Polycyclic Phenol Derivatives from the Leaves of Spermacoce latifolia and Their Antibacterial and α-Glucosidase Inhibitory Activity

Three new polycyclic phenol derivatives, 2-acetyl-4-hydroxy-6H-furo [2,3-g]chromen-6-one (1), 2-(1′,2′-dihydroxypropan-2′-yl)-4-hydroxy-6H-furo [2,3-g][1]benzopyran-6-one (2) and 3,8,10-trihydroxy-4,9-dimethoxy-6H-benzo[c]chromen-6-one (8), along with seven known ones (3–7, 9 and 10) were isolated for the first time from the leaves of Spermacoce latifolia. Their structures were determined by spectroscopic analysis and comparison with literature-reported data. These compounds were tested for their in vitro antibacterial activity against four Gram-(+) bacteria: Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC), Bacillus subtilis (BS), and the Gram-(−) bacterium Escherichia coli. Compounds 1, 2, 5 and 8 showed antibacterial activity toward SA, BC and BS with MIC values ranging from 7.8 to 62.5 µg/mL, but they were inactive to MRSA. Compound 4 not only showed the best antibacterial activity against SA, BC and BS, but it further displayed significant antibacterial activity against MRSA (MIC 1.95 µg/mL) even stronger than vancomycin (MIC 3.9 µg/mL). No compounds showed inhibitory activity toward E. coli. Further bioassay indicated that compounds 1, 4, 5, 6, 8 and 9 showed in vitro α-glucosidase inhibitory activity, among which compound 9 displayed the best α-glucosidase inhibitory activity with IC50 value (0.026 mM) about 15-fold stronger than the reference compound acarbose (IC50 0.408 mM). These results suggested that compounds 4, 8 and 9 were potentially highly valuable compounds worthy of consideration to be further developed as an effective anti-MRSA agent or effective α-glucosidase inhibitors, respectively. In addition, the obtained data also supported that S. latifolia was rich in structurally diverse bioactive compounds worthy of further investigation, at least in searching for potential antibiotics and α-glucosidase inhibitors.


Introduction
The Spermacoce genus in the Rubiaceae family comprises 250-300 plant species that are widely distributed in tropical and subtropical zones around the world, including America, Europe, Africa, Australia and Asia [1]. To date, some species of this genus have been utilized in traditional or folk medicine to treat many human diseases such as malaria, digestive problems, fever, hemorrhage, urinary and respiratory infections, headache and skin diseases [1,2]. Previously, only a small number of plants in the Spermacoce genus were phytochemically studied, but by those studies more than 60 structurally diverse natural compounds, including bioactive iridoids, flavonoids, alkaloids, terpenoids and phenolic compounds, were discovered [3][4][5], which suggested that the plants of Spermacoce

Structure Elucidation of the Compounds
The air-dried and powdered leaf material of S. latifolia was extracted with 95% ethanol, and the resultant crude ethanol extract was then sequentially partitioned with petroleum ether, ethyl acetate (EtOAc) and n-butanol, respectively. The petroleum ethersoluble and EtOAc-soluble fractions of the crude ethanol extract were subjected to a series of column chromatographic fractionation steps over silica gel, ODS and Sephadex LH-20 to afford the three new (1, 2 and 8) and seven known (3-7, 9 and 10) polycyclic phenol derivatives.
Compound 1 was obtained as a yellow powder with molecular formula C13H8O5 as determined by HR-ESI-MS, m/z 243.0300 [M−H] − (calcd. for C13H7O5 − 243.0299), which requires 10 degrees of unsaturation. The 1 H NMR spectrum of 1 (Table 1) showed signals readily recognized for a tertiary methyl group at δH 2.64 (3H, s, H-2′), two aromatic protons at δH 7.07 (1H, s, H-9) and 7.96 (1H, s, H-3), and two cis-oriented vicinal olefinic protons at δH 6.42 (1H, d, J = 9.6 Hz, H-7) and 7.96 (1H, d, J = 9.6 Hz, H-8), respectively. The 13 C NMR and DEPT spectra (Table 1), coupled with HSQC analysis, of 1 supported the above analysis, which showed 13 carbon signals, including the presence of 1 methyl (δC 25.2), 1 carbonyl carbon (δC 188.4), 1 carboxyl carbon (δC 161.2), 4 olefinic methines and 6 olefinic quaternary carbons. These above findings supported 1 to be a highly conjugated compound bearing a basic coumarin skeleton. Careful analysis of the NMR data (Table 1) of 1 with those of the known compound xanthotoxol indicated that they were structurally closely related [18], with the only major difference being the signals for one additional acetyl group located at C-2 in 1 than in xanthotoxol. This assignment was consistent with the molecular formula of 1 and further well-supported by the following HMBC analysis. In the HMBC spectrum (Figure 2), significant correlations from δH 2.64 (Me-2′) to δC 153.4 (C-

Structure Elucidation of the Compounds
The air-dried and powdered leaf material of S. latifolia was extracted with 95% ethanol, and the resultant crude ethanol extract was then sequentially partitioned with petroleum ether, ethyl acetate (EtOAc) and n-butanol, respectively. The petroleum ether-soluble and EtOAc-soluble fractions of the crude ethanol extract were subjected to a series of column chromatographic fractionation steps over silica gel, ODS and Sephadex LH-20 to afford the three new (1, 2 and 8) and seven known (3-7, 9 and 10) polycyclic phenol derivatives.

Antibacterial Activity Evaluation
All of the 10 isolated compounds were evaluated for their in vitro antibacterial activity against 5 bacterial strains, including 4 Gram-(+) bacteria Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC) and Bacillus subtilis (BS), and one Gram-(−) bacteria, Escherichia coli (EC), using a method as described in the experimental section. The resulting MIC values of these compounds, as compared to reference compounds of kanamycin and vancomycin, are listed in Table 3. Compounds 1, 2, 5 and 8 were found to show moderate or weak antibacterial activity against three tested Gram-(+) bacteria of SA, BC and BS with MIC values ranging from 7.8 to 62.5 µg/mL, but they were inactive towards MRSA. Compound 4 was revealed to display the strongest antibacterial activity against all the four Gram-(+) bacteria (including MRSA) with MIC values 1.95~3.9 µg/mL, which were comparable to the two reference compounds of kanamycin and vancomycin. None of the test compounds displayed antibacterial activity against the Gram-(−) bacteria E. coli in this bioassay. It is noteworthy that compound 4 was found to show significant antibacterial activity against MRSA. Compound 4 structurally belonged to the group of prenylated xanthones, and some known structurally similar compounds of this group were reported to show several biological activities, including cytotoxic, antimicrobial, etc. [27]. MRSA infection is responsible for a rapidly increasing number of serious infectious diseases severely threatening global public health [28,29], and it has surpassed hepatitis B and AIDS, ranking first among the three most intractable infectious diseases throughout the world [30]. Although several antibiotics such as vancomycin, teicoplanin and daptomycin had been recommended for the treatment of MRSA infections [31,32], they showed a series of drawbacks such as slow bactericidal activity, low tissue penetration and increasing reports of resistance, which greatly restricted their clinical utility [33][34][35][36][37]. Accordingly, MRSA infection is urgently lacking effective and safe antimicrobial agents for its control and therapy. The discovery of even stronger in vitro anti-MRSA activity of compound 4 (MIC 1.95 µg/mL) than the reference compound of vancomycin (MIC 3.9 µg/mL) suggests that this compound could be worthy of consideration to be developed as an effective anti-MRSA agent.

α-Glucosidase Inhibitory Activity Evaluation
α-Glucosidase inhibitors are capable of inhibiting the conversion of carbohydrates into small-intestine-absorbable monosaccharides, and therefore they have the potential for the treatment of diabetes mellitus type 2 (DM2) by controlling blood sugar levels [38,39]. In this study, compounds 1-10 were then further tested for their in vitro α-glucosidase inhibitory activity, with acarbose employed as a reference compound. The resulting data (Table 4) revealed that compounds 1, 4, 5, 6, 8 and 9 were biologically active to show in vitro α-glucosidase inhibitory activity with IC 50 values ranging from 0.026 to 0.525 mM, which are close to or more potent than positive control of acarbose (IC 50 0.408 mM). In particular, compound 9 showed the best α-glucosidase inhibitory activity with an IC 50 value of 0.026 mM, which is about 15-fold stronger than the reference compound. Compounds 4 and 8 also showed good α-glucosidase inhibitory activity, with IC 50 values more than 2-fold lower than acarbose. In addition, a comparison of the chemical structures and the activities of 9 and 10 indicated that the existence of free hydroxy groups at C-7 and C-4 would be essential for this type of flavonoids to display α-glucosidase inhibitory activity. A negative effect on the α-glucosidase inhibitory activity of coumarin derivatives was also evident when a free hydroxy group was located at C-8 of the basic coumarin skeleton, as supported by comparison of the structures and activities of compounds 5, 6 and 7. Generally, the bioassay data not only indicated that rich phenolic derivatives with αglucosidase inhibitory activity were existing in this plant, but also revealed that compounds 4, 8 and 9 were potentially highly valuable α-glucosidase inhibitors worthy to be further developed as effective hypoglycemic agents for the treatment of DM2 patients [14]. Values represent mean ± SD (n = 3) based on three individual experiments. Different letters indicating significant differences labeled at the inhibitory activity at different compounds (p < 0.01).
As a successful invasive plant, S. latifolia normally grows very fast, and annually it can produce a huge plant biomass at its invasion habitats. However, due to the lack of essential study to discover its potential values, the abundant biomass resources of this plant have not been well-developed so far. In a recently conducted phytochemical investigation, nine ursane and oleanane triterpenoids and five anthraquinone compounds were discovered from this plant species, and some of them were revealed to selectively show antibacterial, cytotoxic or α-glucosidase inhibitory activity [16,17]. This research further identified 10 structurally diverse bioactive polycyclic phenol derivatives from the leaves of S. latifolia, among which compounds 4, 8 and 9 were addressed with significant antibacterial and (or) α-glucosidase inhibitory activity. Collectively, the present study provided new data to support that S. latifolia is a plant worthy of further development in searching for structurally new and bioactive natural compounds. Moreover, compounds 4, 8 and 9 were addressed as bioactive constituents of S. latifolia highly valuable to be further developed in medicinal or healthcare applications, at least as potential antibacterial agents or effective α-glucosidase inhibitors.

Plant Material
The leaf material of S. latifolia was collected around the campus of the South China Agricultural University, Guangzhou, China, in September 2019, identified by Dr. Hong-Feng Chen at South China Botanical Garden, the Chinese Academy of Sciences (CAS). A voucher specimen (No. 20190925) was deposited at the Laboratory of Phytochemistry at the College of Forestry and Landscape Architecture, South China Agricultural University.

Extraction and Isolation
Air-dried leaf material (13.5 kg) of S. latifolia was mechanically powdered at room temperature by a grinder. Then the powdered material was extracted three times (2 days each) with 95% EtOH (13.0 L × 3) at room temperature. The collected extraction solution was then evaporated under reduced pressure by a 20L-type Buchi evaporator to provide a black residue, which was then suspended in H 2 O (4.5 L) and successively partitioned with petroleum ether (4.5 L × 3), ethyl acetate (4.5 L × 3) and n-BuOH (4.5 L × 3) to afford petroleum ether-soluble (1.1 kg), EtOAc-soluble (610 g) and n-BuOH-soluble (570 g) fractions after condensation to dryness in vacuo.

Antibacterial Assay
The antibacterial assay was monitored in 96-well plates by using a method as described previously [40,41]. Briefly, 100 µL indicator solution (resazurin in sterile water, 100 µg/mL) was first placed into each of the sterility control wells (11th column) on the 96-well plates, and the indicator solution (about 7.5 mL, 100 µg/mL) was mixed with test bacteria (5 mL, 10 6 cfu/mL, OD 600 = 0.07) followed by transferring (100 µL, each) to growth control wells (12th column) and all test wells (1-10th column). Then, each of 100 µL of test compounds (1 mg/mL) in beef extract peptone medium, along with the positive control solutions (prepared by adding kanamycin sulfate and vancomycin instead of the samples) and the negative control solution (3% DMSO of beef extract peptone medium), were applied to the wells in the 1st column of the plates. Once all samples and controls were properly applied to the 1st column of wells on the plate, half of the homogenized content (100 µL solution) was then parallel-transferred from the 1st column wells to the 2nd column wells, and each subsequent well was treated similarly (doubling dilution) up to the 10th column, followed by discarding the last 100 µL aliquot. Finally, the plates were incubated at 37 • C for 5-6 h until the color of growth control change to pink. The lowest concentration for each test compound at which color change occurred was recorded as the MIC value of the test compound. In the bioassay, Gram-(+) bacterial strains of Staphyloccocus aureus (CMCC26003), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (CMCC63302), Bacillus subtilis (CMCC63501), and Gram-(−) bacterial strain of Escherichia coli (CMCC44102) were used for the test, and they were all purchased from the Guangdong Institute of Microbiology (Guangzhou, China).

α-Glucosidase Inhibition Assay
The a-glucosidase inhibitory activity of the ten isolated compounds was determined in 96-well microtiter plates following the method as described previously [15,42]. In brief, α-glucosidase (20 µL, 0.5 U/mL) and various concentrations (500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9 µg/mL) of tested compounds (120 µL) in 67 mM phosphate buffer (pH 6.8) were mixed in 96-well microtiter plates at room temperature for 10 min. Reactions were initiated by addition of 20 µL of 5.0 mM p-nitrophenyl-α-d-glucopyranoside (PNPG). The reaction mixture was incubated for 15 min at 37 • C in a final volume of 160 µL. Then, 0.2 M Na 2 CO 3 (80 µL) was added to the incubation solution to stop the reaction and the absorbance was determined at 405 nm (for p-nitrophenol). The negative blank was set by adding phosphate buffer instead of test compounds via the same experimental procedure. Acarbose was utilized as positive control. The blank was set by adding phosphate buffer instead of the α-glucosidase using the same method. Inhibition rate (%) = [(ODnegative control − ODblank) − (ODtest − ODtest blank)]/(ODnegative blank − ODblank) × 100%. IC 50 values were calculated and expressed as means ± standard deviations (SD) and SPSS 23.0 software was used for the analysis of variances.

Conclusions
Three new polycyclic phenol derivatives (1, 2 and 8) along with seven known ones (3-7, 9 and 10) were isolated from the leaves of S. latifolia. Their structures were determined by extensive spectroscopic analysis and comparison with literature-reported data. All the compounds were obtained from S. latifolia for the first time. Bioassays indicated that compounds 1, 2, 5 and 8 were active to show antibacterial activity toward three test Gram-(+) bacteria SA, BC and BS, but they were inactive to MRSA. Compound 4 was revealed to display the best antibacterial activity against all the four tested Gram-(+) bacteria (including MRSA) with MIC values comparable to reference compounds of kanamycin and vancomycin, and 4 in particular showed antibacterial activity against MRSA even stronger than vancomycin. No compounds showed inhibitory active toward the Gram-(−) bacteria E. coli. Further bioassay indicated that compounds 1, 4, 5, 6, 8 and 9 showed in vitro α-glucosidase inhibitory activity with IC 50 values close to or more potent than acarbose (IC 50 0.408 mM), especially for compound 9 which displayed αglucosidase inhibitory activity (IC 50 0.026 mM) about 15-fold stronger than the reference compound. The current research provided new data to support that S. latifolia is a plant rich in structurally diverse chemicals worthy of further development in medicinal or healthcare applications. In particular, compounds 4, 8 and 9 were discovered to be the three most highly valuable compounds worthy to be developed as an effective anti-MRSA agent or effective α-glucosidase inhibitor, respectively.