Three new constituents from the Tujia ethnomedicine Swertia punicea Hemsl.

Abstract Three new constituents: 1,5R-dihydroxy-3,8S-dimethoxy-5,6,7,8-tetrahydroxanthone (1), (3S,4R,16S,17R)-3,16,23-trihydroxyoleana-11,13(18)-dien-28-aldehyde-3-O-β-D-glucopyranoside (2), and new natural product (S)-gentiandiol (3), along with 41 known compounds were isolated from Tujia ethnomedicine Shuihuanglian, namely, the whole plant of Swertia punicea. Structures of all these compounds were established through extensive spectroscopic techniques, namely 1D, 2D-NMR spectroscopy, HRESIMS analysis, and the absolute configuration of the new compounds was discerned by circular dichroism (CD) spectroscopy. Antioxidative effects of these compounds were evaluated by using the DPPH radical scavenging method, compounds 7, 9 and 14 showed antioxidant activities with IC50 values of 68.9, 50.8 and 48.2 μM, respectively. Graphical Abstract


Introduction
Swertia punicea belongs to the genus Swertia of gentianaceae family. There are 170 species of genus Swertia, of which 79 are found in China and 35 species are used as folk medicine. S. punicea is mainly distributed in Hunan, Yunnan, Sichuan and Guizhou provinces (Mou et al. 2020). It is also called 'Shuihuanglian', 'Tuhuanglian', 'Kudancao', 'Caolongdan' and so on in folk. It is widely used in the treatment of various diseases, such as jaundice hepatitis, cholecystitis, pneumonia, osteomyelitis, etc., and because of its unique curative effect in treating liver disease, it is also known as 'Ganyancao'. Modern pharmacological research shows that the plant has a variety of activities, including neuroprotective, hepatoprotective, anti-HBV, antioxidant, anti-bacterial, etc. (Zafar and Wang 2018).

Results and discussion
Compound 1 was obtained as yellow powder, the molecular formula of 1 was elucidated as C 15 Figure S4) inferred that there was a fragment of -CH-CH 2 -CH 2 -CH-in 1. The 13 C NMR data showed 15 carbons attributed to one carbonyl carbon (d C 182.8), two methoxy carbons (d C 56.5 and 57.7), eight olefinic and aromatic carbons (d C 93.4, 99.1, 106.2, 118.3, 159.1, 163.3, 167.4 and 168.3), two methylene carbons (d C 25.2 and 27.2), and two oxygenated methine carbons (d C 67.6 and 70.5). The 1 H and 13 C NMR data suggested that the structure of 1 may be one of tetrahydroxanthone, and showed resemblances with that of the reported compound tetrahydrobellidifolin (Hase et al. 1997), except for the obvious differences observed at the substitution of C-8, it was a methoxy group in 1, but a hydroxyl in the reported compound. Additionally, the HMBC cross peaks ( Figure S6) of H-8 (d H 4.50) to C-8a (d C 118.3), C-4b (d C 168.3) and C-7 (d C 25.2), and of 11-OCH 3 (d H 3.47) to C-8 (d C 70.5) confirmed the location of the methoxy group. The relative configuration of 1 was determined by J-value analysis of 1 H NMR and ROESY ( Figure S7) spectrum. The small coupling constant (J ¼ 3.2 Hz) between H-7 and H-8 revealed H-8 is quasi-equatorial and the 11-OCH 3 is quasi-axial, on the other hand, H-5 is quasi-axial because it has a large coupling constant (dd, J ¼ 10.2, 6.8 Hz). Therefore, the cyclohexene ring (C-8a, C-5-C-8, and C-4b) possess a pseudo-chair conformation. H-5 and H-8 were in the cis configuration that was assigned by ROESY cross peaks of H-8 (d H Figure S9) spectrum, which is similar to the calculated spectrum of (5R, 8S) configuration, rather than (5S, 8R) configuration. So, 1 was ultimately determined as 1,5R-dihydroxy-3,8S-dimethoxy-5,6,7,8-tetrahydroxanthone ( Figure 1).
Compound 3 was obtained as a colorless oil. The molecular formula of 3 was determined as C 10 H 11 NO 4 from the HRESIMS spectrum on the basis of [M þ H] þ ion at m/z 210.0762 (210.0766 calculated for C 10 H 12 NO 4 ). UV spectrum absorptions (k max, ) at 205 and 268 nm, and IR absorptions at 1718 and 3361 cm À1 indicated the presence of hydroxyl and carbonyl groups in the skeleton of 3. The 1 H NMR spectrum of 3 revealed the presence of two olefinic protons at d H 9.02 (s, 1H) and 8.82 (s, 1H), two oxygenated methylene protons at d H 4.58 (dt, J ¼ 7.2, 4.5 Hz, 2H) and 3.73 (m, 2H), one methylene proton at d H 3.23 (dd, J ¼ 7.2, 5.5 Hz, 2H) and one oxygenated methine proton at d H 4.99 (t, J ¼ 5.8 Hz, 1H). The 13 C NMR spectrum of 3 exhibited ten carbon signals, including a carbonyl carbon (d C 165.7), three quaternary carbons (d C 122.8, 136.6 and 149.0), three methylene carbons (d C 25.2, 67.1 and 67.9) and three methine carbons (d C 70.8, 150.5 and 152.9). The NMR data of 3 quite resembled that of gentiandiol (Wang et al. 2012). The HMBC cross peaks ( Figure S29) of H-3 with C-1, C-4 and C-5, H-7 with C-5 and C-6 and H-9 with C-7, C-10 and C-11 confirmed the planar structure of 3. Gentiandiol was obtained as a metabolite of gentianine from the plasma after oral administration to rats, but the stereochemistry of gentiandiol was not described (Wang et al. 2012). Furthermore, the absolute configuration was finally determined by the ECD spectrum ( Figure S31). The ECD spectrum of 3 was found to be similar to the calculated spectrum of S configuration but not to that of R configuration. Hence, the structure of 3 was determined as (S)-gentiandiol (Figure 1), it was a new natural product isolated from the natural plant for the first time.

General experimental procedure
The HRESIMS spectra were performed on Waters UHPLC-H-CLASSXEVO G2-XS Q-tof. The NMR data were recorded on a Bruker AV-600 spectrometer with TMS as an internal standard. Optical rotations were run on a Rudolph Research Analytical Autopol IV automatic polarimeter. UV data were measured in methanol on a TU-1900 UV-visible spectrophotometer. IR spectra were obtained on a LUMOS-Micro infrared spectrometer and a Themo Fisher Nicolet iS5 FT-IR spectrometer. Column chromatographic silica gel (200-300 mesh and 300-400 mesh) was procured from Qingdao Marine Chemical Inc., P. R. China. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatography with an Agilent C 18 (34 mm Â 25 cm) column. Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% vanilline-H 2 SO 4 reagent and heat.

Extraction and isolation
The dried whole plant of S. punicea (30 kg) was ground into powder, then extracted with 95% ethanol three times. After removal of the solvent, a crude extract (600 g) was obtained. The extract was suspended in water (2.0 L) and partitioned sequentially with petroleum ether (PE) 16 L, dichloromethane (DCM) 12 L, ethyl acetate (EtOAc) 16 L, and n-butanol (n-BuOH) 16 L to obtain PE fraction 105.4 g, DCM fraction 11.9 g, EtOAc fraction 134 g, and n-BuOH fraction 60 g.

Acid hydrolysis and HPLC analysis of compound 2
Compound 2 (1.0 mg) was dissolved in hydrochloric acid solution (2 mol/L, 10 mL) and heated in a water bath at 80 C, refluxed for 4.5 h. Then, the reaction solution was dried by a rotary evaporator. The dried reaction mixture was dissolved in water (2 mL) and extracted with ethyl acetate (4 Â 4 mL). After the aqueous layer was dried, anhydrous pyridine (1 mL) was added to dissolve it, and then followed by the addition of Lcysteine methyl ester (2 mg). The reaction mixture was heated at 60 C for 1 h. Then, isothiocyanate was added into the reaction solution and continued reacting at 60 C for 1 h. The standard sugar derivatives were prepared in the same way as the method described above. Lastly, the reaction solution was taken out, cooled and filtered through 0.45 lm microporous membrane, and then analyzed by reversed-phase HPLC. CH 3 CN (A) and H 2 O with 0.1% HOAc (B) were used as mobile phases at a flow rate of 0.8 mL/min with the gradient: 75% A and 25% B. Chromatographic peaks were detected at 250 nm. The standard sugar derivatives were also prepared identically and analyzed by HPLC under similar conditions.
Vitamin C was used as positive control. Briefly, DPPH solution (2 mL, 0.5 mg/mL) was prepared in ethanol, then mixed with sample solution (1 mL, containing 4-200 lg) in ethanol, after 30 minutes of incubation, measure the absorbance at 517 nm and denote it as A1. Using the same method to measure the absorbance value A 2 and A 0 . A 2 is the absorbance of mixture of sample solution in ethanol (1 mL, containing 4-200 lg) and 2 mL ethanol. A 0 is the absorbance of mixture of DPPH solution (2 mL, 0.5 mg/mL) and 1 mL ethanol. Then, the free radical scavenging rate (Table S2) was calculated by using the following equation. The IC 50 value of compounds scavenging DPPH radical was calculated by Statistical Product and Service Solutions. For the data of antioxidant activities, please refer Tables S4 and S5.

Conclusion
Our study on the chemical constituents from whole plant of Swertia punicea led to isolation of 44 compounds, including three new constituents: 1,5R-dihydroxy-3,8S-dimethoxy-5,6,7,8-tetrahydroxanthone, (3S,4R,16S,17R)-3,16,23-trihydroxyoleana-11, 13(18)-dien-28-aldehyde-3-Ob-D-glucopyranoside, and (S)-gentiandiol. Specially, (S)-gentiandiol was discovered as another new type of compound in this plant. It is noted that 23 compounds were isolated from this plant for the first time, which provided the reference for the phytochemistry research of the Tujia ethnomedicine Shuihuanglian. On the other hand, this study revealed that the compounds (7, 9 and 14) with the skeleton of tetrahydroxyxanthone from Swertia punicea possess potent antioxidant effects, which may be the starting point for the study of disease treatment.

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