Secondary Metabolites with Antioxidant and Antimicrobial Activities from Camellia fascicularis

Camellia fascicularis has important ornamental, medicinal, and food value. It also has tremendous potential for exploiting bioactivities. However, the bioactivities of secondary metabolites in C. fascicularis have not been reported. The structures of compounds were determined by spectral analysis and nuclear magnetic resonance (NMR) combined with the available literature on secondary metabolites of C. fascicularis leaves. In this study, 15 compounds were identified, including 5 flavonoids (1–5), a galactosylglycerol derivative (6), a terpenoid (7), 4 lignans (8–11), and 4 phenolic acids (12–15). Compounds 6–7 and 9–12 were isolated from the genus Camellia for the first time. The remaining compounds were also isolated from C. fascicularis for the first time. Evaluation of antioxidant and antimicrobial activities revealed that compounds 5 and 8–11 exhibited stronger antioxidant activity than the positive drug ascorbic acid, while compounds 7, 13, and 15 showed similar activity to ascorbic acid. The minimum inhibitory concentration (MIC) of antibacterial activity for compounds 5, 7, 9, 11, and 13 against Pseudomonas aeruginosa was comparable to that of the positive control drug tetracycline at a concentration of 62.50 µg/mL; other secondary metabolites inhibited Escherichia coli and Staphylococcus aureus at concentrations ranging from 125–250 µg/mL.


Introduction
Camellia fascicularis, a genus of Camellia in the family Theaceae, is an endemic plant in Yunnan province, China.C. fascicularis, which is a rare species resource with unique golden petals, also known as "giant panda in the plant kingdom", "queen of the tea family", and "living fossil of plants" was first discovered in Hekou County and only distributed in Gejiu, Maguan, and Hekou counties [1].C. fascicularis leaves possess high amino acid and mineral content and are considered an edible plant resource with high nutritional and health value [2,3].There is limited literature on the metabolites of Camellia, with polyphenols, flavonoids, saponins, and steroids being the primary active components involved [4][5][6][7][8].It has been proved by experiments that the flowers and leaves of C. fascicularis, in addition to being used as a tea, have many pharmacological effects such as antioxidant [9], antitumor [10,11], and anti-inflammatory [12,13] attributes.Due to its limited distribution in Yunnan, the investigation into secondary metabolites of C. fascicularis commenced relatively late; nevertheless, it harbors immense potential for exploring its biological activities.
The isolation and identification of monomeric active compounds from complex plant components is a pivotal objective in the field of natural product chemistry research [14].However, the bioactivities of secondary metabolites in C. fascicularis have not been reported.Considering the significance of previous research findings in exploring the bioactivities of C. fascicularis, this study aimed to investigate the secondary metabolites and bioactivities (antioxidant and antimicrobial) present in the acetate fraction of the methanol extract derived from C. fascicularis leaves.This investigation facilitated a comprehensive understanding of the composition of monomeric compounds within C. fascicularis leaves, while also anticipating the isolation of novel compounds with remarkable bioactive properties.

Plant Material
The voucher specimen (52,860) of C. fascicularis was identified by taxonomist Min Tianlu and preserved in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.The leaves of C. fascicularis used in this experiment were obtained from Dawei Mountain Nature Reserve, Hekou County, Yunnan Province, China, in December 2019, and were identified as C. fascicularis by taxonomist Prof. Xiang Jianying of Southwest Forestry University.
After obtaining the monomeric compounds through purification, the organic solvent was evaporated and quantified.A suitable deuterium substitute reagent was selected for dissolution and sent for testing, with TMS used as an internal standard.The isolation and purification process of compounds 1-15 is illustrated in Figure 1.

Chemical Structure Analysis
Secondary metabolites investigation on the leaves of C. fascicularis, afforded 15 compounds, including 5 flavonoids (1-5), a galactosylglycerol derivative (6), a terpenoid (7), 4 lignans (8)(9)(10)(11), and 4 phenolic acids (12)(13)(14)(15).The structures of these compounds were established via spectroscopic analysis and comparison of their NMR data with the literature.Compounds 6-7 and 9-12 were isolated from the genus Camellia for the first time.The remaining compounds were also isolated from this plant for the first time.The structures of the known compounds (Figure 2).

Antioxidant Activity
The ABTS free radical scavenging capacity of all isolated compounds was measured, and the procedure followed a method with minor modifications [30].The experiment was conducted using a 96-well plate, with each well containing a total volume of 210 µL.Equal volumes of ABTS solution (7 mM) and potassium persulfate solution (5 mM) were thoroughly mixed and allowed to react at room temperature for 12 h in the absence of light to generate the ABTS radical cation.The mixture was then diluted with anhydrous methanol to achieve an absorbance value of about 0.7 ± 0.02 units at 734 nm.Then, 180 µL of ABTS working solution was added to each well, followed by 30 µL samples of varying concentrations (0.5, 0.1, 0.05, 0.01 µg/mL) were dissolved and diluted with DMSO.After thorough mixing, the samples were incubated at room temperature for 6 min in a light-free environment.Subsequently, the absorbance values of each well were measured at 734 nm using a microplate reader, and the results were obtained from a minimum of three independent experiments.Ascorbic acid was employed as the positive control.DMSO was used to replace the sample solution as a blank and absolute methanol was used to replace the ABTS solution as a control.All tests were performed in triplicate, and the obtained results were processed by analysis of variance (ANOVA) with 95% confidence (p ≤ 0.05).
The percentage radical cation scavenging rate (%) of each test sample for ABTS was calculated as follows (1):

Antimicrobial Activity
The antibacterial activity capacity of all isolated compounds was measured, and the procedure followed a method with minor modifications [31,32].Antimicrobial drug mother liquor preparation: dissolve the drug with DMSO, and its mass concentration was 0.5 mg/mL.Preparation of the bacterial solution to be tested: Thaw the frozen bacteria from a −80 • C low-temperature storage box at room temperature, and sterilize the Nutrient Broth (NB) medium in a 37 • C shaking bed for overnight culture.Then take 2 mL of the overnight culture of the bacterial solution and inoculate it into the NB medium.Incubate it at 37 • C until A 600 = 0.5, and then dilute it 100 times with the NB medium.Micro broth dilution method: Take a sterile 96-well plate, add 75 µL of the NB medium dilution solution to the A2-A11 wells, and add 75 µL of the drug solution to the A1-A2 wells.Take 75 µL of the mixture from the A2 wells to the A3 wells, 75 µL of the mixture from the A3 wells to the A4 wells, and so on up to the A10 wells.Take up 75 µL of the mixture to the A10 wells, discard it, and add 75 µL of 5% DMSO to the 12 wells.Add 75 µL of the bacterial solution to well A12 and mix well.The inoculated 96-well plates were incubated at 37 • C for 12 h.After inoculation, the 96-well plates were incubated at 37 • C for 14 h to observe the growth.The minimum inhibitory concentration (MIC) was calculated by measuring A 600 with an enzyme marker.All tests were performed in triplicate, and the obtained results were processed by (ANOVA) with 95% confidence (p ≤ 0.05).

Antioxidant Activity
In the ABTS assay, the antioxidant activity is measured as the ability of test compounds to decrease the color by reacting directly with the radical ABTS [33].The antioxidant activities of all the isolates were assessed in vitro using the ABTS assay method, and the corresponding results are presented in Table 2.The results of these experiments demonstrated that compounds 5, and 8-11 exhibited superior antioxidant activity compared to the positive drug ascorbic acid, while the activities of compounds 7, 13, and 15 were comparable to that of ascorbic acid.The other compounds had weaker or no significant antioxidant activities.This finding once again confirms that flavonoids [34][35][36], terpenoids [37,38], lignans [39][40][41], and phenolic acid [42] compounds have good antioxidant activities.The compounds 1-5 are all flavonoid glycosides; however, compound 5 demonstrates significantly superior antioxidant activity in comparison to compounds 1-4.An analysis of the structure-activity relationship for these five flavonoid glycosides reveals a direct correlation between the free radical scavenging activity and the number of hydroxyl groups on the B ring.Increasing the number of hydroxyl groups on the B ring enhances the free radical scavenging activity.Moreover, it is noteworthy that C-3 possesses an alcohol hydroxyl group, which exhibits greater stability and reduced susceptibility to electron loss compared to a phenol hydroxyl group.This characteristic contributes to increased water solubility of compounds without significantly affecting their antioxidant activity.These findings are consistent with the previous literature analyses regarding the conformational relationship between flavonoid antioxidant activities [43][44][45].Meanwhile, the antioxidant activities of compounds 1-4 also differed, which was mainly related to the number and position of hydroxyl groups and the spatial site resistance of glycosides [46].The lignan compounds 8-11 possess two phenolic hydroxyl groups, and their antioxidant activities exhibit minimal variation among each other.This similarity may be attributed to the positive impact of the methoxy group, located adjacent to the phenolic hydroxyl group, on the antioxidant activity of lignans in the power supply [47].Compounds 12-15 are phenolic acids derived from hydroxybenzoic acid, with compound 14 exhibiting significantly lower antioxidant activity compared to compounds 12-13 and 15.Furthermore, the introduction of o-hydroxy and o-methoxy groups enhances the antioxidant activity of phenolic acids [48].The findings suggest that flavonoids, phenols, lignans, and terpenoids are the primary antioxidant secondary metabolites in C. fascicularis.Due to the limited quantity of certain compounds and their inadequate initial concentration of 500 µg/mL for effective antioxidant activity, experiments were not conducted on these compounds considering the requirement for subsequent sequential activity determination.The utilization of only ABTS as an indicator for assessing antioxidants may not comprehensively reflect the complete antioxidant activity of compounds.

Antibacterial Activity
The utilization of botanical extracts as natural antimicrobial agents in the food industry is an emerging trend [49].The antimicrobial activity testing of the samples was performed against microorganism strains from the laboratory collection.The Gram-positive bacteria used for the tests were Staphylococcus aureus (ATCC 6538).The Gram-negative bacteria were Escherichia coli (ATCC 6538) and Pseudomonas aeruginosa (CGMCC 1.10712).The antimicrobial activity of C. fascicularis has not been reported, but the antimicrobial activity of researchers in Camellia proved that Camellia has good antimicrobial activity [50][51][52].The findings provide additional evidence to substantiate the potent antibacterial activity of secondary metabolites derived from C. fascicularis.
As shown in Table 3, the results of the antimicrobial activity test showed that compounds 1-15 all showed some degree of inhibition against E. coli at 125-250 µg/mL, with compound 7 showing better antimicrobial activity than the other compounds, but still lower than the positive control drugs penicillin (MIC 31.25 µg/mL) and tetracycline (MIC 7.81 µg/mL).Compounds 3, 5-9, 11, and 14-15 showed a certain degree of inhibition of S. aureus at 125-250 µg/mL, which was still weaker compared with the positive control drug.The antibacterial activity of compounds 5, 7, 9, 11, and 13 against P. aeruginosa was comparable to that of the positive control drug tetracycline (MIC 62.50 µg/mL) and superior to that of penicillin (MIC 125.00 ug/mL), whereas the antibacterial activity of compound 15 against P. aeruginosa showed antimicrobial activity comparable to the positive control drug penicillin (MIC 125.00 µg/mL) and weaker than tetracycline (MIC 62.50 µg/mL).The findings suggest that flavonoids, phenolics, and terpenoids serve as the primary secondary metabolites responsible for the antimicrobial activity in C. fascicularis.The MIC data of 250.00 µg/mL in antibacterial activity indicates a range of true valuest from 125.00 to 500.00 µg/mL.It is important to note that this method provides an approximation rather than an absolute accurate value for the MIC.If it were possible to provide microstructures of different bacteria at optimal inhibitory concentrations and scanning electron microscopy images, it would further support our study; unfortunately, all compounds were completely consumed during the series of bioactivity assays." a " positive control; " b " minimum inhibitory concentration."-" indicates that the experiment was not performed.
The presence of compounds (1-5) in the Theaceae family has been previously demonstrated through chemical studies [17,53,54], highlighting the abundant and diverse flavonoid content in Camellia.Compound 6 was initially derived from plants belonging to the genus Ginger in the Zingiberaceae family.It serves as a crucial metabolite involved in various physiological processes, including germination, growth, flowering, senescence, and fruit ripening in higher plants.Additionally, it plays a significant role in evaluating the effects of glucose-lowering [55][56][57][58].Compound 7 is a tetraterpene with notable anti-inflammatory and anti-complementary activities.Moreover, this study demonstrates its remarkable antioxidant and antibacterial properties [21,59].In addition, lignans (8)(9)(10)(11) have been recognized as bioactive components with applications in the pharmaceutical and nutritional

Figure 1 .
Figure 1.Flow chart for the isolation and purification of compounds 1-15.

Figure 1 .
Figure 1.Flow chart for the isolation and purification of compounds 1-15.

Table 1 .
Weight in (Fr.A-I) of C. fasciculata.