Chemical Constituents from the Stems of Tinospora sinensis and Their Bioactivity

Fifty-seven compounds were purified from the stems of Tinospora sinensis, including three new compounds characterized as a lignan (1), a pyrrole alkaloid (11), and a benzenoid (17), respectively. Their structures were elucidated and established by various spectroscopic and spectrometric analytical methods. Among the isolates, fifteen compounds were examined for their anti-inflammatory potential in vitro. The results showed that several compounds displayed moderate inhibition of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.


Introduction
Inflammation is the first response of the immune system to infection or irritation. Neutrophils play an important role in eliminating most of the exogenous pathogens. Various autoimmune diseases are linked to neutrophil overexpression, such as rheumatoid arthritis, ischemia, and asthma, etc. [1][2][3]. According to response of diverse stimuli, activated neutrophils will secrete a series of cytotoxins. The superoxide anions and neutrophil elastase are the major secreted products of stimulated neutrophils in infected tissues and organs, which contribute to the destruction of tissue in chronic inflammatory diseases [4][5][6]. Therefore, inhibition of superoxide anion generation and elastase release by natural compounds is considered to be an effective screening platform to evaluate anti-inflammatory drug candidates.
Compound 12 displayed very similar 1 H and 13 C-NMR signals ( Table 2) as those of 11 except an additional aldehyde signal (δ H 9.42 (s, H-6) and δ C 180.9 (C-6)) and one methoxy group (δ H 3.66 (s, OCH 3 ) and δ C 52.2 (OCH 3 )). Its HMBC spectrum exhibited the correlations from methoxy protons to butanoic acid C-4 (δ C 175.1), as shown in Figure 3. The molecular formula of 12 was proposed as C 11 H 15 NO 4 also based on the GC-MS analytical data of the molecular ion peak at m/z 225 (see Supplementary Materials). Accordingly, the structure of 12 was established as methyl 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoate. Compound 13 was shown to possess the molecular formula of C 12 H 17 O 4 N by GC-MS measurement. An additional methoxy group (δ H 3.36) was observed in 13 by comparison of its 1 H-NMR spectra with that of 12. The structure of compound 13 was elucidated as a methyl 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoate. Furthermore, compounds 14 and 15 were determined as 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid and 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid, respectively, by comparison of their spectral data with those reported [22,23]. According to the above results, pyrrole alkaloids 10-15 were reported from Tinospora genus for the first time. The connection of the butanoic acid moiety on the nitrogen atom was suggested by observing long range correlation peaks from δH 4.37 (H-1′) to δC 136.9 (C-5) and δC 121.6 (C-2) in the HMBC spectrum ( Figure 3). These spectral data clearly determined that a butanoic acid moiety was attached to N-1 of the pyrrole ring. An oxomethylene group connected to C-5 of pyrrole ring was proved by the HMBC correlation of δH 4.43 (H-7) and δC 136.9 (C-5). Two additional methoxy groups (δH 3.34, δC 51.6; δH 3.67, δC 57.7) were also observed and deduced to be located at C-7 and C-4′ by HMBC analysis (Figure 3). However, the HR-ESI-MS analytical data was unavailable due to the sample lability. Therefore, the molecular formula of 11 was proposed as C12H17NO5 according to the above-mentioned NMR spectral analysis and gas chromatograph-mass spectrometer (GC-MS) analytical results which exhibited a molecular ion peak at m/z 255 (see Supporting Information). On the basis of these data, the structure of 11 was determined as 1-(4-methoxy-4-oxobutyl)-5-(methoxymethyl)-1H-pyrrole-2-carboxylic acid and named trivially as tinosporin A. Compound 12 displayed very similar 1 H and 13 C-NMR signals ( Table 2) as those of 11 except an additional aldehyde signal (δH 9.42 (s, H-6) and δC 180.9 (C-6)) and one methoxy group (δH 3.66 (s, OCH3) and δC 52.2 (OCH3)). Its HMBC spectrum exhibited the correlations from methoxy protons to butanoic acid C-4′ (δC 175.1), as shown in Figure 3. The molecular formula of 12 was proposed as C11H15NO4 also based on the GC-MS analytical data of the molecular ion peak at m/z 225 (see Supporting Information). Accordingly, the structure of 12 was established as methyl 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoate. Compound 13 was shown to possess the molecular formula of C12H17O4N by GC-MS measurement. An additional methoxy group (δH 3.36) was observed in 13 by comparison of its 1 H-NMR spectra with that of 12. The structure of compound 13 was elucidated as a methyl 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoate. Furthermore, compounds 14 and 15 were determined as 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid and 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid, respectively, by comparison of their spectral data with those reported [22,23]. According to the above results, pyrrole alkaloids 10-15 were reported from Tinospora genus for the first time.   8 and 115.5). A 3,4disubstituted cinnamoyl group linked with a hexanoyl alcohol was deduced from the NMR data which described above (Table 1). This was further confirmed by the key HMBC correlations from δH 3.67 (OCH3) to 173.9 (C-1′), from δH 4.19 (H-6′) to δC 167.5 (C-9), and 28.1 (C-5′), as shown in Figure 4. Therefore, compound 16 was confirmed as rhodiolate by comparison of its spectral data with those   (Table 1). This was further confirmed by the key HMBC correlations from δ H 3.67 (OCH 3 ) to 173.9 (C-1 ), from δ H 4.19 (H-6 ) to δ C 167.5 (C-9), and 28.1 (C-5 ), as shown in Figure 4. Therefore, compound 16 was confirmed as rhodiolate by comparison of its spectral data with those reported [24]. Compound 17 displayed closely related 1D NMR spectroscopic and mass spectrometric characteristics to 16 and was determined to have a similar structure to 16. However, a pair of olefinic protons at δ H 6.80 (d, J = 12.9 Hz, H-7) and 5.81 (d, J = 12.9 Hz, H-8) suggested the cis double bond feature. However, 2D NMR spectral analysis of 17 could not be furnished because of the rapid transformation of cis-trans double bond. Thus, the structure of compound 17 was concluded to be methyl 6-((Z)-3-(4-hydroxy-3-methoxyphenyl)acryloyloxy)-hexanoate and assigned the trivial name as tinosporin B.      Fifteen purified compounds were examined for their inhibition bioactivity of superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB (Table S2) [63,64]. However, most displayed weak inhibition percentages at the test concentration (10 µM). Among these, 1, 16, and 17 displayed higher inhibitions of superoxide anion generation at 10 µM with inhibition percentages ranged from 10.2 ± 7.1 to 20.2 ± 5.1%. In addition, compound 39 (10 µM) also exhibited inhibitory effect on elastase release with inhibition percentage of 22.3 ± 10.0% (Table S2). Columbin, an important furanoditerpenoid isolated from several Tinosporae Radix, exhibited significant anti-inflammatory activities in a dose-dependent manner [65]. However, based on our research data the related furanoid bisnorditerpenoid, malabarolide (36), was not the predominant component, maybe due to the different parts of plant materials. The conventional use of T. sinensis in traditional Chinese medicine is for relieving rigidity of muscles and activating collaterals, and the mechanism of action may be related to anti-inflammatory bioactivity. The present experimental data not only suggest that the extracts and purified compounds of the stems of T. sinensis have the potential to be developed as novel anti-inflammatory lead drugs or health foods, but also merit further investigation of the anti-inflammatory mechanism.

Materials
The stems of T. sinensis were collected from Vietnam in August 2009, and the plant material was identified and authenticated by Assoc. Prof. Dr. Vu Xuan Phuong, Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology. A voucher specimen (Viet-TSWu-2009-1801-001) was deposited in the herbarium of the Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hanoi, Vietnam.

Extraction and Isolation
The dried stems of T. sinensis (10 kg) was refluxed with methanol (30 L × 8 × 8 h) and then filtered and concentrated under reduced pressure to obtain the methanol extract (400 g). The extract was suspended in distilled water and successively partitioned with chloroform to yield a chloroform layer (60 g) and water soluble (340 g). The chloroform layer was chromatographed directly on silica gel and eluted with a gradient of n-hexane and acetone to afford 10 fractions (CF 1-10). Fractions CF 1, 2, and 4 did not show any significant spots under TLC check and therefore were not purified further. Fraction CF 3 was isolated by CC on silica gel with a step gradient with benzene and acetone mixtures and the subfraction CF 3-6 was further purified by TLC using n-hexane-ethyl acetate (50:1) to yield cycloabyssinone (40, 3 mg). Fraction CF 5 was purified using silica gel CC eluted with gradient mixtures of n-hexane and acetone to afford thirteen subfractions (CF 5-1 to 5-13). CF 5-2 was fractionated by silica gel CC eluted with benzene ethyl acetate and then lupeol (37, 8 mg), cycloeucalenol (39, 15 mg), and a mixture of β-sitosterol (43) and stigmasterol (44) (364 mg), respectively, was purified from the minor fractions by TLC using n-hexane-ethyl acetate (50:1). CF 5-5 was performed on silica gel CC with gradient mixtures of hexane and acetone to produce ten minor fractions. One minor fraction CF 5-5-7 was purified by silica gel CC with mixture of benzene and acetone and further purification by TLC using chlorofrom-acetone (9:1) yielded a mixture of 7α-hydroxysitosterol (45) and 7α-hydroxystigmasterol (46) (6 mg). CF 5-7 was subjected to silica gel CC eluted with a gradient mixture of benzene ethyl acetate to afford ten minor fractions. CF 5-7-4 was further isolated by silica gel CC, eluted with hexane ethyl acetate and subsequent TLC using hexane ethyl acetate (6:1) to afford 3-O-acetyloleanolic acid (38, 4 mg).

Preparation of Human Neutrophils
The use of human neutrophils was approved by the Institutional Review Board at Chang Gung Memorial Hospital, Taoyuan, Taiwan, and the study was conducted according to the Declaration of Helsinki (2013). Written informed consent was obtained from each healthy donor before blood was drawn. The details of the preparation of human neutrophils are provided in the Supplementary Materials.

Measurement of Superoxide Anion Generation and Elastase Release
The assay of the generation of superoxide anion was based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Degranulation of azurophilic granules was determined by elastase release as described previously [63,64]. The details of measurement of superoxide anion generation and elastase release were provided in the Supplementary Materials.

Conflicts of Interest:
The authors declare no conflicts of interest.