# Anti-Inflammatory Compounds from VietnamesePiper bavinum

Authors: Viet Dung Hoang; Phi Hung Nguyen; Minh Thu Doan; Manh Hung Tran; Nhu Tuan Huynh; Huu Tung Nguyen; Byung Sun Min; Dao Cuong To
Journal: Journal of Chemistry
 (2020)

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## Abstract

This study reports the anti-inflammatory activity-guided fractionation of the aerial part ofPiper bavinum C. CD. (Piperaceae) that led to the isolation of eight secondary metabolites (1–8). The chemical structures of 1–8 were established mainly by NMR and mass spectra. Compound 5 was isolated from P. bavinum for the first time. All the isolated compounds were evaluated against LPS-induced NO production in macrophage RAW 264.7 cells in vitro. Among them, compound 4 showed the most potent inhibitory activity against the LPS-induced NO production with an IC50 value of 5.2 μM followed by compound 5 that inhibited NO production with an IC50 value of 13.5 μM. In the protein levels, compound 4 suppressed LPS-induced COX-2 and iNOS expressions in a dose-dependent manner. The results suggested that P. bavinum and its constituents might exert anti-inflammatory effects.


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## Body

## 1. Introduction

Inflammation is a protective response that occurs following trauma, infection, or tissue injury [1]. In this process, activated inflammatory cells include increased amounts of nitric oxide (NO) and prostaglandin E2 (PGE2). NO is a major product that is controlled by nitric oxide synthases (NOSs). NOSs included inducible nitric oxide synthase (iNOS) that is extremely expressed in macrophage cells, and the activation of iNOS generally leads to some autoimmune diseases [2]. PGE2 is another important inflammatory mediator, produced by arachidonic acid metabolites via the catalysis of cyclooxygenase-2 (COX-2) [2]. Lipopolysaccharide (LPS) plays an important role in activating immune cells to upregulate to inflammatory states. The overproduction of NO by iNOS regulation has been concerned in the pathology of several inflammatory disorders, including septic shock, tissue damage, and rheumatoid arthritis [3–5]. NO and PGE2 production induced by LPS through iNOS and COX-2, respectively, can reflect the degree of inflammation.During a screening program to discover inflammation inhibitors from natural sources, we have found that then-hexane and ethyl acetate (EtOAc) fractions of Vietnamese P. bavinum exhibited appreciable inhibitory activity in LPS-induced NO production in macrophage RAW 264.7 cells. P. bavinum is a member of the Piper genus, which is the largest genus in the Piperaceae family. The Piper genus included over 700 species and is identified in the tropical regions [6, 7]. Piper species have been used in traditional medicine to treat the gynecological ailment, gastrointestinal problem, and depression [7]. Some of these from India, Southeast Asia, and Africa are of high commercial, medicinal, and economic importance since they are used as spices and traditional medicines [8]. Otherwise, Piper species have also been found to possess biological activities, including antioxidative, antimicrobial, antiproliferative/anticancer, antiparasitic, and neuropharmacological activities [9]. Especially, in vivo and in vitro studies showed the Piper genus against inflammation such as P. nigrum, P. crocatum, P. betle, P. umbellatum, P. gaudichaudianum, P. arboreum, P. umbellata, P. fuligineum, P. longum, and P. methysticum [9]. The most widely recognized species of the genus Piper is P. nigrum followed by P. longum, P. chaba, P. mullesa, P. umbellatum, P. hymenophyllum, P. argyrophyllum, P. attenuatum, P. colubrinum, P. galeatum, and P. bavinum [10]. P. bavinum is a liana, distributed in forests of India, China, and Vietnam at attitudes of 1300–1700 m [11]. P. bavinum is known as an oriental medicinal plant and had been reported to possess various pharmacological activities such as antibacterial [11] and anticholinesterase [6]. Previous studies on chemical constituents of P. bavinum showed the presence of essential oil [11], terpenoids, phenolics, and flavonoids [6]. However, there has been no investigation in chemical constituents and inhibitory activity of NO production of P. bavinum. This paper describes the isolation and structural elucidation of the isolated compounds as well as evaluates their inhibitory activity on NO production.


## 2. Results and Discussion

### 2.1. Determination and Elucidation of Isolated Compounds

The methanol extract was partitioned withn-hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (BuOH) to obtain n-hexane, CHCl3, EtOAc, and BuOH soluble fractions, respectively. Based on bioactivity-guided fractionation (data not are shown), the n-hexane and EtOAc fractions were subjected to column chromatography on a silica gel and C18-RP silica gel column to obtain eight compounds (1–8) (see Figure 1).Figure 1
Structure of compounds1–8 from P. bavinum.Compound1 was obtained as a light yellow powder. The presence of two oxymethine groups (δH 4.82 (H-2)/δC 83.2 (C-2), and δH 4.45 (H-2)/δC 71.6 (C-3))and a ketone at δC 197.7 (C-4) in the 1H- and 13C-NMR spectra indicated 1 to be a flavanonol (see Figure 1 and Supplementary Materials (available here)). The 1H-NMR spectrum of 1 further showed signals of two tetrasubstituted benzene rings (δH 5.88 (1H, s, H-6), 5.91 (1H, s, H-8), and 6.52 (2H, s, H-2′/6′)) [12]. The 13C-NMR spectrum exhibited fifteen signals due to twelve sp2 carbons of two benzene rings, a ketone at δC 197.7 (C-4), and two oxymethine carbons (δC 83.2 (C-2) and 71.6 (C-3) (see Figure 1 and Supplementary Materials)). The HRESIMS spectrum of 1 showed the pseudomolecular ion at [M + H]+ at m/z 321.0597, indicating the molecular formula C15H12O8. The positive optical rotation αD25 + 11.2° (c 0.2, MeOH) hinted that 1 probably shared the (R, R) configuration. Based on this evidence and in comparison with the published data, compound 1 was identified as (2R, 3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one (ampelopsin or dihydromyricetin) [6,13]. This compound was found to possess anticholinesterase [6], antiatherosclerosis (anti-inflammatory), antidiabetes, and antitumor activities and effects on cardioprotection, hepatoprotection, neuroprotection, and dermatoprotection [14].Compound2 was obtained as a yellow powder. The presence of a methine group (δH 6.54 (H-3)/δC 102.6 (C-3)), an oxygenated carbon at δC 165.3 (C-2), and a carbonyl carbon at δC 182.6 (C-4) in the 1H- and 13C-NMR spectra indicated 2 to be a flavone [12]. The 1H-NMR of 2 showed signals the presence of 1,4-disubstituted benzene ring characterized with the A2B2 system (δH 77.91 (2H, d, J = 8.4 Hz, H-2′/6′), 6.89 (2H, d, d, J = 8.4 Hz, H-3′/5′)) together with two sugar (δH 5.05 (1H, d, J = 9.6 Hz, H-1″), 5.16 (1H, br s, H-1‴)) for the glucose and rhamnose moieties (see Figure 1 and Supplementary Materials). The 13C-NMR spectrum exhibited twenty-seven signals due to twelve sp2 carbons of two benzene rings, a ketone, and twelve carbons of two sugar moieties. In the HMBC spectrum, the correlations of proton H-1″ at δH 5.05 (1H, d) to carbon signals at δC 109.1 (C-6), 157.6 (C-5), and 163.1 (C-7), as well as the correlations of proton H-1‴ at δH 5.16 (1H, br s) to carbon signals at δC 101.8 (C-8), 155.9 (C-9), and 163.1 (C-7), were observed suggesting the glucose and rhamnose moieties were located at C-6 and C-8, respectively (see Figure 1 and Supplementary Materials). Therefore, compound 2 was identified as violanthin in comparison with literature data [6, 15]. Violanthin has been shown to possess antioxidant [16] and anticholinesterase activities [6].Compounds3 and 6 were isolated as a white amorphous powder. Their 1H-NMR spectra displayed characteristic signals due to aromatic protons of two benzene rings and a carbinol methylene (2H-7′), while their 13C-NMR spectra revealed the signals of a carbonyl carbon (C-7), a carbinol methylene carbon (C-7′), and twelve sp2 carbons of two benzene rings (see Figure 1 and Supplementary Materials). The above observation indicated these compounds were benzyl benzoate derivatives [17]. Detailed analysis of the 1H- and 13C-NMR spectra revealed that 3 possessed ten aromatic protons of two benzene rings, a carbinol methylene (δH 5.39 (2H, s, H-7′)/δC 66.6 (C-7′)), and a carbonyl carbon at δC 166.3 (C-7) (see Figure 1 and Supplementary Materials). Compound 6 also possessed one carbonyl carbon (C-7) and a carbinol methylene (C-7′) but showed only nine aromatic protons and an additional one methoxy (see Figure 1). The HMBC correlated from H-4′/H-6′/H-7′ and protons of a methoxy group (δH 3.86) to C-2′ (δC 158.0), suggested the methoxy group was located at C-2′ (see Figure 1 and Supplementary Materials). The HRESIMS spectrum of 6 showed the pseudomolecular ion at [M − H]− at m/z 241.1213, indicating the molecular formula C15H14O3. Comparison of the 1H- and 13C-NMR data of these compounds with those published in the literature led to the structural identification of compounds 3 and 6 to be benzyl benzoate [18] and 2-methoxybenzyl benzoate [19], respectively. Compound 3 (benzyl benzoate) has been found to exhibit antibacterial activity [19], and compound 6 (2-methoxybenzyl benzoate) exhibits inhibitory activity against the nuclear factor of activated T cells (NFAT) transcription factor [6].Compound4 was obtained as a colorless oil. The HRFABMS spectrum of 4 showed the pseudomolecular ion at [M]+ at m/z 232.1829, indicating the molecular formula C16H24O. The 1H NMR data of 4 revealed the presence of an OH group (δH 5.91, 1H, br s) and 1,4-disubstituted benzene ring characterized by the A2B2 system, confirming the presence of a para-substituted phenol. The 1H NMR signals showed the presence of an OH group, a benzylic methylene (H-1′), a double bond (H-2′ and H-3′), allylic methylene (H-4′), five methylenes (H-5′ to H-9′), an A2B2 system of benzene ring, and an additional one methyl group (H-10′), indicating a decenyl chain (see Figure 1 and Supplementary Materials). The 13C NMR and DEPT spectra revealed the signals of two olefinic carbons, six aromatic carbons, seven methylene carbons, and a methyl carbon (see Figure 1 and Supplementary Materials). The stereochemistry of C-2′ was determined as cis by comparison of the 13C NMR chemical shifts of the allylic carbons (δC 32.7 (C-1′) and 27.2 (C-4′)) in 4 with those of the E form in myricanene B (δC 34.3 (C-1′) and 32.3 (C-4′)). Analysis of these signals by the COSY, HMQC, and HMBC spectra led to the partial structures of 4 (see Figure 1). Therefore, compound 4 was identified as 4-(2′-(Z)-decenyl) phenol (bavinol A) in comparison with literature data [6]. Bavinol A was found to exhibit anticholinesterase activity [6].Compound5 was also obtained as a colorless oil. The 1H NMR spectrum of 5 displayed signals due to five methyls (H-16, H-17, H-18, H-19, and H-20), nine methylenes, an oxygenated methylene at δH 4.11 (H-1), three methines (H-7, H-11, and H-15), and an olefinic δH 5.37 (H-3) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of two olefinic carbons (δC 123.3 (C-2), 140.5 (C-3)), five methyl carbon (δC 22.8 (C-16), 22.9 (C-17), 19.9 (C-18), 19.9 (C-19), and 16.4 (C-20)), nine methylene carbons, three methane carbons at δC 33.0 (C-7), 32.9 (C-11), and 28.2 (C-15), and an oxygenated methylene carbon at δC 59.6 (C-1) (see Figure 1 and Supplementary Materials). The correlations from H-20 to C-2, C-3, and C-4; H-2 to C-1, C-3, C-4, and C-20; and 2H-1 to C-2 and C-3 were observed in the HMBC. Further analysis of these signals by the COSY, HMQC and HMBC spectra led to the partial structures of 5 (see Figure 1). The stereochemistry of C-2 was determined as trans by comparison of the 13C NMR chemical shifts. The positive optical rotation αD25 + 9.7° (c 0.05, MeOH) hinted that 5 probably shared the [R, R] configuration. Thus, the structure of compound 5 was assigned to be (2E, 7R, and 11R)-3,7,11,15-tetramethyl-2-hexadecen-1-ol (trans-phytol) [19] and isolated from P. bavinum for the first time. This compound was also isolated from Piper kadsura [20]. Trans-phytol was found to possess antinociceptive and antioxidant [21], cytotoxic [22], antischistosomal [23], and arthritis activities [24] and was also found to inhibit neutrophil migration [25].Compound7 was isolated as a colorless solid with negative optical rotation, αD25 − 42.3°. The HRFABMS spectrum of 7 showed the pseudomolecular ion at [M + H]+ at m/z 427.1393, indicating the molecular formula C23H22O8. The 1H NMR spectrum of 7 displayed characteristic signals due to ten aromatic protons of two benzene rings, three oxygenated methines (δH 4.29 (H-2), 5.77 (H-3), and 5.68 (H-6)), two olefinic (δH 5.95 (H-4) and 5.87 (H-5)), an oxygenated methylene (δH 4.70 (H-7a) and 4.67 (H-7b)), an acetyl group (δH 2.02 (6-OAc)), and an OH group at δH 4.90 (1-OH) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of two carbonyl carbons (C-7′ and C-7″), two olefinic carbons (C-4 and C-5), twelve sp2 carbons of two benzene rings, three oxygenated methines (C-2, C-3, and C-6), an oxygenated methylene (C-7), a quaternary carbon (C-1), and an acetyl group at δC 171.0 and 20.9 (see Figure 1 and Supplementary Materials). The long-range correlations between H-4 and C-7″, as well as between 2H7 and C-7′, were observed in the HMBC spectrum, suggesting that two benzoyl groups were located at C-7 and C-4. In addition, the HMBC correlation from H-6 to carbonyl carbon at δC 171.0 was also observed, indicating the acetyl group was located at C-6. Further analysis of these signals by the COSY, HMQC, and HMBC spectra led to the partial structures of 7 (see Figure 1). The CD Cotton effects ∆ε236 – 6.24 and ∆ε282 + 4.32 indicated that compound 7 probably shared the [S, S, R, S] configuration. Based on this evidence and in comparison with the published data, compound 7 was identified as (–)-6-acetylzeylenol [26]. This compound has been shown to exhibit cytotoxic activity [27].Compound8 was obtained as a white powder with positive optical rotation, αD25 + 67.5°. The 1H NMR data of 8 revealed the presence of three methyl groups (H-11, H-12, and H-13), two trans olefinic protons (H-7 and H-8), two methylene (H-2 and H-10), and two methine protons (H-4 and H-9) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of a ketone (C-3), three methyl carbons (C-11, C-12, and C-13), four olefinic carbons (C-4, C-5, C-7, and C-8), two methylene carbons (C-2 and C-10), an methine (C-9), and two quaternary carbons (C-1 and C-6) (see Figure 1 and Supplementary Materials). The HRESIMS spectrum of 8 showed the pseudomolecular ion at [M + Na]+ at m/z 263.1244, indicating the molecular formula C13H20O4. The CD spectrum of 8 exhibited a positive Cotton effect (∆ε242 + 11.56), indicating that the 6-position was shown to have an S-configuration. The absolute configuration at the 9-position was elucidated to be S when comparing with the 1H, 13C NMR, and the positive optical rotation value [27]. The 1D and 2D NMR spectra indicated this compound had megastigmane skeleton and identified as cucumegastigmane I in comparison with the literature [28].


### 2.2. NO Production Inhibition and the Cell Viability Assay

In the first experiment, the cytotoxic assay was performed to determine the safe and nontoxic concentration of isolated compounds (1–8) for the next assay. Nontoxicity of the isolated compounds indicated by over 90% of cell viability by MTS assay [29]. The isolated compounds in the concentration of 100 μM were toxic toward RAW 264.7 cells; therefore, the respective concentration was not used for the treatments, and the concentrations of 30, 10, and 3 μM were chosen to further study (Figure 2).Figure 2
Effect on cell viability by LPS stimulation in the presence of compounds1–8. Cell viability was determined by the MTS assay and expressed as a percentage of the control without the addition of indicated compounds 1–8.To determine the effects of these compounds on the LPS-induced production of NO in RAW 264.7 cells, a cell culture medium was harvested, and the production of nitrite was measured using the Griess reaction [30–32]. As shown in Table 1, bavinol A (4) showed the strongest inhibitory on NO production among the isolated compounds with an IC50 value of 5.2 μM, followed by trans-phytol (5) exhibiting inhibitory effects with an IC50 value of 13.5 μM. Interestingly, the other compounds showed weak inhibitory activity with IC50 values over 30 μM (see Table 1). In this assay, celastrol, a natural secondary metabolite, was used as a positive inhibitor. This compound expressively withdrew LPS-induced NO production with an IC50 value of 1.0 μM [30, 31].Table 1
NO production inhibitory activity of isolated compounds1–8.
Compound
IC50 value (μM)a
1
>30
2
>30
3
>30
4
5.2 ± 0.4
5
13.5 ± 1.5
6
>30
7
>30
8
>30
Celastrol
b
1.0 ± 0.1
aThe inhibitory effects are represented as the molar concentration (μM) giving 50% inhibition (IC50) relative to the vehicle control. These data represent the average values of three repeated experiments (mean ± SD). bPositive control for NO.Neither LPS nor the samples were added to the control group. Thus, the inhibitory effects of these compounds on NO production were not attributable to any cytotoxic effect. As shown in Figure3, after LPS (1 µg/mL) stimulation, the NO production increased by approximately 13-fold after 24 h in the control. Compounds 4 and 5 reduced the NO production 24 h after LPS stimulation, in a dose-dependent manner (Figure 3).Figure 3
Inhibitory effect of compounds4 and 5 on the LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations (1, 3, 10, and 30 μM) of the tested compounds for 1 h and then with LPS (1 μg/mL) and incubated for 24 h. Control values were obtained in the absence of LPS and compounds. The data are expressed as the mean ± SD (n = 3). Statistical significance was assessed by two-tailed unpaired Student’s t-test, and P<0.05 was considered statistically significant.It is well-known that COX-2 is induced by cytokines and other activators such as LPS, resulting in the release of large amounts of PGE2 in macrophage cells [2]. Among the tested compounds, bavinol A (4) exhibited the strongest inhibitory effect on the LPS-induced production of NO in macrophage RAW 264.7 cells. Therefore, western blot was performed to determine the inhibitory effects of bavinol A (4) on the modulation of iNOS and COX-2 expression [33] (Supplementary Materials). As shown in Figure 4, bavinol A (4) (0–30 μM) showed a dose-dependent reduction in LPS-induced iNOS and COX-2 expressions but did not change the alpha-tubulin expression (see Figure 4). The results showed that bavinol A (4) inhibited iNOS and COX-2 activities in LPS-stimulated RAW 264.7 cells at the transcription level.Figure 4
Inhibition of LPS-induced iNOS expression in RAW 264.7 cells by compound4 (C4). RAW 264.7 cells were pretreated for 30 min at indicated concentrations of 0, 3, 10, and 30 μM, followed by stimulation with LPS (1 μg/mL) for 24 h. Whole-cell lysates were blotted with the indicated antibodies. α-tubulin level was used as a loading control. The expression levels of iNOS and COX-2 were determined by the analysis of immunoblot.Macrophage cells are principally concerned in acute and chronic inflammatory responses. Among the inflammation stimuli, LPS is known as lipoglycans and endotoxins that stimulated macrophages to induce the expression of iNOS protein to produce NO. In the meantime, NO plays as an activator of macrophages to kill microorganisms through signal transduction [34]. On the contrary, the overproduction of NO in the immune system might cause immune hypersensitivity reactions followed by tissue or cell injury [3–5]. NO is mostly synthesized by iNOS; however, the extensively higher amount of NO coactively activates inflammatory processes in conjunction with other inflammatory mediators [35]. Thus, the inhibition of iNOS activity or downregulation of iNOS expression could be beneficial to inhibit inflammatory responses. From the results, flavonoid glycosides (violanthin, 2), a derivative of apigenin, did not significantly inhibit NO production at 30 μM, regardless of the aglycones (flavone) present and the glycoside linkages (C- or O-glycosides) (Figure 1). The glycosylation of apigenin to violanthin (2) resulted in a loss of nitrite inhibitory production in RAW 264.7 murine macrophages. In general, flavones showed strong inhibition of NO production. Previous studied showed that luteolin and apigenin (have a C-2,3 double bond) inhibited NO production via iNOS downregulation [36]. These results powerfully suggested that the C-2,3-double bond could be an essential factor for inhibiting NO production. It was found that flavanone derivatives (ampelopsin 1), which do not have a C-2,3 double bond (Figure 1), were inactive up to 30 μM. These results also indicated that a planar ring system in the flavonoid molecule might be important for NO inhibition [37].Lv et al. tested the effects of the megastigmane derivatives fromLyonia ovalifolia on NO production. The in vitro results indicated that megastigmane derivatives such as abscisic acid-β-D-glucopyranosyl ester and pisumionoside inhibited NO production from LPS-activated RAW 264.7 cells with inhibition rates of 54.5% and 83.3% at the concentration of 10−5 M, respectively. Pisumionoside was also found to possess better inhibitory activity than the other megastigmane derivatives tested [38]. Both of them revealed the presence of the conjugated double bond of the olefinic group with carbonyl carbon in the structures. Similarly, Trang et al. performed a study regarding the effect of megastigmane derivatives on NO production, and the results showed that these megastigmane derivatives were inactive [39]. In our experiment, cucumegastigmane I (8), a derivative of megastigmane, was also inactive on NO production, presumably because of the absence of the conjugated double bond of the olefinic group with carbonyl carbon in 8 (Figure 1).Phenolics including alkenyl phenols are essential compounds for the suppression of inflammation among phytochemicals. Cuong et al. studied the anti-inflammatory effects of phenolic compounds, and the results showed a strong inhibitory on NO production of isolated compounds because of the presence of the conjugated double bond of the olefinic group, and the 3,4-hydroxylation(s) of benzene ring gave favorable results [31, 32, 40]. In our study, bavinol A (4), which belongs to the alkenyl phenol class, exhibited the strongest inhibitory on NO production among the isolated compounds. However, the inhibitory activities on NO production of bavinol A (4) were weaker than active compounds presumably because of the lack of 3-hydroxylation of the benzene ring [31, 32, 40]. Compound 5 (trans-phytol) containing the conjugated double bond of the olefinic group in the structure also exhibited a strong inhibitory on NO production. However, the inhibitory activities on NO production of trans-phytol (5) were significantly reduced presumably because of the absence of the benzene ring contained the 3,4-hydroxylation(s) (Figure 1). In opposite, other phenolic compounds (3, 6, and 7) were inactive presumably because of the absence of the conjugated double bond of the olefinic group as well as 3,4-hydroxylation(s) of benzene ring (Figure 1). Finally, our obtained data suggested that phenolic compounds, particularly the alkenyl phenols bearing the conjugated double bond as well as 3,4-hydroxylation(s) of the benzene ring, could be considered as new lead compounds for the development of agents against NO production. Moreover, the alkenyl phenol-enrich extracts may be applied as supplemental and/or functional foods having a beneficial effect against inflammation.


## 2.1. Determination and Elucidation of Isolated Compounds

The methanol extract was partitioned withn-hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (BuOH) to obtain n-hexane, CHCl3, EtOAc, and BuOH soluble fractions, respectively. Based on bioactivity-guided fractionation (data not are shown), the n-hexane and EtOAc fractions were subjected to column chromatography on a silica gel and C18-RP silica gel column to obtain eight compounds (1–8) (see Figure 1).Figure 1
Structure of compounds1–8 from P. bavinum.Compound1 was obtained as a light yellow powder. The presence of two oxymethine groups (δH 4.82 (H-2)/δC 83.2 (C-2), and δH 4.45 (H-2)/δC 71.6 (C-3))and a ketone at δC 197.7 (C-4) in the 1H- and 13C-NMR spectra indicated 1 to be a flavanonol (see Figure 1 and Supplementary Materials (available here)). The 1H-NMR spectrum of 1 further showed signals of two tetrasubstituted benzene rings (δH 5.88 (1H, s, H-6), 5.91 (1H, s, H-8), and 6.52 (2H, s, H-2′/6′)) [12]. The 13C-NMR spectrum exhibited fifteen signals due to twelve sp2 carbons of two benzene rings, a ketone at δC 197.7 (C-4), and two oxymethine carbons (δC 83.2 (C-2) and 71.6 (C-3) (see Figure 1 and Supplementary Materials)). The HRESIMS spectrum of 1 showed the pseudomolecular ion at [M + H]+ at m/z 321.0597, indicating the molecular formula C15H12O8. The positive optical rotation αD25 + 11.2° (c 0.2, MeOH) hinted that 1 probably shared the (R, R) configuration. Based on this evidence and in comparison with the published data, compound 1 was identified as (2R, 3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one (ampelopsin or dihydromyricetin) [6,13]. This compound was found to possess anticholinesterase [6], antiatherosclerosis (anti-inflammatory), antidiabetes, and antitumor activities and effects on cardioprotection, hepatoprotection, neuroprotection, and dermatoprotection [14].Compound2 was obtained as a yellow powder. The presence of a methine group (δH 6.54 (H-3)/δC 102.6 (C-3)), an oxygenated carbon at δC 165.3 (C-2), and a carbonyl carbon at δC 182.6 (C-4) in the 1H- and 13C-NMR spectra indicated 2 to be a flavone [12]. The 1H-NMR of 2 showed signals the presence of 1,4-disubstituted benzene ring characterized with the A2B2 system (δH 77.91 (2H, d, J = 8.4 Hz, H-2′/6′), 6.89 (2H, d, d, J = 8.4 Hz, H-3′/5′)) together with two sugar (δH 5.05 (1H, d, J = 9.6 Hz, H-1″), 5.16 (1H, br s, H-1‴)) for the glucose and rhamnose moieties (see Figure 1 and Supplementary Materials). The 13C-NMR spectrum exhibited twenty-seven signals due to twelve sp2 carbons of two benzene rings, a ketone, and twelve carbons of two sugar moieties. In the HMBC spectrum, the correlations of proton H-1″ at δH 5.05 (1H, d) to carbon signals at δC 109.1 (C-6), 157.6 (C-5), and 163.1 (C-7), as well as the correlations of proton H-1‴ at δH 5.16 (1H, br s) to carbon signals at δC 101.8 (C-8), 155.9 (C-9), and 163.1 (C-7), were observed suggesting the glucose and rhamnose moieties were located at C-6 and C-8, respectively (see Figure 1 and Supplementary Materials). Therefore, compound 2 was identified as violanthin in comparison with literature data [6, 15]. Violanthin has been shown to possess antioxidant [16] and anticholinesterase activities [6].Compounds3 and 6 were isolated as a white amorphous powder. Their 1H-NMR spectra displayed characteristic signals due to aromatic protons of two benzene rings and a carbinol methylene (2H-7′), while their 13C-NMR spectra revealed the signals of a carbonyl carbon (C-7), a carbinol methylene carbon (C-7′), and twelve sp2 carbons of two benzene rings (see Figure 1 and Supplementary Materials). The above observation indicated these compounds were benzyl benzoate derivatives [17]. Detailed analysis of the 1H- and 13C-NMR spectra revealed that 3 possessed ten aromatic protons of two benzene rings, a carbinol methylene (δH 5.39 (2H, s, H-7′)/δC 66.6 (C-7′)), and a carbonyl carbon at δC 166.3 (C-7) (see Figure 1 and Supplementary Materials). Compound 6 also possessed one carbonyl carbon (C-7) and a carbinol methylene (C-7′) but showed only nine aromatic protons and an additional one methoxy (see Figure 1). The HMBC correlated from H-4′/H-6′/H-7′ and protons of a methoxy group (δH 3.86) to C-2′ (δC 158.0), suggested the methoxy group was located at C-2′ (see Figure 1 and Supplementary Materials). The HRESIMS spectrum of 6 showed the pseudomolecular ion at [M − H]− at m/z 241.1213, indicating the molecular formula C15H14O3. Comparison of the 1H- and 13C-NMR data of these compounds with those published in the literature led to the structural identification of compounds 3 and 6 to be benzyl benzoate [18] and 2-methoxybenzyl benzoate [19], respectively. Compound 3 (benzyl benzoate) has been found to exhibit antibacterial activity [19], and compound 6 (2-methoxybenzyl benzoate) exhibits inhibitory activity against the nuclear factor of activated T cells (NFAT) transcription factor [6].Compound4 was obtained as a colorless oil. The HRFABMS spectrum of 4 showed the pseudomolecular ion at [M]+ at m/z 232.1829, indicating the molecular formula C16H24O. The 1H NMR data of 4 revealed the presence of an OH group (δH 5.91, 1H, br s) and 1,4-disubstituted benzene ring characterized by the A2B2 system, confirming the presence of a para-substituted phenol. The 1H NMR signals showed the presence of an OH group, a benzylic methylene (H-1′), a double bond (H-2′ and H-3′), allylic methylene (H-4′), five methylenes (H-5′ to H-9′), an A2B2 system of benzene ring, and an additional one methyl group (H-10′), indicating a decenyl chain (see Figure 1 and Supplementary Materials). The 13C NMR and DEPT spectra revealed the signals of two olefinic carbons, six aromatic carbons, seven methylene carbons, and a methyl carbon (see Figure 1 and Supplementary Materials). The stereochemistry of C-2′ was determined as cis by comparison of the 13C NMR chemical shifts of the allylic carbons (δC 32.7 (C-1′) and 27.2 (C-4′)) in 4 with those of the E form in myricanene B (δC 34.3 (C-1′) and 32.3 (C-4′)). Analysis of these signals by the COSY, HMQC, and HMBC spectra led to the partial structures of 4 (see Figure 1). Therefore, compound 4 was identified as 4-(2′-(Z)-decenyl) phenol (bavinol A) in comparison with literature data [6]. Bavinol A was found to exhibit anticholinesterase activity [6].Compound5 was also obtained as a colorless oil. The 1H NMR spectrum of 5 displayed signals due to five methyls (H-16, H-17, H-18, H-19, and H-20), nine methylenes, an oxygenated methylene at δH 4.11 (H-1), three methines (H-7, H-11, and H-15), and an olefinic δH 5.37 (H-3) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of two olefinic carbons (δC 123.3 (C-2), 140.5 (C-3)), five methyl carbon (δC 22.8 (C-16), 22.9 (C-17), 19.9 (C-18), 19.9 (C-19), and 16.4 (C-20)), nine methylene carbons, three methane carbons at δC 33.0 (C-7), 32.9 (C-11), and 28.2 (C-15), and an oxygenated methylene carbon at δC 59.6 (C-1) (see Figure 1 and Supplementary Materials). The correlations from H-20 to C-2, C-3, and C-4; H-2 to C-1, C-3, C-4, and C-20; and 2H-1 to C-2 and C-3 were observed in the HMBC. Further analysis of these signals by the COSY, HMQC and HMBC spectra led to the partial structures of 5 (see Figure 1). The stereochemistry of C-2 was determined as trans by comparison of the 13C NMR chemical shifts. The positive optical rotation αD25 + 9.7° (c 0.05, MeOH) hinted that 5 probably shared the [R, R] configuration. Thus, the structure of compound 5 was assigned to be (2E, 7R, and 11R)-3,7,11,15-tetramethyl-2-hexadecen-1-ol (trans-phytol) [19] and isolated from P. bavinum for the first time. This compound was also isolated from Piper kadsura [20]. Trans-phytol was found to possess antinociceptive and antioxidant [21], cytotoxic [22], antischistosomal [23], and arthritis activities [24] and was also found to inhibit neutrophil migration [25].Compound7 was isolated as a colorless solid with negative optical rotation, αD25 − 42.3°. The HRFABMS spectrum of 7 showed the pseudomolecular ion at [M + H]+ at m/z 427.1393, indicating the molecular formula C23H22O8. The 1H NMR spectrum of 7 displayed characteristic signals due to ten aromatic protons of two benzene rings, three oxygenated methines (δH 4.29 (H-2), 5.77 (H-3), and 5.68 (H-6)), two olefinic (δH 5.95 (H-4) and 5.87 (H-5)), an oxygenated methylene (δH 4.70 (H-7a) and 4.67 (H-7b)), an acetyl group (δH 2.02 (6-OAc)), and an OH group at δH 4.90 (1-OH) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of two carbonyl carbons (C-7′ and C-7″), two olefinic carbons (C-4 and C-5), twelve sp2 carbons of two benzene rings, three oxygenated methines (C-2, C-3, and C-6), an oxygenated methylene (C-7), a quaternary carbon (C-1), and an acetyl group at δC 171.0 and 20.9 (see Figure 1 and Supplementary Materials). The long-range correlations between H-4 and C-7″, as well as between 2H7 and C-7′, were observed in the HMBC spectrum, suggesting that two benzoyl groups were located at C-7 and C-4. In addition, the HMBC correlation from H-6 to carbonyl carbon at δC 171.0 was also observed, indicating the acetyl group was located at C-6. Further analysis of these signals by the COSY, HMQC, and HMBC spectra led to the partial structures of 7 (see Figure 1). The CD Cotton effects ∆ε236 – 6.24 and ∆ε282 + 4.32 indicated that compound 7 probably shared the [S, S, R, S] configuration. Based on this evidence and in comparison with the published data, compound 7 was identified as (–)-6-acetylzeylenol [26]. This compound has been shown to exhibit cytotoxic activity [27].Compound8 was obtained as a white powder with positive optical rotation, αD25 + 67.5°. The 1H NMR data of 8 revealed the presence of three methyl groups (H-11, H-12, and H-13), two trans olefinic protons (H-7 and H-8), two methylene (H-2 and H-10), and two methine protons (H-4 and H-9) (see Figure 1 and Supplementary Materials). The 13C NMR spectrum revealed the signals of a ketone (C-3), three methyl carbons (C-11, C-12, and C-13), four olefinic carbons (C-4, C-5, C-7, and C-8), two methylene carbons (C-2 and C-10), an methine (C-9), and two quaternary carbons (C-1 and C-6) (see Figure 1 and Supplementary Materials). The HRESIMS spectrum of 8 showed the pseudomolecular ion at [M + Na]+ at m/z 263.1244, indicating the molecular formula C13H20O4. The CD spectrum of 8 exhibited a positive Cotton effect (∆ε242 + 11.56), indicating that the 6-position was shown to have an S-configuration. The absolute configuration at the 9-position was elucidated to be S when comparing with the 1H, 13C NMR, and the positive optical rotation value [27]. The 1D and 2D NMR spectra indicated this compound had megastigmane skeleton and identified as cucumegastigmane I in comparison with the literature [28].


## 2.2. NO Production Inhibition and the Cell Viability Assay

In the first experiment, the cytotoxic assay was performed to determine the safe and nontoxic concentration of isolated compounds (1–8) for the next assay. Nontoxicity of the isolated compounds indicated by over 90% of cell viability by MTS assay [29]. The isolated compounds in the concentration of 100 μM were toxic toward RAW 264.7 cells; therefore, the respective concentration was not used for the treatments, and the concentrations of 30, 10, and 3 μM were chosen to further study (Figure 2).Figure 2
Effect on cell viability by LPS stimulation in the presence of compounds1–8. Cell viability was determined by the MTS assay and expressed as a percentage of the control without the addition of indicated compounds 1–8.To determine the effects of these compounds on the LPS-induced production of NO in RAW 264.7 cells, a cell culture medium was harvested, and the production of nitrite was measured using the Griess reaction [30–32]. As shown in Table 1, bavinol A (4) showed the strongest inhibitory on NO production among the isolated compounds with an IC50 value of 5.2 μM, followed by trans-phytol (5) exhibiting inhibitory effects with an IC50 value of 13.5 μM. Interestingly, the other compounds showed weak inhibitory activity with IC50 values over 30 μM (see Table 1). In this assay, celastrol, a natural secondary metabolite, was used as a positive inhibitor. This compound expressively withdrew LPS-induced NO production with an IC50 value of 1.0 μM [30, 31].Table 1
NO production inhibitory activity of isolated compounds1–8.
Compound
IC50 value (μM)a
1
>30
2
>30
3
>30
4
5.2 ± 0.4
5
13.5 ± 1.5
6
>30
7
>30
8
>30
Celastrol
b
1.0 ± 0.1
aThe inhibitory effects are represented as the molar concentration (μM) giving 50% inhibition (IC50) relative to the vehicle control. These data represent the average values of three repeated experiments (mean ± SD). bPositive control for NO.Neither LPS nor the samples were added to the control group. Thus, the inhibitory effects of these compounds on NO production were not attributable to any cytotoxic effect. As shown in Figure3, after LPS (1 µg/mL) stimulation, the NO production increased by approximately 13-fold after 24 h in the control. Compounds 4 and 5 reduced the NO production 24 h after LPS stimulation, in a dose-dependent manner (Figure 3).Figure 3
Inhibitory effect of compounds4 and 5 on the LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were pretreated with different concentrations (1, 3, 10, and 30 μM) of the tested compounds for 1 h and then with LPS (1 μg/mL) and incubated for 24 h. Control values were obtained in the absence of LPS and compounds. The data are expressed as the mean ± SD (n = 3). Statistical significance was assessed by two-tailed unpaired Student’s t-test, and P<0.05 was considered statistically significant.It is well-known that COX-2 is induced by cytokines and other activators such as LPS, resulting in the release of large amounts of PGE2 in macrophage cells [2]. Among the tested compounds, bavinol A (4) exhibited the strongest inhibitory effect on the LPS-induced production of NO in macrophage RAW 264.7 cells. Therefore, western blot was performed to determine the inhibitory effects of bavinol A (4) on the modulation of iNOS and COX-2 expression [33] (Supplementary Materials). As shown in Figure 4, bavinol A (4) (0–30 μM) showed a dose-dependent reduction in LPS-induced iNOS and COX-2 expressions but did not change the alpha-tubulin expression (see Figure 4). The results showed that bavinol A (4) inhibited iNOS and COX-2 activities in LPS-stimulated RAW 264.7 cells at the transcription level.Figure 4
Inhibition of LPS-induced iNOS expression in RAW 264.7 cells by compound4 (C4). RAW 264.7 cells were pretreated for 30 min at indicated concentrations of 0, 3, 10, and 30 μM, followed by stimulation with LPS (1 μg/mL) for 24 h. Whole-cell lysates were blotted with the indicated antibodies. α-tubulin level was used as a loading control. The expression levels of iNOS and COX-2 were determined by the analysis of immunoblot.Macrophage cells are principally concerned in acute and chronic inflammatory responses. Among the inflammation stimuli, LPS is known as lipoglycans and endotoxins that stimulated macrophages to induce the expression of iNOS protein to produce NO. In the meantime, NO plays as an activator of macrophages to kill microorganisms through signal transduction [34]. On the contrary, the overproduction of NO in the immune system might cause immune hypersensitivity reactions followed by tissue or cell injury [3–5]. NO is mostly synthesized by iNOS; however, the extensively higher amount of NO coactively activates inflammatory processes in conjunction with other inflammatory mediators [35]. Thus, the inhibition of iNOS activity or downregulation of iNOS expression could be beneficial to inhibit inflammatory responses. From the results, flavonoid glycosides (violanthin, 2), a derivative of apigenin, did not significantly inhibit NO production at 30 μM, regardless of the aglycones (flavone) present and the glycoside linkages (C- or O-glycosides) (Figure 1). The glycosylation of apigenin to violanthin (2) resulted in a loss of nitrite inhibitory production in RAW 264.7 murine macrophages. In general, flavones showed strong inhibition of NO production. Previous studied showed that luteolin and apigenin (have a C-2,3 double bond) inhibited NO production via iNOS downregulation [36]. These results powerfully suggested that the C-2,3-double bond could be an essential factor for inhibiting NO production. It was found that flavanone derivatives (ampelopsin 1), which do not have a C-2,3 double bond (Figure 1), were inactive up to 30 μM. These results also indicated that a planar ring system in the flavonoid molecule might be important for NO inhibition [37].Lv et al. tested the effects of the megastigmane derivatives fromLyonia ovalifolia on NO production. The in vitro results indicated that megastigmane derivatives such as abscisic acid-β-D-glucopyranosyl ester and pisumionoside inhibited NO production from LPS-activated RAW 264.7 cells with inhibition rates of 54.5% and 83.3% at the concentration of 10−5 M, respectively. Pisumionoside was also found to possess better inhibitory activity than the other megastigmane derivatives tested [38]. Both of them revealed the presence of the conjugated double bond of the olefinic group with carbonyl carbon in the structures. Similarly, Trang et al. performed a study regarding the effect of megastigmane derivatives on NO production, and the results showed that these megastigmane derivatives were inactive [39]. In our experiment, cucumegastigmane I (8), a derivative of megastigmane, was also inactive on NO production, presumably because of the absence of the conjugated double bond of the olefinic group with carbonyl carbon in 8 (Figure 1).Phenolics including alkenyl phenols are essential compounds for the suppression of inflammation among phytochemicals. Cuong et al. studied the anti-inflammatory effects of phenolic compounds, and the results showed a strong inhibitory on NO production of isolated compounds because of the presence of the conjugated double bond of the olefinic group, and the 3,4-hydroxylation(s) of benzene ring gave favorable results [31, 32, 40]. In our study, bavinol A (4), which belongs to the alkenyl phenol class, exhibited the strongest inhibitory on NO production among the isolated compounds. However, the inhibitory activities on NO production of bavinol A (4) were weaker than active compounds presumably because of the lack of 3-hydroxylation of the benzene ring [31, 32, 40]. Compound 5 (trans-phytol) containing the conjugated double bond of the olefinic group in the structure also exhibited a strong inhibitory on NO production. However, the inhibitory activities on NO production of trans-phytol (5) were significantly reduced presumably because of the absence of the benzene ring contained the 3,4-hydroxylation(s) (Figure 1). In opposite, other phenolic compounds (3, 6, and 7) were inactive presumably because of the absence of the conjugated double bond of the olefinic group as well as 3,4-hydroxylation(s) of benzene ring (Figure 1). Finally, our obtained data suggested that phenolic compounds, particularly the alkenyl phenols bearing the conjugated double bond as well as 3,4-hydroxylation(s) of the benzene ring, could be considered as new lead compounds for the development of agents against NO production. Moreover, the alkenyl phenol-enrich extracts may be applied as supplemental and/or functional foods having a beneficial effect against inflammation.


## 3. Conclusion

This study demonstrated for the first time the NO production inhibitory activities of isolated compounds fromP. bavinum. NO production inhibitory guided fractionation led to the isolation of eight compounds, ampelopsin (1), violanthin (2), benzyl benzoate (3), bavinol A (4), trans-phytol (5), 2-methoxybenzyl benzoate (6), (–)-6-acetylzeylenol (7), and cucumegastigmane I (8), from the aerial part of P. bavinum. Trans-phytol (5) was isolated from P. bavinum for the first time. Bavinol A (4) and trans-phytol (5) showed inhibitory effects with IC50 values of 5.2 and 13.5 μM, respectively, but the others were inactive. In addition, bavinol A (4) showed a dose-dependent reduction in LPS-induced iNOS and COX-2 expressions but did not change the alpha-tubulin expression. The results showed that bavinol A (4) inhibited iNOS and COX-2 activities in LPS-stimulated RAW 264.7 cells at the transcription level. This result provides experimental evidence to support that bavinol A may serve as a useful anti-inflammation agent.



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*Source: 1038636-2020-02-08.xml*