Study on Chemical Constituents of Panax notoginseng Leaves

Panax notoginseng (Burk.) F. H. is a genuine medicinal material in Yunnan Province. As accessories, P. notoginseng leaves mainly contain protopanaxadiol saponins. The preliminary findings have indicated that P. notoginseng leaves contribute to its significant pharmacological effects and have been administrated to tranquilize and treat cancer and nerve injury. Saponins from P. notoginseng leaves were isolated and purified by different chromatographic methods, and the structures of 1–22 were elucidated mainly through comprehensive analyses of spectroscopic data. Moreover, the SH-SY5Y cells protection bioactivities of all isolated compounds were tested by establishing L-glutamate models for nerve cell injury. As a result, twenty-two saponins, including eight dammarane saponins, namely notoginsenosides SL1-SL8 (1–8), were identified as new compounds, together with fourteen known compounds, namely notoginsenoside NL-A3 (9), ginsenoside Rc (10), gypenoside IX (11), gypenoside XVII (12), notoginsenoside Fc (13), quinquenoside L3 (14), notoginsenoside NL-B1 (15), notoginsenoside NL-C2 (16), notoginsenoside NL-H2 (17), notoginsenoside NL-H1 (18), vina-ginsenoside R13 (19), ginsenoside II (20), majoroside F4 (21), and notoginsenoside LK4 (22). Among them, notoginsenoside SL1 (1), notoginsenoside SL3 (3), notoginsenoside NL-A3 (9), and ginsenoside Rc (10) showed slight protective effects against L-glutamate-induced nerve cell injury (30 µM).


Introduction
Panax notoginseng (Burk.) F. H. Chen, also called "Sanqi" in Chinese, which belongs to the Panax genus, family Araliaceae [1]. P. notoginseng is a valuable traditional Chinese medical herb, which has also been used as medicines for a long time, such as Yun-Nan-Bai-Yao, Xuesaitong capsules, and Xuesaitong injections [2]. As a genuine medicinal material in Yunnan, P. notoginseng is cultivated extensively in Wenshan on account of its unique geological and climatic conditions. Its roots have been widely used as tonic and main components in a great deal of compound preparations of Chinese medicine. P. notoginseng is one of the most widely used Chinese herbal drugs for the treatment of cardiovascular diseases, such as occlusive vasculitis, coronary diseases, atherosclerosis, and cerebral infarction in China and other overseas countries [3]. As resource accessories, few studies have been published on the leaves of P. notoginseng in the early stage [4]. However, P. notoginseng leaves play vital roles in medicinal and edible value. Modern pharmacological studies reveal that P. notoginseng leaves have shown remarkable effects as promising tranquilization [5], antidepressant [6,7], antioxidant [8], and anticancer treatments [9][10][11], as well as have shown multiple benefits on the blood system, cardiovascular system [12], nervous system [13], and metabolic system. Chinese patent medicine "QiyeShenAnPian" takes P. notoginseng leaves as raw material, and it has remarkable effects on invigorating qi, tranquilization, stimulating blood circulation, relieving pain, etc. Approximately 3 Mt P. notoginseng stem-leaves are produced in China annually, while they are used for forage or discarded in the local environment [14]. Therefore, the studies of P. notoginseng leaves have become a hot topic nowadays.
In this paper, dammarane triterpenoid saponins from P. notoginseng leaves were isolated and identified. Moreover, the neuroprotective effect of saponins was tested in SH-SY5Y cells induced by L-glutamate.
In this paper, dammarane triterpenoid saponins from P. notoginseng leaves were isolated and identified. Moreover, the neuroprotective effect of saponins was tested in SH-SY5Y cells induced by L-glutamate.

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Compound 2 (24.0 mg) was obtained as a white amorphous powder. Its molecular formula was determined as C 47 Supplementary  Materials). The absolute configuration of the hydroxyl at C-24 was determined by its chemical shift of 13 C-NMR. The chemical shift of a relatively low field corresponded to the R configuration of C-24, and a relatively high field corresponded to the S configuration of C-24. As shown in the 13 C-NMR spectrum, a hydroxyl existed in the relatively low field (δ C : 76.6), and indicated the configuration of C-24 was R. The 1 H-NMR and 13 C-NMR data of side chain was similar to majoroside F 1 [21]. Three anomeric carbon signals (δ C : 106.8 (Glc C-1 ), 97.9 (Glc C-1 ), 109.8 (Ara(f) C-1 ) and hydrogen signals [δ H : 4.97 (d, J = 7.8 Hz, Glc H-1 ), 5.17 (d, J = 7.7 Hz, Glc H-1 ), 4.69 (br.s, Ara (f) H-1 ] were observed, and it was revealed that the configurations of two glucoses were β, and that arabinose (f) was α. In the HMBC spectrum, the correlations from Glc H-1 (δ H : 4.97) to C-3 (δ C : 88.6), Glc H-1 (δ H : 5.17) to C-20 (δ C : 83.2), and Ara (f) H-1 (δ H : 4.69) to C-6 (δ C : 68.3), respectively, indicated that Glc C-1 was connected with C-3, and Glc C-6 was connected with Ara (f) C-1 , and finally, Glc C-1 was connected with C-20 (shown in Figure 2). Three glycosides were β-D-glucoses and α-L-arabinose determined by hydrolysis, derivatization, and GC analysis. The 1 H-NMR and 13 C-NMR data of sugars was highly consistent with notoginsenoside Fe [22]. Consequently, the structure of compound 2 was determined and named as notoginsenoside SL 2  3) indicated that C-24 of this compound was substituted, and its lateral chain was changed. Combining the 13 C-NMR (δ C : 89.8) with the data of MS, it revealed that C-24 of this compound was replaced by hydroxyperoxy. Besides, the HMBC correlations from H 2 -26/H 3 -27 to C-25, and H 2 -23/H 2 -26/H 3 -27 to C-24 verified that an alkene proton signal existed between C-25 and C-26 (shown in Supplementary Materials). Furthermore, a hydroxyperoxy existed at C-24. As for the configuration of hydroxyperoxy at C-24, it was necessary to convert hydroxyperoxy into hydroxyl. The 1 H-NMR and 13 C-NMR data of side chain was similar to ginsenoside II [23].
Combining MS spectrum with carbon spectrum (δ C : 81.1), a hydroperoxyl was presented at C-25.    Figure 2), respectively, from which indicated Glc C-1 was connected with C-3, Glc C-1 was connected with Glc C-2 , Glc C-1 was connected with Xyl C-2", and Glc C-1 was connected with C-20, finally Xyl C-1 was connected with Glc C-6 . Five glycosides were determined as β-D-glucoses and β-D-xyloses by same methods above. The 1 H-NMR and 13 C-NMR data of sugars was highly consistent with notoginsenoside Fc [27]. Finally, the structure of compound 6 was elucidated and named as notoginsenoside SL 6 Table 7). Compound 7 was a protopanaxadiol saponin substituted by sugars at C-3 and C-20 from above. (δ H : 5.00) was correlated with Glc C-6 (δ C : 68.7) (shown in Figure 2), respectively, from which indicated Glc C-1 was connected with C-3, Glc C-1 was connected with Glc C-2 , Glc C-1 was connected with Xyl C-2", and Glc C-1 was connected with C-20, and finally, Ara (p) C-1 was connected with Glc C-6 . Five glycosides were β-D-glucoses, β-D-xylose and α-L-arabinose, which was determined by same methods above. The 1 H-NMR and 13 C-NMR data of sugars was highly consistent with notoginsenoside Fz [4]. Accordingly, the structure of compound 7 was determined and named as notoginsenoside SL 7 13 C-NMR data of the side chain was similar to quinquefoloside-Lb [28], whose 1 H-NMR and 13 C-NMR data was assigned by comparing it with that in the literature.  (δ H : 5.68) was correlated with Glc C-6 (δ C : 67.2) (shown in Figure 2), respectively, from which it was indicated that Glc C-1 was connected with C-3, Glc C-1 was connected with Glc C-2 , Glc C-1 was connected with Xyl C-2", and Glc C-1 was connected with C-20, finally Ara (f) C-1 was connected with Glc C-6 . Five glycosides were β-D-glucoses, β-D-xylose and α-L-arabinose determining by same methods above. The 1 H-NMR and 13 C-NMR data of sugars was highly consistent with notoginsenoside NL-A 3 [29]. Consequently, the structure of compound 8 was elucidated and named as notoginsenoside SL 8 .

Bioactivity Assays
In order to clarify the neuroprotective effect of saponins from P. notoginseng leaves, all the isolates were tested on L-glutamate-induced cellular damage in SH-SY5Y neuroblastoma cells by using MTT assays, and VPA was used as a positive control. The concentrations of L-glutamate and VPA were determined with gradient screening method. As a result, N-SL 1 (1), N-SL 3 (3), N-NL-A 2 (9), and G-Rc (10) displayed slight activities at 30 µM ( Figure 3). Then, under this concentration, in vitro potential neuroprotective activities of those compounds were investigated.
all the isolates were tested on L-glutamate-induced cellular damage in SH-SY5Y neuroblastoma cells by using MTT assays, and VPA was used as a positive control. The concentrations of L-glutamate and VPA were determined with gradient screening method. As a result, N-SL1 (1), N-SL3 (3), N-NL-A2 (9), and G-Rc (10) displayed slight activities at 30 µ M ( Figure 3). Then, under this concentration, in vitro potential neuroprotective activities of those compounds were investigated.

Discussion
Studies have reported that protopanaxadiol saponins are the main active ingredient in P. notoginseng leaves. In recent years, more and more rare saponins with changed side chains in P. notoginseng leaves have been continuously discovered, and most of the rare saponins retain intact sugar chains, mainly characterized by their side chains.
As shown in results, 22 triterpene saponins from P. notoginseng leaves were isolated and identified. Eight saponins were identified as new compounds, and they all were saponins with changed side chains. Among them, compound 1, 2, 4 featured 25-hydroxyl, 25-ene-24hydroxyl, and 23-ene-25-hydroxyl. Compounds 3 and 5 featured 25-ene-24 hydroxyperoxy. Compounds 6-7 featured 23-ene-25 hydroxyperoxy. Additionally, compound 8 featured an oxygen ring. These eight new compounds are all protopanaxadiol saponins, which further verifies the theory that protopanaxadiol saponins are mainly contained in P. notoginseng leaves. The rare variant ginsenosides in P. notoginseng leaves are more abundant than those in P. notoginseng roots. The growth environment of P. notoginseng leaves and P. notoginseng roots is different, which may be the main reason why P. notoginseng leaves are more abundant in rare variant ginsenosides. The Yunnan area, with a high-altitude and strong sunshine climate, provides strong ultraviolet environment, and the chlorophyll is used as a photosensitizer. Therefore, it is speculated that the main component saponins of P. notoginseng leaves may be oxidized due to strong ultraviolet environmental factors, and the structures of the side chains are changed.
The saponins in P. notoginseng have neuroprotective effects. Because the main active components of P. notoginseng leaves are also saponins, it is speculated that the saponins in P. notoginseng leaves also have neuroprotective activity. In this study, the neuroprotective activity of P. notoginseng leaves was described, and the study found that the monomeric compounds 1, 3, 9, and 10 had neuroprotective activity (30 µM). Indeed, the investigation of P. notoginseng leaves will provide valuable information in understanding the chemical constituents of P. notoginseng leaves and searching new candidates for neuroprotection agents. The studies of the plant itself and the isolates are now in progress, which may provide the basic theory for following research.

Plant Materials
P. notoginseng plants were collected from a market. The voucher specimen was identified by associate Prof. Haizhou Li and Prof. Min Xu, and was kept in the Department of Pharmaceutical Chemistry and Biology's labratory at Kunming University of Science and Technology (Kunming, China). Yunnan Weihe Pharmaceutical Co., Ltd. (Yuxi, China) was entrusted to extract a sample with 60% ethanol, and made desugaring treatment with microporous adsorption resin which was then refined to get the total saponin of P. notoginseng leaves (Total yield was about 4%).

Extraction and Isolation
Dried parts of P. notoginseng (1.35 kg) leaves were dissolved with EtOH, then filtered and concentrated to a certain concentration to get a sample solution, which was subjected to a silica gel column with dichloromethane-methanol-water to afford Fr.1-Fr.14. Fr.5 (40.0 g) was separated by a silica gel column with the elution of dichloromethane-methanol-water, providing the subfractions Fr.5.1-Fr.5.5. Fr.5.3 (8.2 g) was subjected to a ODS column with methanol-water to afford Fr.

Acid Hydrolysis
Each compound (2.0 mg each) was dissolved in 2% HCl-dioxane (1:1) for a total of 4 mL solvent, and 80 • C under the condition responded for 5 h. After the reaction, the reactants were extracted with chloroform for 3 times (3 × 2 mL). Then, the water layer was neutralized with Amberlite IRA-401 and finally filtered and vacuum concentrated to obtain monosaccharide mixture.

Determination of Absolute Configuration
Monosaccharide mixture made the solvent in 2 mL pyridine and added L-Cysteine methyl ester hydrochloride (1.5 mg), then reacted at 60 • C for 1 h. Then, 1.5 mL N-(Trimethylsilyl) imidazole was added under the condition of an immediate ice-, and reacted at 60 • C for 30 min to obtain derivates of monosaccharide. Next, monosaccharide derivatives were prepared by the same method. The derivative of monosaccharide and the standard were analyzed by GC. By comparing the retention time of monosaccharide derivatives of samples and standards, the types and absolute configuration of sugar in samples were determined. The retention time of D/L-glucose, D/L-xylose, and D/L-arabinose is 19

Neuroprotective Effect
Glutamate (Glu) is the main transmitter of excitatory synapses in the central nervous system (CNS), and it plays an important role in excitatory of the dielectric synapse and flexibility of the synapse. Besides, it is pivotal in facilitating calcium transportation and the growth, differentiation, and recondition of intracerebral neurons [30]. Under normal circumstances, the release, ingestion, and reabsorption of glutamate are in dynamic equilibrium, whereas when glutamate is released or malabsorption occurs excessively, glutamate accumulates in the brain and causes the concentration ascension, resulting in a series of pathological reactions, finally leading to degeneration and necrosis of nerve cells [31]. Studies have confirmed that neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease (PD) are closely related to the neurotoxicity of glutamate [32]. Furthermore, it has been proposed that valproic acid (VPA), which is used in epileptic and bipolar disorders, may be protective against excitotoxicity insult [33]. The aerial part of P. notoginseng mainly contains protopanaxadiol saponins that mainly present the effect of central inhibition. Protopanaxadiol saponins are beneficial to tranquilizing, allaying excitement, and eliminating inflammation and analgesic effect. The protective effect of P. notoginseng leaves extract on glutamate induced SH-SY5Y cell injury was investigated in this paper.

Cell Culture
The study was performed using SH-SY5Y human neuroblastoma cells that were grown in Dulbecco's modified Eagle's medium with F12 (1:1) containing 10% fetal bovine serum (Dalian Meilun Biotech Co., Ltd., Dalian, China) and 0.1% penicillin-streptomycin solution. The cells were incubated at 37 • C with 5% CO 2 . The SH-SY5Y cells were seeded into 96-well plates (1 × 10 4 cells/well) and incubated in complete culture medium for 24 h prior to the addition of L-glutamate or VPA.

Treatment of Cell Damage by Glutamate
Cells were treated with 9 different concentrations of L-glutamate (8,9,10,11,12,13,14,15, and 16 µM; Shanghai Titan Scientific Co., Ltd., Shanghai, China) to determine the glutamate toxicity in the cultured SH-SY5Y cells. The glutamate concentrations that caused a significant reduction in cell viability were determined by drawing dose-cell viability curves. The glutamate concentration of 13 µM caused a~30% decrease in cell viability after 24 h, then used this for subsequent experiment. Cell viability was determined by MTT assays, as described below.

Screening of VPA Concentration
SH-SY5Y cells were treated with 6 different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µM) of VPA (Depakin, 400 mg/4 mL; lyophilized powder; Sanofi S.A., Paris, Britain) for 2 h prior to exposure to 13 µM glutamate. The effect of VPA treatment also was tested by MTT assays. The VPA concentrations caused a significant increase in cell viability determined by MTT assays. Finally, the VPA concentration of 13 µM was used as the optimum concentration.

Cell Viability Assay
The logarithmic phase cells were rinsed in phosphate-buffered saline (PBS) and digested with trypsin to make a single cell suspension. Then, the 96 well plate was made into a planked cell suspension and the cell concentration per well was 1 × 10 4 . Next, all models were randomly divided into three groups: the control group, model group (L-glutamate treated), positive control group (VPA treated), P. notoginseng leaves extract treated group, and 30 µM P. notoginseng leaves extract was added to administration group.
Finally, equivalent DEME culture medium was added to the control group and model group. Six multiple wells were set in each group and all cells were incubated for 15~20 h with 5% CO 2 at 37 • C. Then, the cell viability was evaluated by MTT assays.

MTT Assay
An MTT (Sigma-Aldrich; Merck KGaA) assay was applied to evaluate cell viability. After adding MTT solution (5 mg/mL) to each well, cells were incubated for 4 h with 5% CO 2 at 37 • C. Then, following the removal of the culture medium, 200 µL dimethyl sulfoxide was used to dissolve the formazan product. Finally, absorbance values were measured at 490 nm using a microplate reader (Tecan Trading Co., Ltd., Männedorf, Switzerland). Cell viability was calculated by considering the controls as 100%.

Conclusions
In conclusion, for the consideration of rational utilization of P. notoginseng resources, the chemical compositions of P. notoginseng leaves were studied mainly, especially rare saponins with altered side chains. Furthermore, their neuroprotective activities were further investigated. According to the results of the study, 22 saponins were isolated and purified, including 8 new compounds, which were all rare saponins with altered side chains. The neuroprotective activities of these saponins were determined by establishing a model of L-glutamate-induced nerve cell injury. The new compounds 1 and 3, and known compounds 9 and 10 showed slight functions on neuroprotection. Our results not only increase the molecular diversity and bioactive diversity, but also provide a theoretical basis for promoting the rational utilization of P. notoginseng leaves. In addition, the study of P. notoginseng leaves can also promote the sustainable utilization of P. notoginseng medicinal resources and the economic development of the western region of China.