A new polysaccharide from leaves of Sabia parviflora

Abstract A new polysaccharide (SPT1) was isolated from Sabia parviflora Wall. ex Roxb., and the structure was identified by GPC, 1D and 2D NMR spectroscopy, SEM and AFM. The results showed that the average molecular weight (Mn) of SPT1 was 4.057 × 103 Da, and it was composed of α-glucose with a connection mode of 1→6. The SEM showed that the particle size of SPT1 was 1–200 μm and there were small gaps between the crystals. SPT1 was mainly spherical aggregates in AFM, each aggregate was 0.550–0.983 μm long, 1.059–2.275 μm wide and 208–450 nm high. Furthermore, its liver-protective and PTP1B inhibitory activities were evaluated, and the results showed that SPT1 exhibited moderate effects of liver-protective and PTP1B inhibitory activity. The above results provided experimental evidence for the folk application of S. parviflora in the treatment of hepatitis. Graphical Abstract


Introduction
Sabia parviflora Wall.ex Roxb. is an evergreen wood climbing vine of Sabia.It is mainly distributed in natural forests or bushes in karst areas in Guizhou, Yunnan, Guangxi and other provinces in China.The medicinal parts of S. parviflora included roots, stems and leaves.It tastes bitter, slightly cold and acts on the liver meridian, and it had the functions of clearing heat, promoting dampness, promoting choleretics and stopping bleeding.There were some reports on the chemical constituents and pharmacological activities of S. parviflora, and research results showed that S. parviflora had pharmacological activities such as liver protection (Liu et al. 2008), antiviral (Qu et al. 2015), rheumatoid arthritis (Xu et al. 2018), inhibiting lipid accumulation (Fan et al. 2018) and antioxidant activities (Sun et al. 2021).According to the literature, the chemical composition of S. parviflora included triterpenes (Chen et al. 2002a), alkaloids (Chen et al. 2002b), benzophenones (Fan et al. 2018), benzoic acids (Zhou et al. 2022) and flavonoids (Sun et al. 2021;Zhou et al. 2022).The above research works on the chemical composition of S. parviflora were mainly focused on the alcohol extract.However, there were few reports about chemical composition and pharmacological activity focusing on its water extracts.
During our efforts to explore novel active natural products, we reported bioactive steroidal saponins, furanones and alkaloids from plants or fungi obtained from the three gorges region in China (Liu et al. 2013;Yu et al. 2018;Xu et al. 2022).In recent years, we had done some research works on the chemical constituents and pharmacological activities of S. Parviflorai, especially its water extraction (Li et al. 2019;2020;2021;Zhang et al. 2021).
We had previously isolated a polysaccharide SPS60 from 60% ethanol-precipitated polysaccharides which could significantly improve the survival rate of LO2 hepatocytes damaged by CCl 4 (Zhang et al. 2021).However, 30% ethanol-precipitated polysaccharides showed more significant hepatoprotective activity with cell survival rate 93.00% than 60% ethanol-precipitated polysaccharides with cell survival rate 78.52% (Zhang et al. 2021).So, our team continued to purify and separate 30% ethanol precipitated polysaccharides in order to obtain the homogeneous polysaccharide with better hepatoprotective activity.Fortunately, in this paper, another new polysaccharide SPT1 from 30% ethanol-precipitated polysaccharides with significant hepatoprotective and PTP1B inhibitory activity was isolated by repeated Sephadex G-100 and Sephadex G-200, and its structural characteristics were determined by PMP-HPLC, NMR ( 1 H-NMR, 13 C-NMR, DEPT135, 1 H-1 H COSY, HSQC, HMBC, HSQC-TOCSY and NOESY), SEM and AFM.Comparing polysaccharides SPS60 and SPT1, we found that although they came from different concentrations of ethanol-precipitated polysaccharides, their monosaccharide composition was the same, the only difference was the molecular weight.The results of this paper provided a basis for the folk medicine treatment of hepatitis, and provided a scientific basis for further research or can find potential new PTP1B inhibitor for the treatment or improvement of type 2 diabetes and obesity.

Results and discussion
In this article, a new polysaccharide (SPT1) was isolated from S. parviflora, its structure was identified by GPC, hydrolysis and derivatization of polysaccharides, 1D and 2D NMR, SEM and AFM.And SPT1 exhibited significant effects in protecting the liver and PTP1B inhibitory activity.Herein, we presented the detailed results.

Molecular weight of SPT1
The molecular weight of polysaccharide was determined by GPC.The results (see Supplemental material, Table S1) revealed that the average molecular weight (Mn) of SPT1 was 4.057 Â 10 3 Da, the average molecular weight (Mw) of weight was 4.596 Â 10 3 Da and the polydispersity index was 1.133.

Monosaccharide composition of SPT1
After hydrolysis and derivatization of SPT1, by comparing the retention time in HPLC, it was proved (see supplemental material Figure S2) that SPT1 was composed of glucose.

NMR analysis of SPT1
SPT1 was dried in a vacuum, and then dissolved in a nuclear magnetic tube with deuteroxide (D 2 O) and measured by Bruker avance 400 MHz NMR spectrometer for 1 H-NMR, 13 C-NMR, DEPT135, 1 H-1 H COSY, HSQC, HMBC and NOESY.The 13 C NMR showed six carbon signals of glucose, d C 97.8, 73.5, 71.5, 70.3, 69.7 and 65.7 (see Supplemental material Table S2).The 1 H NMR showed a terminal proton signal d H 4.98 (J ¼ 2.5 Hz) (see Supplemental material Table S2).So the conformation of glucose was determined to be a-type by the correlation between H-6 and H-1 in NOESY and proton coupling constant.By analyzing the HMBC of SPT1, we could see that H-1 was related to C-6 and H-6 was related to C-1, indicating that the connection mode of SPT1 was 1!6 (Figure 1).

SEM analysis of SPT1
When SPT1 was magnified 50 times, the sample was granular and aggregated; when SPT1 was magnified 200 times, the surface part resembled a powder-like or irregular flake-like structure; And furthermore, magnify SPT1 to 1000 times, the surface of polysaccharide particles was uneven, and some had powdery or flake-like structures; when magnified to 10,000 and 20,000 times, SPT1 showed a flat and smooth planar structure (see Supplemental material, Figure S11).Even if the magnification was further increased, the surface was still smooth and there was no clear detail, and the micro-morphological information of SPT1 aggregates couldn't be obtained.SEM showed the particle size was 1-200 lm, and there were small gaps between the crystals, so SPT1 were not completely assembled.This indicated that there was mutual repulsion between polysaccharide molecules, and the intermolecular attraction was relatively weak.

AFM analysis of SPT1
Topographical AFM 2-and 3-D images of the SPT1 are shown in Supplemental material Figure S12, SPT1 was mostly spherical aggregates, each aggregate was 0.550-0.983lm long, 1.059-2.275lm wide and 208-450 nm high (see Supplemental material, Figure S12).

The protective effect of SPT1 on L-O2 cells damaged by CCl 4
The L-O2 cells were damaged by CCl 4 , and the results showed that the cell survival rate of model group was significantly lower than normal group, indicating that the model was successfully established (see supplemental material, Figure S13).Different concentrations of drugs were added to the injured cells.After 24 h, the survival rate of the cells in the administration group was significantly higher than model group, indicating that SPT1 had a statistical significance on protecting the injured L-O2 cells in a dose-dependent manner.

The result of the PTP1B inhibitory activity
The measured data showed that SPT1 had an inhibitory effect on PTP1B, and its IC 50 was 29.68 lmol/L, positive drug (MVO 4 ) was 26.94 lmol/L.

Plant material
S. parviflora was collected from Lapo area, Baise City, Guangxi Province of P. R.China and identified by Prof. Yu-Bin Wang.A voucher specimen (No. 20181022) had been deposited in the Herbarium of Department of Medicinal Plants, College of Biological & Pharmaceutical Sciences, China Three Gorges University, China.

Extraction and isolation
The stems and leaves of S. parviflora were dried and crushed, after that 25.6 kg of sample was weighed.It was extracted with water at a material-to-liquid ratio of 1:20, heated and refluxed at 100 C three times, and each extraction time was 1 h.After the extract was obtained, it was concentrated to a suitable concentration by a rotary evaporator, and then spray-dried to obtain the dry powder of total extract of 1.5 kg. 100 g of dry powder was respective precipitated with 30%, 60% and 90% ethanol, and the 30% ethanol precipitation was obtained after centrifugation.The precipitate was dissolved through adding water, the pigment was removed with hydrogen peroxide and the protein was removed by the sevag reagent to obtain crude polysaccharide.The crude polysaccharide was separated and purified by Sephadex G-100 and eluted with ultrapure water at a flow rate of 100 mL/h into Fr.1-50.Fr.29-41 were combined and further purified by Sephadex G-100, and eluted with a pure water flow rate of 4.8 mL/h into Frs.1-40.Frs.26 was purified with Sephadex G-200, and eluted with a pure water flow rate of 1.8 mL/h to obtain SPT1.

Monosaccharide composition analysis
According to the reference (Huo et al. 2021), SPT1 was hydrolyzed and acetylated.The monosaccharide composition of SPT1 was analyzed by comparing the retention time of the sample and the standard monosaccharide by HPLC.The standard monosaccharides used were mannose, rhamnose, glucose and galactose.
Liquid phase conditions: samples were determined by HPLC with C-18 column, the mobile phase was 0.1 mol/L phosphate buffer (pH 6.8) and acetonitrile with a ratio of 78:22, flow rate was 1 mL/min.

Scanning electron microscope observation (SEM)
Take an amount of dry polysaccharide sample STP1 with different purification degrees and stick it on the sample stage with copper tape.The sample was placed in an ion sputterer and coated with conductive gold powder.It was placed under a SIGMA300 SEM.Working conditions: The acceleration voltage was 3 kV, the observation multiples were 50 times, 200 times, 1000 times, 10,000 times and 20,000 times.Carry out the corresponding clarity adjustment until the ideal observation field was obtained, and select the appropriate field of view to take pictures and recorded, repeated the pictures for each sample 3 times to eliminate sample interference and system errors.

Atomic force microscope observation (AFM)
Took an appropriate amount of SPT1, dissolved it with ultrapure water, dropped the solution onto the surface of mica, absorbed it for a certain period of time, then rinsed with ultrapure water, used filter paper to absorb excess liquid on the surface of mica, and gently purged the surface of mica with nitrogen to obtain a dry sample.The dried sample was placed on the sample stage and observed with an atomic force microscope.
The AFM instrument used in this article was innova (Bruker, USA).In order to obtain high-quality images, the sample size was required to be within 30 mm Â 30 mm Â 10 mm, and the difference in surface relief was within 500 nm.An analysis software with the instrument was used to smoothly process and analyze the obtained pictures, and the information such as the shape, height, and width of the sample were obtained.

Cell recovery and culture
Preheated the medium.After 30 min, added 8.0 mL of the preheated medium to the culture flask, took out the L-O2 cell frozen in liquid nitrogen, and quickly threw it into a 37 C water bath with constant shaking.Took out the cryopreservation tube, scrubbed with 75% alcohol for disinfection, sucked the frozen cell suspension into a culture flask, mixed it evenly and moved it to an incubator at 37 C and 5% CO2 for culture.When the cell monolayer grew to 80%, it was digested with 0.25% trypsin for passage.Hepatocytes L-O2 in the logarithmic growth phase were digested with 0.25% trypsin, then suspended in 1640 medium containing 10% fetal bovine serum, and gently pipetted into a single cell suspension with a glass tube.Placed it upside down under a microscope, counted with a cell counting plate and adjusted the cell concentration to 1 Â 10 5 cells/mL.The cells were seeded in a 96-well plate, 100 lL per well, and placed in a 37 C, 5% CO 2 incubator for 24 h.

Cell viability test
The experiment was divided into normal group (blank control group), model group and administration group (high, medium and low dose).Aspirated 10 mL of CCl 4 and dissolved it in 100 mL of 1640 medium containing 10% FBS to make a saturated CCl 4 medium solution.Used saturated CCl 4 medium solution to dissolve the drug to different concentrations.Set 3 multiple holes for each concentration.The drug concentration was 25, 50 and 100 lg/mL.Added 100 lL/well of 1640 medium containing 10% FBS to the normal group.Added 100 lL/well of saturated CCl 4 medium solution to the model group, and added 100 lL/well of sample solution to the administration group.Placed the culture plate with the solution added in a 37 C, 5% CO 2 incubator for 24 h.
After the crystal was completely dissolved, the absorbance (OD value) of each well was measured at 492 nm with automatic enzyme spectrophotometer, so as to calculate the survival rate of L-O2 hepatocytes.In this study, the above methods were used to track the activity of the whole separation process.
In this experiment, the MTT colorimetric method was used to determine the cell survival rate.The culture was terminated after adding 5 mg/ml MTT solution (20 lL/ well) to each group.After 4 hours, aspirated the supernatant and added 150 lL of DMSO to each well.After the crystals were completely dissolved, measured the absorbance (OD value) of each well at 492 nm with an automatic enzyme spectrophotometer to calculate the survival rate of L-O2 hepatocytes.

Evaluated for PTP1B inhibitory activity
The measurement method of PTP1B was fine-tuned according to literature method (Xu et al. 2018).Using p-NPP as the reaction substrate, the activity of PTP1B was screened at 405 nm of the microplate reader.SPT1 and positive control NaVO 4 were dissolved in the reaction system, added 10 lL of sample solution (concentration gradient: 500, 1000 and 2000 lg/mL) and 150 lL enzyme solution (prepared with the reaction system) to the reaction system (pH 6.6, 2 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L DTT and 50 mmol/L citric acid), then placed in a 37 C incubator, incubate for 30 min, add 10 lL of substrate p-NPP, placed in a 37 C incubator, incubate for 15 min, set up blank control group (without enzyme solution).Added 20 lL of 2 mmol/ L NaOH to stop the reaction.

Conclusions
We previously tested the liver protective activities of 30%, 60% and 90% ethanol precipitated polysaccharides with cell survival rate 93.00%, 78.52% and 55.87, respectively, of which 30% showed the strongest activity.We had previously isolated a polysaccharide SPS60 from 60% ethanol-precipitated polysaccharides which could significantly improve the survival rate of LO2 hepatocytes damaged by CCl 4 (Zhang et al. 2021).Based on above research, the 30% alcohol precipitation polysaccharides were further separated and purified.Finally, polysaccharide SPT1 was obtained, and its structure, surface characteristics, hepatoprotective and PTP1B inhibitory activities were explored.The average molecular weight (Mn) of SPT1 was 4.057 Â 10 3 Da, and it was composed of a-glucose with connection mode of 1!6.The SEM showed that the particle size of SPT1 was 1-200 lm and there were small gaps between the crystals, and SPT1 was mostly spherical aggregates in AFM, SPT1 was mostly spherical aggregates, each aggregate was 0.550-0.983lm long, 1.059-2.275lm wide and 208-450 nm high.Comparing polysaccharides SPS60 and SPT1, their monosaccharide composition was

Figure 1 .
Figure 1.The structure of SPT1 and key correlations of HMBC and NOESY.