Comparison of Composition, Free-Radical-Scavenging Capacity, and Antibiosis of Fresh and Dry Leave Aqueous Extract from Michelia shiluensis

Numerous plants of medicinal value grow on Hainan Island (China). Given the lack of knowledge on the phytochemical and pharmacological properties of Michelia shiluensis Chun and Y. F. Wu (M. shiluensis), the application of natural antioxidants and antimicrobials in the food industry has attracted increasing interest. This study aimed to compare the chemical composition, free-radical-scavenging capacity, and antibiosis of aqueous extracts of the fresh and dried leaves of M. shiluensis. The aqueous extract of the leaves of M. shiluensis was obtained using steam distillation, and its chemical components were separated and identified via gas chromatography–mass spectrometry (GC-MS). The free-radical-scavenging capacity and antibiosis were determined. Further, 28 and 20 compounds were isolated from the fresh leaf aqueous extract of M. shiluensis (MSFLAE) and dried leaf aqueous extract of M. shiluensis (MSDLAE), respectively. The free-radical-scavenging capacity of MSFLAE and MSDLAE was determined by the 2,2-diphenyl-1 picrylhydrazyl (DPPH) method, which was 43.43% and 38.74%, respectively. The scavenging capacity of MSFLAE and MSDLAE determined by the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS)) method was 46.90% and 25.99%, respectively. The iron ion reduction capacity of MSFLAE and MSDLAE was determined by the ferric-reducing antioxidant power (FRAP) method as 94.7 and 62.9 μmol Fe2⁺/L, respectively. This indicated that the two leaf aqueous extracts had a certain free-radical-scavenging capacity, and the capacity of MSFLAE was higher than that of MSDLAE. The antibiosis of the two leaf aqueous extracts on the three foodborne pathogenic bacteria was low, but the antimicrobial effects on Gram-positive bacteria were better than those on Gram-negative bacteria. The antibiosis of MSFLAE on Escherichia coli and Staphylococcus aureus was greater than that of MSDLAE. Finally, MSFLAE and MSDLAE both had certain free-radical-scavenging capacities and antibiosis, confirming that the use of this plant in the research and development of natural antioxidants and antibacterial agents was reasonable. Plant aqueous extracts are an essential source of related phytochemistry and have immense pharmacological potential.

Significant differences were found in the chemical composition of MSFLAE and MD-LAE. MSDLAE contained more compounds, and the relative content of compounds also changed. In general, numerous compounds revealed an increasing trend. Some highboiling-point compounds were retained, while others were lost or transformed. This was due to the evaporation or conversion of leaf components into new compounds during the drying process, resulting from heat, enzymatic action, oxidation, and other factors [31]. Only four components were shared between MSFLAE and MSDLAE: cryptomeridiol, cis-Zα-bisabolene epoxide, (-)-spathulenol, and 2,6-di-tert-butyl-4-methylphenol. After drying, the content of these compounds fluctuated, increasing and decreasing to varying extents. The relative contents of alcohols and oxides in the dried aqueous extract increased by 3% and 7.07%, respectively. Sesquiterpene alcohols and sesquiterpene oxides, intrinsic to both MSFLAE and MSDLAE, were the characteristic components and served as crucial indicators distinguishing them from the components in other plant species.

Antioxidant Activity
DPPH is a stable organic free radical widely used to evaluate the antioxidant capacity of compounds in plants by quenching the stable purple DPPH to yellow with a spectrophotometer [32]. The principle of antioxidant activity is based on the ability to neutralize free radicals by electrons, and phytochemicals such as pentacyclic triterpenoids have been recorded to have this effect [33]. The free-radical-scavenging capacity of DPPH gradually increases with increasing VC and BHT concentrations ( Figure 3A). The standard curve equations for VC and BHT were defined as Y = 13.84X + 11.80 (R 2 = 0.996) and Y = 10.63X + 0.58 (R 2 = 0.997), respectively. The DPPH free-radical-scavenging capacity of MSFLAE was recorded at 43.43%, equivalent to 2.28 µg/mL VC and 4.1 µg/mL BHT, respectively. The DPPH free-radical-scavenging capacity of MSDLAE was measured at 38.74%, equivalent to 1.95 µg/mL of VC and 3.7 µg/mL of BHT, respectively. The antioxidant activity exhibited by MSFLAE and MSDLAE does not significantly deviate from that observed in other medicinal plants and herbs [34,35]. At the same concentration, the free-radical-scavenging capacity of MSFLAE was significantly higher than that of MSDLAE, which is consistent with the finding of Singh et al. [36]; fresh sample extracts have higher antioxidant potential. Kessy et al. [37] reported that the radical-scavenging capacity of FRAP and DPPH of dried Litchi Pericarp peel extracts decreased significantly by 52.53% and 25.55%, respectively, which corresponds to the high degradation of bioactive phenolic compounds. In addition, many plant aqueous extracts contain high levels of sesquiterpene components, which have similar antioxidant activity to phenolic compounds, disrupting free radical chain reactions and causing their irreversible oxidation to inert compounds [38]. The ferric iron reduction antioxidant potential assay is useful for evaluating the antioxidant activity of MSFLAE and MSDLAE. The method is based on the reduction of a  The ferric iron reduction antioxidant potential assay is useful for evaluating the antioxidant activity of MSFLAE and MSDLAE. The method is based on the reduction of a colorless iron complex (Fe 3+ -tripyridyltriazine) to a blue ferrous complex (Fe 2+ -tripyridyltriazine) by the antioxidant action of the donor at low PH [39]. As illustrated in Figure 3B, the standard curve equation is defined as Y = 0.7835X − 0.00568 (R 2 = 0.997). Based on this standard curve, the iron reduction capacities of MSFLAE and MSDLAE are quantified as 94.7 and 62.1 µmol Fe 2+ /L, respectively. The iron reduction capacity of MSFLAE surpasses that of MSDLAE, an observation in agreement with the DPPH measurements. Aldogman et al. [40] conducted a study on the biological activity of Mentha suaveolens leaf volatile oil and discovered that the antioxidant capacity of fresh leaf volatile oil (94.50%) was also superior to that of the dry leaf volatile oil (90%). This was attributed to the high content of rosmarinic acid, ferulic acid, and other sesquiterpene phenolic compounds present in the fresh leaf volatile oil.
The free-radical-scavenging capacities of MSFLAE and MSDLAE were evaluated using the ABTS method. The standard curve for VC was established as Y = 3.2844X − 17.8464 (R 2 = 0.98). The free-radical-scavenging capacities of MSFLAE and MSDLAE were 46.90% and 25.99%, respectively, which is equivalent to 19.71 and 13.34 µmol/L of VC, respectively. The IC 50 of VC was found to be 20.65 µmol/L. When compared to VC, both MSFLAE and MSDLAE demonstrated a weaker free-radical-scavenging capacity. This is consistent with previous findings. Aazza et al. [41] reported similar antioxidant activity (282.6 µmol/L) on aqueous extract of Salvia miltiorrhiza. The free-radical-scavenging ability of MSFLAE and MSDLAE was measured using DPPH, ABTS, and FRAP methods. The results show that MSFLAE has a higher free-radical-scavenging capacity than MSDLAE, which may be due to the fact that MSFLAE contains more sesquiterpene alcohols and oxides. However, the free-radical-scavenging capacity of MSFLAE and MSDLAE is not significantly high, which can be attributed to the lower concentration of chemical constituents in their aqueous extracts. Essentially, they only encompass the water-soluble fraction of the respective essential oils. Consequently, while their biological activity is lower, so is their toxicity level, rendering them simpler to apply [42].

Determination of Antibacterial Activity
In order to study the antimicrobial potential of MSFLAE and MSDLAE, three foodborne pathogens, E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria), as well as the fungus C. albicans, were selected. The results revealed variable antimicrobial activities of MSFLAE and MSDLAE against these foodborne pathogens, with MSFLAE showing slightly superior inhibition against E. coli and C. albicans compared to MSDLAE ( Figure 4). The antimicrobial circle of MSFLAE against E. coli, S. aureus, and C. albicans was 8.44 ± 0.10, 8.71 ± 1.08, and 7.41 ± 0.19 mm, respectively. The antimicrobial circle of MSDLAE against E. coli, S. aureus, and C. albicans was 7.11 ± 0.89, 8.10 ± 1.36, and 8.40 ± 0.36 mm, respectively. MSFLAE and MSDLAE were less susceptible to Gramnegative bacteria than Gram-positive bacteria, which may be due to the presence of an outer membrane in the cell membrane of Gram-negative bacteria, which can act as a barrier to reduce or prevent the penetration of various antimicrobial drugs [43]. There was no significant difference in the antimicrobial effect of the MSFLAE and MSDLAE against the three foodborne pathogens, and both showed weak antibiosis. The antimicrobial activity of aqueous extract may be related to the high content of oxygenated sesquiterpenes and sesquiterpene alcohols in aqueous extracts [44] because the active sesquiterpenes belong to the guaiane and elemene-type skeletons, and these compounds all contain hydroxyisopropyl. Wu et al. [45] documented that guaiacol, along with four other sesquiterpenes isolated from Michelia formosana, exhibited significant antibacterial effects against wood rot bacteria. Conversely, Feng et al. [46] reported that the four monoterpenoids with a seven-membered ring system, isolated from the flower buds of Magnolia biondii, did not demonstrate antibacterial activity within the tested range. E. coli and S. aureus were extremely sensitive to PGSS, with inhibitory zones of 21.2 ± 0.34 and 49.78 ± 0.99 mm, respectively. The antibacterial effect of PGSS was significantly higher than that of MSFLAE and MSDLAE (Table 2). There were significant differences in the antibacterial effect of aqueous extracts of different plants. Ali Shtayeh et al. [47] reported that the aqueous extracts of Phagnalon rupestre and Micromeria nervosa showed significant antibacterial effect against C. albicans. However, Huseyin et al. [48] reported that aqueous extracts of Citrus aurantium flowers did not exhibit antibacterial activity against the tested bacteria in the concentration range used.

Plant Materials and Extraction
M. shiluensis leaves were collected from Haikou City, Hainan Province, in Octo 2021 (20° N, 110° E). Dr. Zhao Ying from the School of Forestry at Hainan University co firmed the source plant as M. shiluensis. The voucher specimens were stored in the herb ium of Danzhou Campus of Hainan University (SCUTA 2965). Aqueous extracts were rived using a previously established procedure [49] with minor alterations. MSFLAE w

Plant Materials and Extraction
M. shiluensis leaves were collected from Haikou City, Hainan Province, in October 2021 (20 • N, 110 • E). Dr. Zhao Ying from the School of Forestry at Hainan University confirmed the source plant as M. shiluensis. The voucher specimens were stored in the herbarium of Danzhou Campus of Hainan University (SCUTA 2965). Aqueous extracts were derived using a previously established procedure [49] with minor alterations. MS-FLAE was extracted via steam distillation. Furthermore, 500 g fresh leaves was connected to the distillation device for heating for 120 min according to the solid-liquid ratio of 1:7. Finally, the steam-condensed distillate was collected, and the upper trace of essential oil was separated to isolate MSFLAE, which was then used for the detection of biological activity. The preparation of MSDLAE was consistent with the aforementioned conditions except for drying to constant weight at 60 • C. Additionally, 500 mL of the aqueous extract was taken and concentrated to 10 ml using rotary evaporation for the determination of components through gas chromatography-mass spectrometry (GC-MS).

Chemical Analysis
The GC-MS test/experiment was conducted following the procedure given below: a HP-5MS 5% phenyl methyl siloxane (30 m × 0.25 mm × 0.25 µm) elastic quartz capillary column was used; the heating program was: column temperature 50 • C, 5 • C/min to 310 • C, keeping 10 min; gasification chamber temperature of 250 • C; the carrier gas was high purity He (99.999%); precolumn pressure of 43 kPa, carrier gas (He) flow rate of 1.0 mL/min; the injection volume was 1.0 µL; and the solvent delay time was 4 min. The mass spectrometry detection conditions were as follows: electron bombardment (EI) was used as the ion source, with an electron energy of 70 eV, interface temperature of 280 • C, ion source temperature of 230 • C, four-bar temperature of 150 • C, the tuning mode was standard tuning, electron multiplier voltage of 1718 kV, and mass scanning range of 40-800 m/z. By using the Data Analysis chemical workstation and following the above chemical analysis methods, combined with the Nist2005 and Wiley275 mass spectrometry libraries, and drawing on the identification methods of previous researchers and our research group, the detected chromatographic peaks were retrieved and manually analyzed with reference to the relevant literature.

Assay of DPPH Radical-Scavenging Activity
The DPPH assay was performed by a previously described method [50] with some modifications: 0.1 mmol/L DPPH solution was prepared by dissolving 5.91 mg of DPPH in 150 mL of 75% ethanol and the mixture was left to react in the dark for 30 min for further use. The 1 mg/mL VC and BHT solutions were diluted to different concentrations as control. Further, 2 mL of the sample was poured into a centrifugation tube and 1 mL of DPPH solution was added to it; the mixture was then left to react in the dark for 30 min. The absorbance of the reaction solution was measured at 517 nm and 75% ethanol was used as blank control. The DPPH free-radical-scavenging rate was calculated based on the formula used in previous studies.

Assay of FRAP Radical-Scavenging Activity
The FRAP assay was performed based on a previous procedure [51] with minor modifications. The FRAP working solution was prepared as follows: we took 10 mmol/L TPTZ solution with a solvent of 40 mmol/L hydrochloric acid (HCl) solution, 300 mmol/L sodium acetate buffer, and 20 mmol/L FeCl 3 solution. The three solutions were mixed in a ratio of 1:10:1 and the mixture was used immediately. Further, 27.8 mg FeSO 4 ·7H 2 O was diluted to 10 mL with deionized water at a concentration of 100 mmol/L and then diluted to different concentrations. Subsequently, 1 mL of FRAP working solution was made to react with 2 mL of different concentrations of FeSO 4 solution, and the absorbance A value of the solution after the reaction was measured. The standard curve of FeSO 4 was drawn using the mixed solution of ethanol and FRAP working solution as blank control. Finally, the absorbance of the solution after the reaction of the sample with the FRAP working solution was measured and substituted into the regression equation of the standard curve, which was repeated three times.

Assay of ABTS Radical-Scavenging Activity
The ABTS assay was performed based on the method proposed by Thaipong et al. [52] with some modifications. First, the acetic acid buffer with pH value of 4.3 was prepared; 0.075 mmol/L ABTS and 2 mmol/L AAPH solutions were mixed with this buffer. This mixture was then reacted in a 45 • C water bath for 60 min. The 1 mol/L VC solution was prepared as a control and diluted to different concentration gradients. Further, 2 mL of the mixture was mixed with 1 mL of the sample solution, and the absorbance value was determined at 734 nm. The standard curve was drawn with VC solution as the standard reference and deionized water as the blank control. The standard curve was prepared based on the following formula: where A 0 was 1 mL of the sample solution added with 2 mL of mixed solution and A 1 was 1 mL of deionized water added with 2 mL of the mixed solution.

Preparation of Bacterial Suspension and Drug Solution
Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC27217), and Candida albicans (ATCC10231) were inoculated into the corresponding liquid media. The bacteria were cultured at 120 rpm and 37 • C for 24-48 h, and the fungi were cultured at 120 rpm and 25 • C for 48-72 h. Finally, the bacterial suspension was diluted to 10 6 -10 7 CFU/mL, and 1 mg/mL PGSS solution and 1 mg/mL Tween-80 solution were used as controls.

Disk Diffusion Method
The antibacterial effect of hydrosol was determined by paper diffusion method [53,54]. Under aseptic conditions, 200 µL of the bacterial suspension used in the experiment was uniformly coated on the surface of the corresponding solid medium to prepare a bacterial plate. Three circular filter papers with a diameter of 6 mm were uniformly adsorbed on the bacterial plate. A total of 10 µL of aqueous extract, Tween-80, and PGSS were added to different filter papers. The bacteria were cultured at 37 • C for 24 h and the fungi were cultured at 25 • C for 48 h. Then, the diameter of their antimicrobial circle was determined.

Statistical Analysis
Data were presented as mean and standard error (SE). Data were submitted to one-way analysis of variance (ANOVA) with a comparison of means using Tukey's test (p < 0.05), using IBM SPSS Statistics 26.0 software, Mapping using Origin 2021.

Conclusions
In this study, 28 and 42 different compounds were isolated from MSFLAE and MS-DLAE, respectively. Significant differences existed in the components of the two, with only four common components. The dried extract had more kinds of compounds, and the relative content also changed. Sesquiterpene alcohols and sesquiterpene oxides were the main components in the extracts of the two aqueous solutions. The total content remained relatively stable after drying, and the aforementioned compounds contributed significantly to free-radical-scavenging ability and antibacterial effect. The free-radicalscavenging ability and trivalent iron-reducing ability of MSFLAE were slightly higher than those of MSDLAE, and both had certain antioxidant activity. No significant differences were observed in the antimicrobial activity of MSFLAE and MSDLAE against the three foodborne pathogenic bacteria, exhibiting approximately the same antimicrobial capacity and lower antimicrobial activity and higher susceptibility to Gram-positive bacteria. The aqueous plant extracts have high pharmacological potential and great application potential. Therefore, M. shiluensis can also be used as a potential source of functional food and medicinal antimicrobials. Moreover, further studies on its toxicity, in vivo efficacy, and mechanism of action are needed to elucidate its potential use.
Author Contributions: W.W. is the experimental designer and executor of this research, completing data analysis and writing the first draft of the paper; G.L., W.Z., E.W., X.Z. and X.S. participated in experimental design and analysis of experimental results; Y.Z. is the designer and director of the project, guiding experimental design, data analysis, thesis writing, and revision. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.