Chemical constituents from the aerial part of Polygala tenuifolia

Abstract Three new compounds, polygalapyrone A (1), tenuiside G (2) and polygalapyrrole A (3), together with two known compounds (4–5) were isolated by silica gel, ODS and preparative HPLC from the aerial part of Polygala tenuifolia. Their structures were elucidated by spectrum analysis and compared with findings from the literature. The anti-inflammatory effects of those compounds were investigated in vitro. Graphical Abstract


Introduction
Polygala tenuifolia Willd., also known as 'Yuanzhi' in Chinese, is an herb from the Polygalaceae family and widely distributed in Asia. P. tenuifolia has been used for thousands of years as an expectorant and stimulant in the traditional Chinese Medicine. Studies have shown that P. tenuifolia has protective effects on the central nervous system and is frequently applied in the treatment of forgetfulness, insomnia and neurasthenia (Deng et al. 2020;Nguyen et al. 2020;Vinh et al. 2020;Zhao et al. 2020). The aerial part of P. tenuifolia, which is prescribed using the name 'Xiaocao', is recorded in the Compendium of Materia Medica as a treatment for amnesia. It contains various bioactive compounds, including flavonoids and phenolic glycosides, which have been reported to have anti-inflammatory and anti-oxidative effects . Recently, studies have also confirmed that the aerial parts of P. tenuifolia exhibit significant biological activities in ameliorating learning ability and memory impairments ).
In the course of this study, as a part of research on P. tenuifolia in our group, a new a-pyrone (1), a new megastigmane glycoside (2), a new 5-acyl-2-methylpyrrole (3), together with two known compounds (4-5) were isolated from the aerial parts of P. tenuifolia ( Figure 1). Moreover, compounds 4 and 5 were isolated from Polygalaceae for the first time. The detailed isolation and structural elucidations of three new compounds were provided in this article and the anti-inflammatory evaluation of these compounds (except 3) on RAW 264.7 macrophages were also measured.
Compound 2 was obtained as a white amorphous.  Figure S9). The 1 H-NMR spectrum of 2 ( Figure S10)  , and the left 13 carbons were attributed to a megastigmane skeleton. From the above analysis, the structure of 2 was deduced to be a megastigmane diglycoside with a benzoyl group. A comparison of NMR data of 2 was essentially similar to those of premnaionoside (5), except an additional benzoyl group shifts were observed (Sudo et al. 2000). The HMBC correlations of H-5 00 to C-7 000 (d H 4.48, 4.40/d C 167.8) further proved the linkage positions of the benzoyl group ( Figure S1). Based on the similar 1D NMR spectra and close biogenetic relationship, the absolute configurations of 2 were assumed to be the same as that of 5. Thus, the structure of 2 was eventually determined and named tenuiside G ( Figure 1). Compound 3 (white amorphous powder) had the molecular formula of C 12 H 15 NO 3 as deduced by analysis of HR-ESI-MS at m/z 222.1121 ([M þ H] þ , calcd. for C 12 H 16 NO 3 þ 222.1130) ( Figure S17). The presence of a disubstituted pyrrole nucleus was deduced from the 1 H-NMR signals at d H 6.98 (1H, d, J ¼ 4.1 Hz) and 6.33 (1H, d, J ¼ 4.1 Hz), and the 13 C-NMR signals at d C 140.9, 131.1, 114.9, 111.8. The small coupling constant (4.1 Hz) between the pyrrolic protons indicated a 2,5-disubstitution pattern. Furthermore, the carbonyl signal at d C 188.1 was a keto group conjugated with the pyrrole nucleus. The remaining 1 H-and 13 C-NMR signals revealed two methine protons at d H 5.63 and 5.13, two oxirane protons at d H 5.07 and 4.49, one hydroxymethyl proton at d H 4.55 (2H), two methylene protons at d H 2.83 and 2.53, one methyl proton at d H 1.68 (3H). Comparison of the 1D NMR data of 3 (Figures S18 and S19) with that of the known 1-(5-methyl-1H-pyrrol-2-yl)-2-(2S Ã ,3R Ã )-3-((E)-prop-1-enyl)oxiran-2-yl)ethenone revealed a high similarity, except for the presence of an hydroxymethyl moiety on the pyrrole instead of a methyl group (Dai et al. 2009). Moreover, the HMBC correlations of H-13 to C-2 and C-3 (d H 4.55/d C 140.9, 111.8) further proved the linkage positions of the hydroxymethyl moiety ( Figure S1). Based on a larger coupling constant (15.2 Hz) between H-10 and H-11, the E stereochemistry of the double bond was also determined. In addition, the cis-configuration of the oxirane protons was confirmed by the correlation between H-7 and H-10 in the NOESY spectrum (Dai et al. 2009). Hence, the structure of 3 was defined and named as polygalapyrrole A (Figure 1).
In addition, two known compounds were isolated and identified as 4-methoxy-6phenyl-2H-pyran-2-one (4) and premnaionoside (5), respectively, based on the spectroscopic data and literature. Moreover, both 4 and 5 were isolated from Polygalaceae for the first time.
The anti-inflammatory effects of compounds 1, 2, 4 and 5 were evaluated by measuring nitric oxide (NO) production on LPS-induced RAW 264.7 cells. However, no antiinflammatory effect was observed at concentrations of 30 lM.

Plant material
The aerial part of Polygala tenuifolia was harvested in Jincheng, Shanxi, China in September 2020, and authenticated by Prof. Xiang-ping Pei (Shanxi University of Chinese Medicine). The voucher specimen (No. 20200925) was deposited in Shanxi Modern Chinese Medicine Engineering laboratory, Shanxi University of Chinese Medicine.

Extraction and isolation
Dried and segmental aerial parts of Polygala tenuifolia (3.0 kg) were extracted with 70% EtOH (24 L Â 3, 2 h each) under reflux. The extracts were filtered, combined and then concentrated to afford the ethanol extract (843 g) under reduced pressure. Then the extract was suspended in H 2 O and sequentially partitioned with petroleum ether, ethyl acetate, and n-butyl alcohol to afford the ethyl acetate fraction (215 g).

Acid hydrolysis and GC analysis of compounds 2 and 5
Compounds 2 (1.0 mg) and 5 (1.0 mg) were acid hydrolyzed according to the earlier literature (Tan et al. 2020a). The hexane layer was analyzed by an Agilent 7890A GC-MS system using a DM-5 column (30 m Â 0.25 mm, 0.25 lm). Finally, the absolute configuration of the monosaccharides was determined by comparing the retention times with those of standard sugars.

Anti-inflammatory effects
RAW 264.7 macrophages were chosen to determine the anti-inflammatory effect of isolated compounds (1, 2, 4, 5) based on the previous literature (Tan et al. 2020b). Namely, the anti-inflammatory effect of isolates was measured by NO production after stimulated with LPS, and the dexamethasone (DEX) was used as the positive control.

Conclusion
In this paper, a new a-pyrone (1), a new megastigmane glycoside (2), a new 5-acyl-2methylpyrrole (3), and two known compounds (4-5) were isolated from the aerial part of Polygala tenuifolia. And their structures were identified by analysis of HR-ESI-MS, 1D, 2D NMR data and comparison with the related existing literature works.

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