Xanthones with Potential Anti-Inflammatory and Anti-HIV Effects from the Stems and Leaves of Cratoxylum cochinchinense

Four new xanthones, cratocochinones A–D (1–4), together with eight known analogues (5–12), were isolated from the stems and leaves of Cratoxylum cochinchinense. The chemical structures of cratocochinones A–D (1–4) were elucidated by comprehensive spectroscopic analyses and the known compounds were identified by comparisons with the spectral data reported in the literature. All isolated compounds 1–12 were evaluated for their anti-inflammatory activities and anti-HIV-1 activities. Compounds 1–12 showed remarkable inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro, with IC50 values in the range of 0.86 ± 0.05 to 18.36 ± 0.21 µM. Meanwhile, compounds 1–12 exhibited significant anti-HIV-1 activities with EC50 which ranged from 0.22 to 11.23 µM. These findings indicate that the discoveries of these xanthones, isolated from the stems and leaves of C. cochinchinense, showing significant anti-inflammatory and anti-HIV-1 effects could be of great importance to the research and development of new natural anti-inflammatory and anti-HIV agents.


Phytochemical Investigation
The stems and leaves of C. cochinchinense were extracted by means of 85% ethanol and then suspended in purified water and extracted successively using petroleum ether and ethyl acetate. The ethyl acetate extract was repeatedly subjected to silica gel column chromatography (CC), Sephadex LH-20 gel CC, ODS gel CC, as well as semi-preparative HPLC, to afford four new xanthones 1-4, along with eight know analogues 5-12, as shown in Figure 1.  indicating the presence of a hydroxyl group, conjugated carbonyl group, benzene ring, double bond, and methyl group functionalities, respectively. Its UV spectrum displayed UV absorption bands at 231, 268, 318, and 377 nm, which were characteristic of xanthones [25]. The 13 C-NMR and DEPT data of 1 (as shown in Table 1) revealed the presence of twentyeight carbons, including nineteen sp 2 carbons, one sp 3 methine, four sp 3 methylenes, and four methyls. Furthermore, the nineteen sp 2 carbons were assigned with one to xanthone group, one to two tri-substituted double bonds, and one to terminal double bond. The above spectral data suggest that the chemical structure of 1 was similar to that of cochinchinone A [25], except that the 2-methylbut-2-ene group at C-4 in cochinchinone A is substituted by a 3-methylbut-3-en-2-ol group in 1, which was confirmed by the HMBC correlations of H-4 to C-2 (δ C 122.5), C-3 (δ C 136.7) and C-9 (δ C 16.2), H-5 to C-3 and C-7 (δ C 147.1), H-6 to C-7 , C-8 (δ C 111.2), and C-10 (δ C 17.9). The planar structure of 1 was unambiguously confirmed via the comprehensive analyses of its 2D NMR (HSQC, HMBC, 1 H-1 H COSY, and ROESY) spectra as shown in Figure 2. Furthermore, the observation of a strong cross-peak in the ROESY spectrum between H 2 -1 and H 3 -9 permitted assignment of the orientation of the olefinic bond between H-2 and H-3 as E. In addition, in view of cratocochinone (1) with a specific rotation of [α] 28 D − 18.6 (c 0.11, CH 3 OH) only containing a chiral center at C-6 , the absolute configuration at C-6 in 1 was assigned to be S by comparison of its specific rotation with that of garcihombronone B, [α] 26 D −16.2 (c 0.29, CH 3 OH) [26]. Accordingly, the chemical structure of 1 was determined as depicted in Figure 1.    Cratocochinone B (2) was separated as a pale white amorphous powder. Its molecular formula was determined as C 19 [11]. The 13 C-NMR and DEPT data of 2 (as shown in Table 1) suggest the presence of nineteen carbons, including thirteen sp 2 carbons, one sp 3 quaternary carbon, one sp 3 methine, one sp 3 methylene, and three methyls. Furthermore, the thirteen sp 2 carbons were assigned to one xanthone group. Since 1 xanthone ring group accounted for ten out of eleven degrees of unsaturation, the remaining one degree of unsaturation was assumed for the presence of another ring system in 2. The above spectral data suggested that the chemical structure of 2 was similar to that of cochinchinone P [11], except that the hydroxyl group at C-6 in cochinchinone P was substituted by a methoxy group in 2, which was verified by the presence of the methoxy group resonating at δ H 3.86 (3H, s, 6-OCH 3 ), and δ C 60.5, which was further confirmed by the HMBC correlations of H-7, H-8, and 6-OCH 3 to C-6 (δ C 134.6), as well as the ROESY correlation of 6-OCH 3 and H-7. Furthermore, the planar structure of 2 was unambiguously confirmed by the comprehensive analyses of its 2D NMR (HSQC, HMBC, 1 H-1 H COSY, and ROESY) spectra as shown in Figure 2. Additionally, in view of the fact that 2 holds a specific rotation of [α] 28 D +58.3 (c 0.13, CH 3 OH) only possessing a chiral carbon at C-2 , therefore, the absolute configuration of 2 at C-2 could be assigned to be 2 R, which is the same as that of pruniflorone M, whose absolute configuration had been unquestionably determined by the X-ray single crystal diffraction method, on the basis of their identical chiral structures and similar rotation values [27,28]. Thus, the chemical structure of 2 was determined as depicted in Figure 1.

ROESY
Cratocochinone C (3) is obtained as a pale-yellow amorphous powder. The molecular formula of 3 was acquired as C 16 [29]. The 13 C-NMR and DEPT data (as shown in Table 2) revealed the presence of sixteen carbon atoms, including thirteen sp 2 carbon atoms, and three methyls. In addition, the thirteen sp 2 carbon atoms were attributable to one xanthone ring group. The above data revealed that the chemical structure of 3 was similar to that of calophymembranol C [29], except that the methyl group located at C-5 and the hydroxyl group located at C-6 in calophymembranol C were substituted by a methyl group and a hydroxyl group in 3, respectively, which was supported the HMBC correlations of H-7, H-8, and 6-OCH 3 to C-6 (δ C 145.2), along with the ROSEY correlation of H-7 with 6-OCH 3 . Comprehensive analysis of its 2D NMR (HSQC, HMBC, 1 H-1 H COSY, and ROESY) spectra confirmed the chemical structure of 3 as shown in Figure 2. Hence, the chemical structure of 3 was established as shown in Figure 1.  13 C NMR and DEPT data of 4 revealed the presence of sixteen carbon atoms, including thirteen sp 2 carbon atoms, and three methyl groups. In addition, the thirteen sp 2 carbon atoms were attributable to one keto carbonyl group and two benzene ring groups. The above data revealed that the chemical structure of 4 was similar to that of 2,4-dihydroxy-3,6-dimethoxy-9H-xanthen-9-one [30]. Comparison of the NMR data of 4 (as shown in Table 2) with those of 2,4-dihydroxy-3,6-dimethoxy-9H-xanthen-9-one suggested that both compounds shared the same basic skeleton, but the molecular weight of 4 is larger than that of 2,4dihydroxy-3,6-dimethoxy-9H-xanthen-9-one by 30 units, namely, the hydrogen atom at C-5 in 2,4-dihydroxy-3,6-dimethoxy-9H-xanthen-9-one was replaced by a methoxy group resonating at δ H 3.79 (3H, s) and δ C 60.9 in 4. The above elucidation was supported by the HMBC correlations of the methoxy hydrogens resonating at δ H 3.79 (3H, s) to C-5 (δ C 144.9), as well as the ROSEY correlations of 6-OCH 3 , and the methoxy group resonating at δ H 3.85 (3H, s) with 5-OCH 3 and H-7. Comprehensive analysis of 2D NMR (HSQC, HMBC, 1 H-1 H COSY, and ROSEY) spectra confirmed the chemical structure of 4 as shown in Figure 2. Thus, the chemical structure of 4 was established as shown in Figure 1.

Anti-Inflammatory Activity
All isolated xanthones 1-12 were evaluated for their anti-inflammatory effects by examining their inhibitory activities against NO production in vitro. Meanwhile, the MTT assay was used for evaluating the cytotoxic activities of those isolated xanthones against mouse macrophage RAW 264.7 cells. As a result, all isolated xanthones 1-12 showed notable inhibitory activities against NO production, thereby holding IC 50 values in range of 0.86 ± 0.05 to 18.36 ± 0.21 µM (as shown in Table 3) which are comparable to that of the positive control (hydrocortisone). No cytotoxicity was observed in the macrophage RAW 264.7 cells treated by those isolated xanthones 1-12 (cell viability > 90%).

Anti-HIV-1 Activity
All isolated xanthones 1-12 were assessed for their anti-HIV reverse transcriptase (RT) effects according to the inhibition assay for the cytopathic activities of HIV-1 (EC 50 ) as well as the cytotoxic activities assay against C8166 cell line (CC 50 ) according to MTT methods. Xanthones 1-12 exhibited notable anti-HIV affects with EC 50 values in the range from 0.22 to 11.23 µM (as displayed in Table 4). No cytotoxicity was observed against the C8166 cell line treated with the isolated xanthones 1-12 (CC 50 > 200.00 µM).

Plant Material
The stems and leaves of C. cochinchinense were collected from Bawangling Nature Reserve, Hainan Province, China, in June 2020, and identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University. A voucher specimen (No. CRCO20200608) has been deposited at the Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University.

Anti-Inflammatory Bioassays
All isolated xanthones 1-12 were appraised for their anti-inflammatory activities on the basis of measuring the inhibitory effects against NO production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. The protocol for this assay is described in detail in the Supplementary Material of our current article [37][38][39], in which hydrocortisone was utilized as the positive control.

Anti-HIV-1 Activity Bioassays
All isolated xanthones 1-12 were assessed for their anti-HIV-1 reverse transcriptase (RT) effects in vitro on the basis of the inhibition assay for the cytopathic effects of HIV-1 (EC 50 ), in light of the protocol which is described in detail in the Supplementary Material of our current article [40][41][42], in which AZT (3 -azido-3 -deoxythymidine) was adopted as the positive control. All isolated xanthones 1-12 and the positive control (AZT) were also evaluated for their cytotoxic activities against C8166 cells (CC 50 ) on the basis of the MTT method [40][41][42].

Conclusions
In this investigation, the chemical study on the stems and leaves of C. cochinchinense was conducted and led to the isolation and identification of four new xanthones, cratocochinones A-D (1-4), together with eight known xanthones 5-12. The discovery of four new xanthones 1-4 is not only a further addition to diverse and highly aromatic array of xanthones, but also, their presence as characteristic markers might be helpful in chemotaxonomical classifications. All isolated xanthones 1-12 were also investigated for their anti-inflammatory effects and anti-HIV-1 activities, and were proven to be very powerful. In particular, among these isolated compounds, compounds 1-3 and 11 displayed stronger inhibitory effects against NO production with IC 50 values ranging from 0.86 ± 0.05 to 3.16 ± 0.18 µM, which were below that of hydrocortisone. These remarkable inhibitory activities against nitric oxide (NO) production of xanthones 1-12 might be used as an explanation of the folk applications of the stems and leaves of C. cochinchinense, which are applied as an anti-inflammatory ethnic drug in China. These findings also indicate that these isolated xanthones, isolated from the stems and leaves of C. cochinchinense with notable inhibitory activities on nitric oxide (NO) production and anti-HIV-1 effects, could be used for the research and development of new anti-inflammatory and anti-HIV agents.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article or its Supplementary Materials.