Bisabolane-type sesquiterpenoids with potential anti-inflammatory and anti-HIV activities from the stems and leaves of Morinda citrifolia

Abstract The phytochemical study on the stems and leaves of Morinda citrifolia L. resulted in the isolation of a new naturally occurring bisabolane-type sesquiterpenoid, morincitrinoid A (1), together with five known analogues (2–6). The chemical structure of 1 was elucidated by comprehensive spectral analyses. The known compounds 2–6 were identified by comparing their spectral data with those reported in the literature, which were isolated from M. citrifolia for the first time. In addition, the anti-inflammatory and anti-HIV activities of compounds 1–6 were evaluated in vitro. Compounds 1–6 displayed significant inhibitory activities on NO (nitric oxide) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with IC50 values ranging from 0.98 ± 0.07 to 6.32 ± 0.11 μM, which was comparable to hydrocortisone. Meanwhile, compounds 1–6 showed remarkable anti-HIV-1 reverse transcriptase (RT) effects with the EC50 values ranging from 0.16 to 6.29 μM. Graphical Abstract


Introduction
Sesquiterpenoids are the largest class of natural products, commonly containing 15 carbon atoms on their carbon skeleton (Fraga et al. 2012(Fraga et al. , 2013Vasas and Hohmann 2011;. Sesquiterpenoids have attracted considerable interest not only due to their structural diversities that include thousands of compounds and more than 100 skeletal types, but also due to their various biological activities including antimalarial, antibacterial, anti-tumor, anti-inflammatory, anti-oxidant, anti-viral and anti-ulcerogen acitivities, and so on (Fraga 2012(Fraga , 2013Vasas and Hohmann 2011;Ezzatzadeh et al. 2012;. Therefore, searching and discovering new sesquiterpenoids displaying remarkable biological activities is very significant for the research and development of new natural drugs. The genus Morinda (Rubiaceae) comprising approximately 102 species is widely distributed in tropical, subtropical and temperate regions of the world. There are approximately 26 species, 1 subspecies and 6 varieties in China, which grows in South China, Southwest, Southeast and Central China (Editorial Committee of Flora of China 1999). Among them, several species had been used as folk medicines. Previous phytochemical investigations on the genus Morinda had caused the isolation of many different types of compounds, such as iridoids, anthraquinones, lignans, triterpenes, flavonoids and their glycosides, which displayed a series of biological activities including antitumor, anti-inflammatory, antibacterial, hypoglycemic, lipoxygenase inhibitory and tyrosine phosphatase 1B inhibitory activities (Sang et al. 2001;Su et al. 2005;Deng et al. 2007;Nguyen et al. 2013;Youn et al. 2016;Zhai et al. 2019;Zandi et al. 2020;Yang et al. 2020). Our preliminary research results exhibited that the 90% ethanol extract of the stems and leaves of M. citrifolia L. showed significant inhibitory effect on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with the IC 50 value of 9.27 ± 0.16 lg/mL as well as anti-HIV-1 reverse transcriptase (RT) activity with an EC 50 value of 16.18 lg/mL. As a part of our ongoing research into natural products with diverse chemical structures and significant biological activities from tropical medicinal plants distributed in China, a phytochemical study on M. citrifolia was therefore carried out and had resulted in the isolation and characterization of a new naturally occurring bisabolane-type sesquiterpenoid, morincitrinoid A (1), along with five known analogues which were isolated from M. citrifolia for the first time. Their structures were elucidated by comprehensive spectral analyses. In addition, the anti-inflammatory and anti-HIV activities of all isolated bisabolane-type sesquiterpenoids 1-6 were evaluated in vitro. Herein, we describe the isolation, structure elucidation and anti-inflammatory and anti-HIV activities of these isolated bisabolane-type sesquiterpenoids.

Results and discussion
The residue of 90% ethanol extract from the stems and leaves of M. citrifolia was suspended in distilled water and extracted successively with petroleum ether (PE) and ethyl acetate (EtOAc). The PE extract fraction was repeatedly subjected to silica gel CC, Sephadex LH-20 CC, ODS gel CC and semi-preparative HPLC, to afford six bisabolane-type sesquiterpenoids (1-6), including a new naturally occurring one, as shown in Figure 1.
Morincitrinoid A (1) was a new naturally occurring bisabolane-type sesquiterpenoid, which had been already synthesized and reported as an intermediate in a chemical reaction in the literature (Zhang et al. 1988(Zhang et al. , 1989. However, the structural elucidation for the planar structure and the configurations of morincitrinoid A (1) has not been reported. This time, morincitrinoid A (1) was obtained as a colorless oil with a specific rotation of [a]-32.6 (c 0.13, CH 3 OH). Its molecular formula, C 15 H 24 O 3 , was established on the basis of its HR-ESI-MS (m/z 253.1799 [M þ H] þ , calcd 253.1798), indicating four degrees of unsaturation. Its IR spectrum displayed the existence of hydroxyl group (3424 cm À1 ), phenyl group (1610, 1509 and 1457 cm À1 ) and methyl group (1383 cm À1 ). Its UV spectrum exhibited the UV absorption bands at 258 nm, which was characteristic of a benzene ring. Its 13 C NMR and DEPT data revealed the existence of 15 carbons, including six sp 2 carbons, two sp 3 quaternary carbon, one sp 3 methine, two sp 3 methylenes and four methyls. Furthermore, the six sp 2 carbon atoms were attributed to the existence of a benzene ring group. The above data suggested that the chemical structure of 1 was similar to that of gypseatriol (2) (Zhao et al. 2016), except that the hydrogen atom located at C-7 and the hydroxyl group situated at C-9 in gypseatriol (2) was replaced by a hydroxyl group and a hydrogen atom in 1, respectively, which was confirmed by the HMBC correlations of H-3, 5, H-8a, H-8b and H 3 -14 to C-1 (d C 145.1), H-2, 6, H-9a, H-8b and H 3 -14 to C-7 (d C 74.5), as well as the 1 H-1 H COSY correlations from H-8a and H-8b to H-10. The chemical structure of 1 was further confirmed on the basis of the comparison of the spectral data of 1 with those reported spectral data of the synthetically prepared compound (Zhang et al. 1988(Zhang et al. , 1989 as well as the detailed analyses of 2 D NMR spectra including 1 H-1 H COSY, HSQC, HMBC and ROESY spectra, as shown in Figure S1, Figure S6- Figure S9. However, the relative and absolute configurations of C-7 and C-10 could be unassigned based on currently available data. Therefore, the chemical structure of morincitrinoid A (1) was determined as shown in Figure 1.
In addition, the anti-inflammatory activities of all isolated bisabolane-type sesquiterpenoids 1-6 were evaluated though measuring the inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. As a result, compounds 1-6 displayed notable inhibitory effects with the IC 50 values in range of 0.98 ± 0.07 to 6.32 ± 0.11 mM, which were comparable to that of hydrocortisone (see Table S1 in supplementary material). While the positive control, hydrocortisone, displayed an inhibitory effect with the IC 50 value of at 4.02 ± 0.11 lM.
No cytotoxicity was detected against the mouse macrophage RAW 264.7 cells treated by compounds 1-6 with the maximum concentration at 40.00 lM (cell viability > 90%).
Meanwhile, all isolated bisabolane-type sesquiterpenoids 1-6 were assessed for their anti-HIV reverse transcriptase (RT) activities using the inhibition assay for the cytopathic activities of HIV-1 (EC 50 ) and cytotoxic activity assay against C8166 cell line (CC 50 ) according to the MTT method. Bisabolane-type sesquiterpenoids 1-6 displayed notable anti-HIV activities with the EC 50 values ranging from 0.16 to 6.29 mM (see Table S2 in supplementary material). No cytotoxicity was observed against the C8166 cell line treated by those evaluated bisabolane-type sesquiterpenoids 1-6 (CC 50 > 200.00 mM).

Anti-inflammatory bioassays
The  Liu et al. 2017). The Griess reagent (50 lL of 1% sulfanilamine in 5% H 3 PO 4 , and 50.0 lL of 0.1% N-1-naphthylethylenediamine dihydrochloride) was added to each well. After 10.0 min, the reaction products were colorimetrically quantitated at 540.0 nm using a microplate reader. The experiments were performed three times in triplicate. Hydrocortisone was used as a positive control; The cytotoxicity assay was performed using the MTT method in 96-well microplates at concentrations of 0.0625, 0.32, 1.6, 8.0 and 40.0 lM of test samples Liu et al. 2017). A MTT solution (200.0 lg/mL) was added after the 24.0 h treatment and then incubated for another 4.0 h at 37.0 C. The reduced MTT-formazan was solubilized with 150.0 lL of DMSO, and the absorbance of the MTT-formazan solution at 570.0 nm was measured by an immunoreader. The percentage of suppression was calculated by comparing the absorbance of sample treated cells with that of nontreated cells.

Anti-HIV activity bioassays
All isolated bisabolane-type sesquiterpenoids (1-6) were evaluated for their anti-HIV-1 reverse transcriptase (RT) effects on the basis of the inhibition assay for the cytopathic effects of HIV-1 (EC 50 ) in the light of the protocol which has been described in detail by us in our previously published paper (Liu et al. 2018. In this bioassay, the experiments were performed three times in triplicate, and AZT (zidovudine) was applied as the positive control. All isolated bisabolane-type sesquiterpenoids (1-6) and the positive control were assessed for their cytotoxic activities against C8166 cells (CC 50 ) by the MTT method (Liu et al. 2018.

Conclusions
In our investigation, the phytochemical study on the stems and leaves of M. citrifolia was implemented and had brought about the separation and characterization of a new naturally occurring bisabolane-type sesquiterpenoid, morincitrinoid A (1), as well as five known bisabolane-type sesquiterpenoids (2-6), which were also isolated from M. citrifolia for the first time. The discovery of 1 is not only a further addition to diverse and complex array of bisabolane-type sesquiterpenoids, but also, the existence of bisabolane-type sesquiterpenoids (2-6) as characteristic marker may be helpful in chemotaxonomical classifications. Furthermore, all isolated bisabolane-type sesquiterpenoids (1-6) were evaluated for their anti-inflammatory and anti-HIV activities in vitro, which were confirmed to be very significant. These findings also indicated that these bisabolane-type sesquiterpenoids with notable anti-inflammatory effects isolated from M. citrifolia could be used for the development of new natural anti-inflammatory and anti-HIV agents.

Disclosure statement
No potential conflict of interest was reported by the authors.