GC-MS based metabolite profiling and antioxidant activity of solvent extracts of Allium chinense G Don leaves

Allium chinense, a main source of “Xiebai” drug in Chinese traditional medicine and commonly known as Ganoderma lucidum belongs to the family Amaryllidaceae. The main focus of this research was to quantify the secondary metabolites, antioxidant potential and study the GCMS based metabolite profile of different solvent leaf extracts of A. chinense. The reports on the bioactive compounds of A. chinense leaves are still insufficient compared to the bulb; hence this study was carried out to understand the bioactive compounds present in A. chinense leaves using different solvents of varying polarity. Our investigation showed that the ethanol extract contained the highest saponin, flavonoid, phenol, and DPPH scavenging activity. Further, metabolite profiling revealed a total of forty-eight compounds, indicating a diverse range of phytochemicals present in the four extracts. The highest number of compounds were observed in ethanol extract (15) followed by chloroform extract (13), petroleum ether extract (11) and methanol extract (9). Some of the major compounds identified in the four solvents are octacosane (27.11%), heptadecane (19.66%), eicosane (18.51%), ethyl palmitate (18.50%), phytol (17.68%) and phytol acetate (17.30%). In conclusion, this study highlights that A. chinense leaf extracts contain high saponins, terpenes and alkanes which could be a potential source of a new beneficial drug.


Introduction
Medicinal herbs are a rich source of a bioactive compound and considered to play a beneficial role in traditional and modern health care delivery systems. The bioactive compounds synthesized from these plants provide the raw material for the cosmetic and pharmaceutical industries (Kretovich, 2005). Allium chinense G Don, is a medicinal herb from the Amaryllidaceae family, commonly known as the oriental onion or Ganoderma lucidum in vegetables. The plant is widely distributed in India's North-East states and grows well in 15-30 °C temperature with moderate fertile soils (Lim et al., 2015). It has a strong onion-like odor and known for its rich organo-sulfur (Pino et al., 2001;Liu et al., 2014) and saponin (Sobolewska et al., 2020) content and is also the original source of the famous Chinese medicine "Xiebai", as it functions like a tonic to the digestive system (Yao et al., 2016). Saponins from edible plants have been reported to have diverse biological

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Notulae Scientia Biologicae functions, including antitumor effects (Wang et al., 2019). The essential oil of A. chinense bulb and leaf has been reported to have sulfide containing compounds (Pino et al. 2001;Liu et al., 2014) which are the main components for odour and it is effective in breaking seed and bud dormancy (Hosoki et al., 1986;Kubota et al., 2000). However, there is no report on quantification, GC-MS based metabolite profiling, and antioxidant potential of different solvents extracts of A. chinense leaves. The investigation on bioactive compounds of A. chinense leaves is still inadequate compared to the research findings on the bulb. Hence, the present study aimed to identify and characterize biomolecules by GC-MS analysis and to determine the antioxidant potential of A. chinense leaf in different solvent extracts.

Quantitative analysis
Determination of total phenols The total phenols in different solvent extracts of A. chinense leaf were evaluated using FC (Folin-Ciocalteu) spectrophotometric method (Khan and Bhat, 2018). The standard curve was plotted using gallic acid at 1-100 µg/ml concentrations. Plant extracts at 1mg/ml were mixed with 5 ml of FC (1:10) and 4 ml of sodium bicarbonate (7.5%) and the mixture was incubated for half an hour in the dark at 20 °C to allow the reaction to take place. The absorbance was measured at a wavelength of 765 nm against blank.

Estimation of total alkaloids
The total alkaloids were quantified using atropine as a reference standard at 200-1000 µg/ml concentration to obtain the calibration curve (Tan, 2018). The extracts (1 mg/ml) were mixed with 2 ml of 2N HCl and washed with 5 ml of chloroform. The solution was vortexed and layers were separated using a micropipette. Later, the separated layer was taken in a test-tube and 5 ml of BCG (Bromocresol Green) solution and phosphate buffer (pH 4.7) was added and vortexed. A yellow color complex observed at the bottom was carefully removed by using pipette and absorbance was measured at a wavelength of 470 nm.

Estimation of total flavonoids
The quantification of total flavonoids content in different solvent extracts was determined using the AlCl3 (Aluminum trichloride) method. Catechin was used as a standard (Aryal et al., 2019) at 20-100 µg/ml concentration to plot the standard calibration graph. Plant extracts (0.5 ml) with 1 mg/ml concentration was suspended in distilled water (2 ml) in a test tube, 0.15 ml of 5% NaNO2 was added to it and incubated for 6 minutes. Thereafter, 0.15 ml of 10% AlCl3 was added and incubated again for 6 minutes. Later, 2 ml of 10% NaOH was added to it and the final volume was made up to 5 ml using distilled water. The solution was then incubated at room temperature for 15 minutes and the absorbance of the solution was read at 510 nm.

Estimation of saponins
The saponin content of the extracts was estimated using the method described by Le et al. (2018). The Plant extract at 1mg/ml concentration was taken in a test tube and 500 µL of 8% vanillin and 72% sulphuric acid was added and incubated for 10 minutes at 60 °C in a water bath. After incubation, it was allowed to cool to room temperature and absorbance was measured at 544 nm. Quillaia (200-1000 µg/ml) was used as a standard to generate the standard calibration curve.
DPPH free radical scavenging assay A. chinense leaf's ability in different solvent extracts to scavenge free radicals was evaluated (Vasundhara et al., 2017) using ascorbic acid as the standard and all the tests were performed in triplicate. One ml of varying plant extract and ascorbic acid with 1-100 μg/ml concentrations were mixed with 3.0 ml of DPPH (0.06 mM) and incubated in dark for 15 minutes at room temperature for reaction to occur. The absorbance was measured at a wavelength of 517 nm using a UV-Vis spectrophotometer. The percentage of inhibition of the extracts and ascorbic acid was calculated using the given formula: DPPH radical scavenging activity (%) = [(A1 -A2) / A1] × 100 Where A1 -Absorbance of control (DPPH), A2 -Absorbance of extracts The calibration curve was generated and 50% inhibition concentration (IC50) values were calculated.

GC-MS analysis and identification of compounds
To obtain the complete chemical profile of A. chinense leaf, GC-MS analysis was performed using SHIMADZU QP2010S GC-MS system. The operating condition was set up as follows: and oven temperature was programmed at 70.0°C; the ion source temperature was set at 200.00 °C sample was injection mode was split less and sampling time 2.00 min equipped with Rxi-5Sil MS column; length: 30 meters, the carrier gas was helium (99.99%) at a flow of 1.00 mL/min; start time 7.00 min; End time 35.75 min; event time 0.50 sec; scan range 50-500 m/z. The chemical compounds were identified based on the peaks observed at different mass-tocharge ratios. Further identification was made by comparing with the standard spectrum existing in the database mass spectral library of the National Institute of Standards and Technology NIST-11 and WILEY 8 library.

Statistical analysis
The data were analysed using a one-way method of Analysis of variance (ANOVA) at a 5% probability (P,0.005) by using Prism V. 5.00 (Graphpad Inc. USA). The total phenol, alkaloid, flavonoids and saponin content was estimated using the linear regression equation obtained from the standard graph. All the analysis was carried out in triplicates and expressed as mean ± SE.

Quantitative analysis
In recent years, plant metabolites have played an important role in alleviating several ailments and multiple health benefits, the knowledge on phyto components is important to obtain active principles for its highest pharmacological significance. The different solvent extract has a significant difference in the concentration of total phenols, alkaloid, flavonoid, and saponin (Table 1). The highest concentration of saponins was recorded in ethanol extract, followed by methanol extract, petroleum ether extract and chloroform extract. Quantitative analysis in leaf extract showed a moderately high concentration of alkaloid in chloroform and petroleum ether extract; however, in methanol and ethanol the concentration was moderate. In quantitative estimation, low phenol and flavonoid content was observed in leaf extract; ethanol extract showed higher phenolic and flavonoid content than the other extracts.
Saponin are widely distributed in monocotyledonous families (Sobolewska et al., 2020), and several studies have confirmed that they have an extensive range of pharmacological activities. Steroidal saponins vernoniamyoside A, B, and B2, possess cytotoxicity activity against BT-549 , steroidal sapogenin 25-R-spirosta-3, 5-dien-12β-ol showed cytotoxicity on 5-8F cells a human nasopharyngeal carcinoma cell line (Chu et al., 2018). Saponin subsides coronary heart disease  and acts as cytotoxic towards the human glioblastoma U87MG and U251 cell lines (Liu et al., 2018). Saponins like diosgenin have great value in the pharma industry and used as substrates in drug and steroid hormone production (Sobolewska et al., 2020). The presence of a high saponin amount might have contributed to the significant use of this plant in traditional medicine and drug discovery. Quantitative analysis of leaf extract showed a moderately high concentration of alkaloid. Plant alkaloids possess anti-inflammatory, anti-depressive, antioxidant, anti-convulsing, anti-amyloid efficacy, antiviral, antifungal, anticancer and antibacterial activity (Hussain et al., 2018;Thawabteh et al., 2019). The analysis showed a moderate concentration of phenol and flavonoid content in the leaf extract. Phenols possess high antioxidant activity (Safari and Ahmady-Asbchin, 2019) and contribute hydrogen and react with nitrogen and reactive oxygen compounds acting as an antioxidant (Pereira et al., 2009). Medicinal plants have a copious amount of flavonoids and are considered potential nutraceuticals, They also regulate numerous pathways for diseases like diabetes, neuro-disease, cancers, and other transmittable diseases .

Antioxidant assay
The free radical scavenging activity was determined in all the four solvent extracts of A. chinense leaf viz ethanol, methanol, chloroform, and petroleum ether by the DPPH method. Scavenging activity against free radicals from leaf extracts is shown in (Figure 1, Table 2).  The A. chinense leaf extracts showed moderate scavenging activity in all the four solvents compared to standard ascorbic acid (IC50=55.096 µg/ml) with a significant difference (p<0.05). Ethanol extract with IC50 value of 228.156 µg/ml was found to exhibit the highest free radical scavenging activity when compared to other solvent extracts, followed by methanol with IC50 value of 391.529 µg/ml, petroleum ether with IC50 of 947.025 µg/ml, and chloroform extract with IC50 value of 1045.287 µg/ml. Free radicals are unstable molecules that cause oxidative stress, triggering cell damage, antioxidants are substances that may prevent or delay cell deterioration, vegetables and fruits are a rich source of antioxidants (Lobo et al., 2010). The percentage of free radical scavenging activity was found to be significantly low, which may be due to a lower concentration of phenol  and flavonoids in the present study (Liu et al., 2018). Lin et al. (2016) observed mild antioxidant activity in the essential oil of A. chinense bulb which supports the present findings.

GC-MS analysis
Identifying plant's chemical constituents is important for finding new therapeutic agents and GC-MS is the key technology for secondary metabolites profiling in plants. Bioactive compounds identified by GC-MS analysis showed that the leaf extracts have a complex combination of numerous compounds; some of which were present in trace quantities. The GC-MS analysis of methanol, ethanol, chloroform, and petroleum ether extracts of A. chinense leaf revealed a total of 48 peaks. The highest number of peaks was observed in ethanol extract. Fifteen compounds were identified in ethanol extract with a run time of 50 min. (Figure 2, Table 3). In the chloroform extract, thirteen peaks were observed with a run time of 45 min. (Figure 3, Table 4). The petroleum ether extract was run for 49 min. (Figure 4, Table 5) where eleven peaks were recorded. A total of nine peaks were detected in the chromatogram of methanol leaf extract with a run time of 49 min. (Figure 5, Table 6). The peaks indicated the number of compounds and the active compounds was confirmed based on retention time, molecular weight, molecular formula, and molecular structure. Among the various phytochemicals identified, phytol and phytol acetate were the most common chemical compounds found in all four extracts.   The most versatile compounds in ethanol leaf extract were ethyl palmitate (18.50%) which is a longchain fatty acid ethyl ester with has nematicide, antioxidant, anti-androgenic, hemolytic, flavor, and hypocholesterolemic (Tyagi and Agarwal, 2017) activity followed by phytol (15.94%) a diterpene mostly used as a fragrance in the pharmacological and biotechnological industry has autophagy, antioxidant, anxiolytic, immune-modulating, anti-inflammatory, cytotoxic, metabolism-modulating, apoptosis-inducing, antinociceptive, antimicrobial effects nematicidal, antibacterial, anti-inflammatory and pesticidal activities (Islam et al., 2018;Adnan et al., 2019). No activity has been reported in cis, cis, cis-7,10,13-hexadecatrienal (15.34%), and Z, Z-6,13-octadecadien-1-ol acetate (11.97%).
All the four extracts of the A. chinense leaf exerted a very high amount of saponin content. The ethanol extract had the highest concentration of saponins, flavonoid, and phenol. The GC-MS report showed a high content of terpenoids and alkanes. The metabolite profile reported from the different solvent extracts of the present study varies from the essential oil extracted from both A. chinense leaves and bulbs (Pino et al., 2001;Liu et al., 2014). The difference in results may be due to the extraction method, climatic conditions, soil pH, seasonal variation and many other environmental factors.

Conclusions
To the best of our knowledge, this is the first GC-MS report from India on the metabolite profile of A. chinense leaf extracts using methanol, ethanol, petroleum ether, and chloroform solvent. The extracts of A. chinense are rich in saponins, terpenes and alkanes representing an important step to understand the phytochemicals constituent of the leaf extracts which could facilitate further use in the pharmaceutical and food industry considering its availability since it is non-toxic and edible. The present results may recommend the use of A. chinense leaf to treat various ailments where this plant may be a natural source of a new drug to the scientific and biomedical communities.

Authors' Contributions
TR; Performed all the experiments and drafted the manuscript. RMS; Participated in carrying out the analysis. SR; Participated in interpretation of the data. SV; Participated in design and critical revision of the manuscript. All authors read and approved the final manuscript. studies and the Central Instrumentation Unit, Kerala Forest Research Institute, Peechi, Thrissur, Kerala, India for GC-MS Analysis of the extracts.