use.
Reflux extraction was used to produce the WE and EE of the A. coriandriodora rhizome.The crushed plant material (0.5 kg) and 70% ethanol or distilled water (2 L) were added to a roundbottomed flask (5 L).Afterward, the mixture was reflux-extracted twice (2 h/reflux).The extraction solution was mixed, filtered, condensed using a rotary evaporator, and dried using a freeze drier.Then, WE and EE were weighed and stored in a brown glass bottle at 4°C until usage.

EO's composition analysis using GC-FID/MS
Quantitative analysis of phytochemical components was performed on GC-FID (Agilent Technologies, 6890N) equipped with an HP-5MS column (60 m × 0.25 mm, 0.25 mm film thickness).The helium was used as a carrier gas (1 mL/min).The injection volumes were 1 µL (split ratio 1:20).The injector temperature was 250°C.The oven temperature was programmed as follows: kept at 70°C (2 min), raised to 180°C (2°C/min), increased to 310°C (10°C/min), and held at 310°C for 14 min (running time: 84 min).Agilent 6890/5975C GC-MS was employed for qualitative analysis, and its GC settings were the same as those of GC-FID.The following conditions were applied in MS: ion source (EI, 70 eV); ion source temperature (230°C); interface temperature (280°C); scan range from 29 to 500 m/z.The peak area revealed the relative percentage of different chemical constituents.A series of n-alkanes (C 8 -C 16 ) were used to determine the retention index (RI).Matching the RI and MS information in Wiley 275 and NIST 2020 databases was done to identify the chemical constituents.

TPC analysis
The TPC in WE and EE were quantified using the Folin-Ciocalteu method (Tian et al., 2020).Folin-Ciocalteu reagent (2.5 mL) and sample solution (0.5 mL) were combined and kept at 25°C for 5 min.The 7.5% Na 2 CO 3 solution (4 mL) was subsequently incorporated and reacted for 1 h at 25°C.The absorbance at 760 nm was measured, and gallic acid equivalents (mg GAEs/g extract) were used to express TPC.

TFC analysis
The TFC in WE and EE were quantified using NaNO 2 -Al(NO 3 ) 3 -NaOH colorimetry with minor modifications (China Pharmacopoeia Committee, 2020).The sample solution (5 mL) and NaNO 2 (0.4 mL, 5%) were blended and kept at 25°C for 5 min, which was reacted with 10% Al(NO 3 ) 3 solution (0.4 mL) for 5 min.Subsequently, 4 mL NaOH solution (4%) and distilled water were added to gain a final volume of 10 mL and reacted 15 min at 25°C.The absorbance at 510 nm was measured, and rutin equivalents (mg REs/g extract) were applied to represent TFC.

DPPH assay
Equal amounts of DPPH solution (0.08 mM) and sample solution (100 mL) were mixed, and the reaction was carried out at 25°C for 30 min away from light.Optical density at 517 nm was measured, and IC 50 values and BHT equivalents (mg BEs/g sample) were calculated for demonstrating the scavenging effect of DPPH.

ABTS assay
In order to produce the ABTS• + solution, 50 mL of ABTS solution (0.7 mM) was reacted with an equal amount of K 2 S 2 O 8 solution (2.45 mM) for 12 h at 25°C without light.In addition, it was further diluted with ethanol prior to use to yield an absorbance of 0.70 ± 0.02 at 734 nm.Then, 0.4 mL of sample solution reacted with 4 mL of diluted ABTS• + solution for 10 min at 25°C away from light.Optical density at 734 nm was gained, and IC 50 values and the equivalents of BHT (mg BEs/g sample) were expressed in the results.

Agar well diffusion assay
The agar well diffusion method assayed the diameter of the inhibition zone (DIZ) (Zhang et al., 2017).EO was dissolved in DMSO and diluted with sterile distilled water (DMSO content was less than 0.5%).Sterile distilled water was used to dissolve the WE and EE.Three sample solutions were brought to a concentration of 100 mg/mL.Streptomycin was dissolved in distilled water to reach a 100 mg/mL concentration, which was used as a positive control.The Mueller-Hinton agar medium surface was spread with 100 mL of the bacterial suspension (1 × 10 7 CFU/mL) and was covered with filter paper discs of 6 mm diameter (containing 20 mL sample solutions).The DIZ was determined after 24 h of incubation at 37°C.

Determination of MIC and MBC
In a 96-well plate, 100 mL of bacterial suspensions (1 × 10 6 CFU/ mL) was added to an equal amount of sample solution.After 24 h of incubation at 37°C, each well was supplied with 20 mL of resazurin aqueous solution (100 mg/mL), which was cultured for 2 h at 37°C without light.The minimal sample concentration without color change was determined as MIC (minimal inhibitory concentration) (Tian et al., 2020).The mixture (10 mL) from the wells that did not change color was subcultured in a Mueller Hinton agar plate for 24 h at 37°C, the least sample concentration at which no bacterial growth was determined as MBC (minimal bactericidal concentration) (Eloff, 1998).

Antifungal capacity
The anti-Aspergillus flavus activity of EO was tested by a previously published method with slight modification (da Silva et al., 2012) using amphotericin B as a positive control.The EO solution (3.125 mg/mL-100 mg/mL) was mixed with Potato Dextrose Agar (PDA) medium (2:18, v:v).Subsequently, the mixture (15 mL) was added to Petri dishes (diameter 90 mm) and solidified.The A. flavus spore suspension (1 × 10 3 CFU/mL, 100 mL) was inoculated into wells (6 mm in diameter) of the culture medium and incubated at 28°C for 36 h.The MIC value was recorded as EO concentrations that inhibited the visible growth of A. flavus, and the diameter of colony growth zone was less than 12 mm (including well diameter of 6 mm) (Yadav et al., 2005).The minimum fungicidal concentration (MFC) was the minimum sample concentration showing no A. flavus growth on the PDA medium.

Enzyme inhibitory properties
The enzyme inhibitory properties of A. coriandriodora rhizome WE, EE, and EO on cholinesterases (AChE and BChE), tyrosinase, and a-glucosidase were tested, and galanthamine, arbutin, and acarbose were used as positive controls, respectively (Tian et al., 2020).

Tyrosinase inhibition
In a 96-well plate, 100 mL of tyrosinase solution (100 U/mL) was mixed with 70 mL of sample solution and incubated for 5 min at 37°C . Then, 80 mL of L-tyrosine solution (5.5 mM) was injected into each well.After 30 min of reaction at 37°C, the absorbance at 492 nm was measured.Data were presented as arbutin equivalents (mg AREs/g sample) and IC 50 values.

a-Glucosidase inhibition
In a 96-well plate, 10 mL of a-glucosidase solution was reacted with 90 mL sample solution for 15 min at 37°C.Next, 10 mL of p-NPG (p-Nitrophenyl-a-D-glucopyranoside) solution (1 mM) was injected into each well and maintained at 37°C or 15 min.Lastly, 80 mL of Na 2 CO 3 (0.2 M) was added for stopping the reaction.After the absorbance detection at 405 nm, a-glucosidase inhibition was represented as acarbose equivalents (mmoL ACEs/g sample) and IC 50 values.

Statistical analysis
All data were collected from three independently performed experiments and were expressed as mean ± standard deviation (SD).In SPSS (version 25) software, the two-tailed unpaired t-test or one-way analysis of variance (ANOVA) and Fischer's LSD post hoc test (p < 0.05) were utilized to compare the significant difference between the two groups.

Chemical composition of WE and EE
Based on the fresh weight of A. coriandriodora rhizome, the yield of WE and EE were 2.04% (w/w) and 1.37% (w/w), respectively.As shown in Figure 2, A. coriandriodora rhizome WE (60.49± 0.24 mg GAEs/g extract) exhibited more TPC compared to EE (52.99 ± 0.16 mg GAEs/g extract) (p < 0.01).The TFC in WE (286.42 ± 2.21 mg REs/g extract) was notably greater than that in EE (260.69 ± 0.44 mg REs/g extract) (p < 0.01).

Antioxidant capacity
In the antioxidant capacity of A. coriandriodora rhizome (Table 3), the scavenging effect of EO on DPPH and ABTS was weak.WE (IC 50 : 6.59 ± 0.88 mg/mL) and EE (IC 50 : 17.70 ± 1.15 mg/mL) possessed Excessive production of reactive oxygen species triggers oxidative stress, which could induce or aggravate many chronic diseases, such as neurodegenerative disorders, cardiovascular disease, asthma, and cancer (Loṕez-Alarcoń and Denicola, 2013; Pisoschi and Pop, 2015).
Past studies have revealed that various phenolic and flavonoid compounds from Alpinia genus plants were natural antioxidants that are effective in scavenging free radicals (Honmore et al., 2016;Tang et al., 2018;da Cruz et al., 2020).Based on the results of UHPLC-Q-Orbitrap-MS, a variety of identified phenolic and flavonoid compounds, such as protocatechuic acid (8), methyl gallate (10), vanillic acid (11), and epicatechin ( 14), has been proved to have DPPH and ABTS scavenging effects (Kakkar and Bais, 2014;Li et al., 2014;Bernal-Mercado et al., 2018;Ryu and Kim, 2022).Hence, the antioxidant activity of A. coriandriodora rhizome WE and EE can be attributed to their richness in phenolics and flavonoids, which can serve as a natural source of antioxidants.

Antibacterial properties
The antibacterial properties of EO, WE, and EE were determined qualitatively using the DIZ and assessed quantitatively using the MIC and MBC.Streptomycin was used as the control drug (Table 4).The DIZ of WE and EE (100 mg/mL) were not observed, and the MIC and MBC of WE and EE were not listed as they were not detected at 25 mg/mL.EO exhibited broad-spectrum antibacterial activity with DIZ values ranging from 12.60 to 22.17 mm.Previous studies suggest that MIC values less than 5 mg/mL have potent antibacterial properties (Monteiro et al., 2021).Hence, the EO displayed a strong antibacterial effect against P. vulgaris (MIC = 1.56 mg/mL, MBC = 3.13 mg/mL), E. faecalis (MIC = 0.78 mg/mL, MBC = 3.13 mg/mL), B. subtilis (MIC = 0.78 mg/mL, MBC = 3.13 mg/mL), E. coli (MIC = 0.78 mg/mL, MBC = 6.25 mg/mL) and S. aureus (MIC = 0.78 mg/mL, MBC = 3.13 mg/mL).In addition, it revealed moderate antibacterial activity against P. aeruginosa (MIC = 6.25 mg/mL, MBC = 6.25 mg/mL).(E)-2-Decenal, as the predominant component, has been proven to possess broad-spectrum antibacterial action, with MIC values ranging from 7.8 to 500 mg/mL against E. coli, S. aureus, S. epidermidis, Salmonella typhi, S. enteritidis, Bacillus cereus, Moraxella catarrhalis, Haemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, and S. pyogenes (Bisignano et al., 2001;Trombetta et al., 2002).(E)-2-Octenal was a potential antibacterial agent that inhibited the growth of a variety of bacteria, including S. aureus, E. coli, P. vulgaris, P. aeruginosa, Enterobacter aerogenes, Propionibacterium acnes, Streptococcus mutans, Brevibacterium ammoniagenes, and Bacillus subtilis (Kubo and Kubo, 1995;Sagun et al., 2016).Thus, the antibacterial action of A. coriandriodora EO can be attributed to these main components.Bacterial infections and the emergence of bacterial resistance have threatened human public health around the world (Fernandes et al., 2022).Therefore, EO with antibacterial properties has gained great attention.Our results suggested that A. coriandriodora EO may be used in the food and pharmaceutical industries as a natural antibacterial agent.

Antifungal capacity
The anti-Aspergillus flavus properties of A. coriandriodora rhizome EO are shown in Figure 3 and Table 5.The EO exhibited   anti-Aspergillus flavus effects in a dose-dependent manner.The growth of A. flavus on the PDA medium was obviously inhibited when the EO concentration reached 0.625 mg/mL (MIC).Besides, EO completely inhibited A. flavus growth at the concentration of 1.250 mg/mL (MFC).(E)-2-Decenal is a potent antifungal agent against several fungi, including Trichophyton mentagrophytes, Penicillium chrysogenum, Pityrosprum ovale, Candida utilis, Saccharomyces cerevisiae, Purpureocillium lilacinum, Isaria fumosorosea, Metarhizium flavoviride, Paecilomyces suffultus, Beauveria bassiana, and Akanthomyces lecanii (Kubo and Kubo, 1995;Bojke et al., 2020).(E)-2-Octenal has been proven to be a substitute for chemical fungicides with antifungal activity against Trichophyton mentagrophytes, Penicillium italicum, Sclerotium rolfsii, Aspergillus niger, Penicillium digitatum, Alternaria alternate, Microsporum canis, Botrytis cinerea, etc. (Battinelli et al., 2006;Liarzi et al., 2016;Liarzi et al., 2020).In particular, (E)-2-octenal   Anti-Aspergillus flavus activity of A. coriandriodora rhizome EO. exerted a potent inhibitory effect on the growth of A. flavus and the production of aflatoxin B1 (Cleveland et al., 2009).Hence, these compounds could explain the remarkable anti-Aspergillus flavus activity of A. coriandriodora rhizome EO. A. flavus is known to cause spoilage of vegetables, grains, fruits, and nuts, which results in huge economic losses worldwide (Hashem et al., 2022).In addition, aflatoxin produced by A. flavus poses a great threat to human health due to its immunosuppressive, genotoxic, and carcinogenic effects (Lasram et al., 2019;Yadav et al., 2019).Recent studies have suggested that essential oils can be used as a greener alternative to protect foods from Aspergillus flavus contamination (Dutta et al., 2020;Kundu et al., 2022).Hence, A. coriandriodora rhizome EO has the exploitation potential as an anti-Aspergillus flavus agent in the food field.

Enzyme inhibitory properties
The enzyme inhibitory properties of A. coriandriodora rhizome WE, EE, and EO on cholinesterases (AChE and BChE), tyrosinase, and a-glucosidase were tested, and the results are shown in Table 6.
AD (Alzheimer's disease) is a chronic disease with memory loss and cognitive impairment caused by central nervous system degeneration, and low levels of cholinergic energy in the human brain play an essential role in the pathogenesis of AD (Sharma, 2019).As shown in Table 6, compared with the positive control galanthamine, EO, WE, and EE of A. coriandriodora rhizome showed weak AChE (IC 50 : 5.84 ± 0.48 mg/mL, 6.67 ± 0.04 mg/mL, and 1.59 ± 0.09 mg/mL, respectively) and BChE (IC 50 : 5.73 ± 0.33 mg/mL, 3.61 ± 0.89 mg/mL, and 0.38 ± 0.02 mg/mL, respectively) inhibitory activity.
In mammals, tyrosinase is a crucial enzyme in the production of melanin, which protects against UV damage, but excessive melanin can lead to many skin problems, such as malignant melanoma, freckles, and age spots (Brenner and Hearing, 2008;Sun et al., 2017).Tyrosinase inhibitors have been extensively applied in cosmetic and medicine industries to solve these problems.According to the results presented in Table 6, EO (IC 50 : 1.01 ± 0.08 mg/mL, 253.12 ± 9.13 mg AREs/g sample) had the most strong tyrosinase inhibition, followed by WE (IC 50 : 8.91 ± 1.69 mg/mL, 29.78 ± 5.61 mg AREs/g sample) and EE (IC 50 : 19.85 ± 3.74 mg/mL, 13.21 ± 2.71 mg AREs/g sample).(E)-2octenal, as the main components of A. coriandriodora rhizome EO, have been demonstrated to possess tyrosinase inhibition (Kubo and Kinst-Hori, 1999).Plant-derived flavonoids and phenolic compounds can serve as natural inhibitors of tyrosinase (Gonccalves and Romano, 2017).A variety of phenolic and flavonoid compounds identified from WE and EE, such as orsellinic acid, arctigenin, and hordenine, have been confirmed to possess tyrosinase inhibitory activity (Kim et al., 2013;Park et al., 2013;Lopes et al., 2018).Thus, the tyrosinase inhibition of A. coriandriodora rhizome can be attributed to the presence of these active ingredients.

Conclusions
In addition to the chemical composition of A. coriandriodora rhizome EO, this study is the first to analyze the phytochemical components of WE and EE and evaluate their antioxidant, antimicrobial, and enzyme inhibitory properties.According to the GC-MS data, the primary components of A. coriandriodora rhizome EO were (E)-2-decenal, (E)-2-decenyl acetate, (Z)-3-dodecenyl acetate, and (E)-2-octenal. A. coriandriodora rhizome WE and EE were abundant in phenolics and flavonoids.UHPLC-Q-Orbitrap-MS analysis further identified sixty-three compounds in WE and EE, including 14 phenolic compounds and 23 flavonoids.WE and EE exhibited a potent DPPH radical scavenging effect, superior to the positive control BHT.Besides, EO displayed powerful antimicrobial activity against E. faecalis, S. aureus, B. subtilis, E. coli, P. vulgaris, and A. flavus.In enzyme inhibitory activity, the EE and WE revealed a remarkable a-glucosidase inhibition, which were higher than positive control acarbose.Hence, A. coriandriodora rhizome may be used as a source of bioactive substances and has excellent potential for exploitation in the pharmaceutical and food fields.

FIGURE 2
FIGURE 2Total phenolic and flavonoid contents of A. coriandriodora rhizome WE and EE, **p < 0.01.

TABLE 1
Chemical components of the EO from A. coriandriodora rhizome.Retention indices (RI) were determined utilizing n-alkanes (C 8 -C 16 ).b RI were obtained through NIST 2020 database.c Identification: MS, comparing MS similarity with NIST 2020 and Wiley 275 databases; RI, comparison of calculated RI with those in NIST 2020 database.d tr, trace < 0.1%.

TABLE 2
Phytochemical compounds of A. coriandriodora rhizome WE and EE detected and characterized using UHPLC-Q-Orbitrap-MS in positive and negative ionization modes.

TABLE 2 Continued
Based on comparison with mzCloud and mzVault databases and references.b "√" means detected from extracts, "-" means undetected from extracts.
a Identification:

TABLE 3
Antioxidant activity of EO, WE and EE from A. coriandriodora rhizome.The concentration of sample that scavenged 50% free radical.2Themg BEs/g sample represents milligrams of BHT equivalent per gram of sample. 3BHT as positive control.a-c Different letters in the same column represent significant difference (p < 0.05).

TABLE 6
The enzyme inhibitory properties of A. coriandriodora rhizome EO, WE, and EE.: The concentration of sample that affords a 50% inhibition in the assay.The mg GALAEs/g sample represents milligrams of galanthamine equivalent per gram of sample; The mg AREs/g sample refers to milligrams of arbutin equivalent per gram of sample; The mmoL ACEs/g sample means mmoL of acarbose equivalent per gram of sample.a-d Different letters in the same column represent significant difference (p<0.05).*Galanthamine: IC 50 (mg/mL).
d IC 50