Neuroprotective triterpene saponins from the leaves of Panax notoginseng

Abstract Two new triterpene saponins, namely notoginsenoside Ng5 (1) and notoginsenoside Ng6 (2) were isolated from the leaves of Panax notoginseng, along with five known ones. Their structures were determined by chemical methods, NMR and X-ray experiments. The absolute configuration of compound 3 with four sugar units was confirmed by single crystal X-ray analysis. Compounds 2–4 and 6 inhibited PC12 cell damage induced by serum deprivation, and increased cell viability from 58.7 ± 6.7% to 66.7 ± 4.5%, 76.1 ± 6.1%, 64.7 ± 5.2% and 67.2 ± 5.0% at 10 μM, respectively. Graphical Abstract


Introduction
Panax notoginseng, also referred to as 'Chinese ginseng', is a perennial herb with dark green leaves widely cultivated in Southwestern China. To date, more than 200 chemical compounds have been isolated from this plant and its endophytic fungi, most of them are the triterpene saponins (Wang et al. 2016;Jin et al. 2017). Recent studies have shown that Panax notoginseng (P. notoginseng) saponins possessed various biological effects, including neuroprotection , anti-cancer (Gu et al. 2017), anti-depression (Zhang et al. 2016), anti-inflammation (Li et al. 2019) and inhibiting vascular smooth muscle cell proliferation (Fang et al. 2018), etc. After firstly reporting the isolation of three novel tricyclic tetranordammarane saponins from the P. notoginseng leaves ), we continue with our investigation to further enrich the diversity of bioactive saponins of this plant. Herein, we present the discovery of seven dammarane-type saponins, including two new saponins, termed notoginsenoside Ng5 (1) and notoginsenoside Ng6 (2), and five known saponins (3-7) from the ethanolic and water extracts of this herb. As an important source of neuroprotective constituents, plants are receiving much attention from science world (Carito et al. 2014;Les et al. 2017;Venditti and Bianco 2019). In this study, we evaluated, in the serum deprivation-induced PC12 cell damage model, the neuroprotective effects of compounds 1-7 at 10 lM. Owing to the difficulty obtaining high quality crystal, single crystal of saponins were seldom reported. Here, for the first time, a single crystal of compound 3 was reported to confirm the absolute configuration of 12(R),23(R)-epoxy dammarane-type saponin.  Na, 1169.6078. The presence of hydroxyl, ester and olefin groups were deduced by the adsorption bands at 3360, 1712 and 1657 cm À1 in its IR spectrum. In 13 C NMR spectrum (Table S1, supplementary material), 57 carbon signals were observed, 30 of them were assigned to the aglycone, including eight methyl carbons (d C 16.0, 16.2, 16.5, 17.4, 17.9, 22.2, 25.8 and 28.0), three oxygen substituted carbons (d C 70.1, 83.5 and 89.2) and a pair of olefinic carbons (d C 125.9 and 131.0). According to the HSQC spectrum, the proton signals could be assigned to the above 30 carbons, indicating the sapogenin of 1 was 20(S)-protopanaxadiol (He et al. 2005 (Table S1, supplementary material). Their absolute configurations were proved to be D-glucose and D-xylose by the acid hydrolysis and GC analysis of 1. Besides, the positions and sequences of the sugar moieties were confirmed by HMBC spectrum according to the correlations ( Figure S6, supplementary material) from H-1 0 to C-3 (d C 89.2), H-1 00 to C-2 0 (d C 84.2), H-1 000 to C-20 (d C 83.5), and H-1 0000 to C-6 000 (d C 69.9). Furthermore, apart from 53 carbons due to the skeleton and sugars, the remaining signals of 1 were assigned to a crotonic group (d C 17.8, 123.2, 144.7 and 166.6) , and it was placed at C-6 00 position of glucose according to the HMBC correlations from H-6 00 [d H 4.99 (d, 11.4), 4.88 (m)] to C-1 00000 (d C 166.6). Consequently, the structure of compound 1 was deduced as 3
Compound 3   singlet at d H 5.54 (1H, d, J ¼ 7.2 Hz). Its 13 C NMR spectrum presented two olefinic carbons (d C 129.2 and 131.1) and four anomeric carbons (d C 99.3, 105.1, 106.0 and 106.3). By comparing their NMR data with those of literature, compound 3 was determined as quinquefoloside-L b (Jiang et al. 2008). Finally, according to the X-ray crystallography analysis [Cu Ka, Flack parameter: 0.03 (8)] of 3, its absolute configuration was unquestionably confirmed, as depicted in Figure 2. Crystallographic data of 3 can be accessed via the Cambridge Crystallographic Data Centre (CCDC: 1897315).

Plant materials
Leaves of P. notoginseng were acquired in Wenshan, Yunnan province of China, in 2015, and authenticated by associate Prof. Lin Ma of our school. A sample (ID-22816) of P. notoginseng leaves has been deposited at our herbarium.

Instruments and chemicals
See SI-1 in supplementary material.

Acid hydrolysis of new saponins (1 and 2)
See SI-3 in supplementary material.

Absolute configuration of sugars
See SI-4 in supplementary material.

Neuroprotection bioassays
Neuroprotection bioassays were carried out as described by Li, Chen, et al (Li et al. 2011), and compounds 1-7 were tested for neuroprotective activity against serum deprivation induced PC12 cell by using MTT method. Results were expressed as the means ± SD.

Conclusions
In this chemical investigation of P. notoginseng leaves led to the discovery of seven triterpene saponins, including two new saponins (1 and 2), together with five known ones (3 2 7). The single crystal data of 12(R),23(R)-epoxy dammarane-type saponin (3) was reported for the first time. Bioactive experiment results revealed that compounds 2-4 and 6 showed moderate neuroprotective effects on serum deficiency treated PC12 cell at 10 lM, but their structure-activity relationship still need to be explored.