Cynomorium songaricum polysaccharide as well as preparation method and application thereof

Abstract

The invention provides cynomorium songaricum polysaccharide as well as a preparation method and application thereof, and belongs to the field of medicines. The cynomorium songaricum polysaccharide is a polysaccharide extracted from succulent stems of cynomorium songaricum, and the total sugar content of the cynomorium songaricum polysaccharide is greater than 90wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%-3wt%. Monosaccharide in total sugar of the cynomorium songaricum polysaccharide comprises 96.00% or above of glucose. The cynomorium songaricum polysaccharide provided by the invention can activate a Keap1-Nrf2 signal channel to increase endogenous antioxidant enzymes and antioxidant substances in cells and remove oxides in the cells, so that the osteoblast oxidative damage protection effect is achieved; the compound has a wide application prospect in preparation of medicines for preventing and / or treating osteoporosis caused by osteoblast oxidative stress caused by homocysteine.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a Cynomorium songaricum polysaccharide and a preparation method and application thereof. Background Art

[0002] Cynomorium songaricum Rupr., commonly known as the elixir of life, is also known as desert ginseng. It is a perennial fleshy parasitic herb of the genus Cynomorium in the family Cynomoriaceae. It has no chlorophyll and the whole plant is reddish brown. It is mostly parasitic on the roots of plants such as Nitraria and Reaumuria. In my country, it is mainly distributed in Qinghai, Xinjiang, Gansu, Inner Mongolia, Ningxia, Shaanxi and other provinces (Flora of China, 1993). It has been used as medicine as early as the Han Dynasty in China and is a precious traditional medicinal material. Cynomorium songaricum only has fleshy stems for medicinal purposes, which can nourish the kidney, benefit the essence, and moisturize dryness. It is mainly used to treat impotence and spermatorrhea, soreness of the waist and knees, dry intestines and constipation, and has a certain effect on paralysis and improving sexual dysfunction (Pharmacopoeia of China, 2020). The compounds of Cynomorium songaricum are mainly flavonoids, steroids, terpenes, polysaccharides and organic acids. Modern pharmacological studies have shown that Cynomorium songaricum has pharmacological activities such as anti-aging, anti-oxidation, anti-hypoxia, anti-fatigue, anti-cancer and immune system regulation (Cui Z et al., 2013; Jin S et al., 2014; Lin Y et al., 2018; Bo Z et al., 2022). It has good prospects for clinical treatment applications.

[0003] Osteoporosis (OP) has a high incidence and mortality rate worldwide (Bing WJ et al., 2021). Osteoporosis is a systemic bone disease characterized by decreased bone mass, damage to bone tissue microstructure, increased bone fragility, and susceptibility to fractures. In 2001, the National Institutes of Health (NIH) pointed out that osteoporosis is a bone disease characterized by decreased bone strength and increased risk of fractures. Recent studies have shown that homocysteine ​​(Hcy) is a new independent risk factor for OP (Jacobsen WD et al., 2000). Osteoporosis is divided into two categories: primary osteoporosis and secondary osteoporosis. Among them, primary osteoporosis includes postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis (including juvenile type) (Guidelines for the Diagnosis and Treatment of Osteoporosis in the Elderly in China (2023)).

[0004] Estrogen can promote osteoblast differentiation and inhibit osteoclast activity. Postmenopausal estrogen deficiency leads to increased osteoclast activity, decreased bone density, increased bone turnover rate, and ultimately osteoporosis. Postmenopausal women can prevent bone loss by using estrogen alone or in combination with progesterone. Bazedoxifene (BZA) is an estrogen drug. Studies have shown that bazedoxifene has good efficacy and safety in the treatment of postmenopausal women with osteoporosis in 3, 5 and 7 years (Claus C et al., 2010; LSS et al., 2012; Santiago P et al., 2015). However, since January 2020, bazedoxifene has been included in the banned list issued by the World Anti-Doping Agency (WADA) (Masato O et al., 2022). Therefore, it is particularly urgent to find a natural product that can replace bazedoxifene for the prevention or treatment of postmenopausal osteoporosis. Summary of the invention

[0005] The purpose of the present invention is to provide a Cynomorium songaricum polysaccharide and a preparation method and application thereof.

[0006] The invention provides a Cynomorium songaricum polysaccharide, which is a polysaccharide extracted from Cynomorium songaricum fleshy stems. The total sugar content of the Cynomorium songaricum polysaccharide is greater than 90wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%-3wt%.

[0007] Furthermore, the total sugar content of the Cynomorium songaricum polysaccharide is 91wt%-97wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%-3wt%; the composition of monosaccharides in the total sugar includes: more than 96.00wt% of glucose.

[0008] Further,

[0009] The Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-50, wherein the total sugar content is 93.45wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%; the composition of the monosaccharides in the total sugar includes: 96.58wt% of glucose, 0.80wt% of mannose, 0.48wt% of glucuronic acid, 0.19wt% of rhamnose, 0.58wt% of galacturonic acid, 0.45wt% of galactose, 0.80wt% of arabinose and 0.12wt% of fucose;

[0010] Or, the Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-70, wherein the total sugar content is 96.79wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0.34wt%; the composition of monosaccharides in the total sugar includes: 96.75wt% of glucose, 0.78wt% of mannose, 0.24wt% of glucuronic acid, 0.15wt% of rhamnose, 0.69wt% of galacturonic acid, 0.61wt% of galactose, 0.66wt% of arabinose and 0.11wt% of fucose;

[0011] Or, the Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-90, wherein the total sugar content is 92.82wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 2.09wt%; the composition of monosaccharides in the total sugar includes: 96.01wt% of glucose, 0.86wt% of mannose, 0.40wt% of glucuronic acid, 0.06wt% of rhamnose, 0.25wt% of galacturonic acid, 1.18wt% of galactose, 1.08wt% of arabinose and 0.16wt% of fucose.

[0012] Furthermore, the molecular weight of the Cynomorium songaricum polysaccharide CSRP-50 is 5.7 kDa;

[0013] Or, the molecular weight of the Cynomorium songaricum polysaccharide CSRP-70 is 4.5 kDa;

[0014] Or, the molecular weight of the Cynomorium songaricum polysaccharide CSRP-90 is 2.0 kDa.

[0015] Furthermore, the primary structure of the Cynomorium songaricum polysaccharide CSRP-50 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain;

[0016] Or, the primary structure of the Cynomorium songaricum polysaccharide CSRP-70 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming the side chain at the O-6 position of α-(1,4)-D-Glcp;

[0017] Alternatively, the primary structure of the Cynomorium songaricum polysaccharide CSRP-90 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming side chains at the O-6 position of α-(1,4)-D-Glcp.

[0018] Furthermore, the microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-50 is honeycomb-shaped;

[0019] Or, the microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-70 is irregular in shape;

[0020] Or, the microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-90 is an aggregated network structure.

[0021] The present invention also provides a method for preparing the above-mentioned Cynomorium songaricum polysaccharide, the method comprising the following steps:

[0022] (1) taking a fleshy stem of Cynomorium songaricum, adding water to extract, collecting the extract, concentrating it, adding ethanol to precipitate it, collecting the precipitate, and obtaining a crude polysaccharide CSRP of Cynomorium songaricum;

[0023] (2) Separate and purify the crude polysaccharide CSRP from Cynomorium songaricum by using a DEAE-Cellulose cellulose chromatography column, collect the effluent containing the polysaccharide, concentrate it, and add ethanol for precipitation to obtain the product.

[0024] Furthermore, in step (1), the fleshy stem of Cynomorium songaricum is from the fleshy stem of fresh Cynomorium songaricum; the solid-liquid ratio of the extraction is 1:(12-18), the extraction temperature is 90-100°C, the number of extractions is 1-6 times, and the time of each extraction is 2-6 hours; the amount of ethanol added is added until the alcohol content of the solution reaches more than 70%;

[0025] In step (2), the eluent used for separation and purification is water, and the method of adding ethanol precipitation is: first add ethanol to an ethanol concentration of 28%-32%, precipitate, centrifuge, collect the supernatant, adjust the ethanol concentration in the supernatant to 48%-52%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-50, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 68%-72%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-70, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 88%-92%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-90.

[0026] Furthermore, in step (1), the Cynomorium songaricum fleshy stem is from a fresh Cynomorium songaricum fleshy stem; the solid-liquid ratio of the extraction is 1:15, the extraction temperature is 95-100° C., the number of extractions is 4 times, and the time for each extraction is 4 hours; the amount of ethanol added is added until the alcohol content of the solution reaches 75%;

[0027] In step (2), the method of adding ethanol precipitation is: first add ethanol to an ethanol concentration of 30%, precipitate, centrifuge, collect the supernatant, adjust the ethanol concentration in the supernatant to 50%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-50, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 70%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-70, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 90%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-90.

[0028] The present invention also provides the use of the above-mentioned Cynomorium songaricum polysaccharide in preparing antioxidant drugs.

[0029] Furthermore, the antioxidant drug is an osteoblast oxidative damage protection drug.

[0030] The present invention also provides the use of the above-mentioned Cynomorium songaricum polysaccharide in preparing a medicine for preventing and / or treating osteoporosis.

[0031] Furthermore, the osteoporosis is postmenopausal osteoporosis.

[0032] Furthermore, the osteoporosis is homocysteine-induced osteoporosis.

[0033] Furthermore, the drug is a drug that increases endogenous antioxidant substances in osteoblasts by activating the Keap1-Nrf2 signaling pathway.

[0034] Furthermore, the drug is a drug that increases the levels of GSH, SOD and T-AOC in osteoblasts, reduces the level of MDA in osteoblasts, and reduces the level of LDH released outside osteoblasts.

[0035] Furthermore, the drug is a drug that increases the levels of the regulatory factor Nrf2 and the antioxidant enzymes HO-1 and NQO1 in osteoblasts, and reduces the level of the regulatory factor Keap1 in osteoblasts.

[0036] The present invention uses Cynomorium songaricum as the research object and adopts water extraction and alcohol precipitation method to extract Cynomorium songaricum polysaccharide CSRP. DEAE-Cellulose anion exchange column chromatography and step precipitation method are used to separate and purify CSRP-N to obtain five polysaccharide homogeneous components CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S. The structure of the obtained Cynomorium songaricum homogeneous polysaccharide component is characterized by monosaccharide composition FT-IR, NMR and other analytical methods. Technical route is as follows Figure 1 shown.

[0037] Chemical composition analysis showed that the total sugar content of CSRP, CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S was 61.35%, 47.57%, 3.62%, 93.54%, 96.79%, 92.82% and 1.75%, respectively. The total phenol content of CSRP, CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S was 14.43%, 2.50%, 1.36%, 0.00%, 0.34%, 2.09% and 0.75%, respectively. Only CSRP contained uronic acid, with a content of 4.63%; CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S did not contain uronic acid. The protein content of CSRP and CSRP-S was 5.02% and 0.10% respectively, and the protein content of CSRP-N, CSRP-30, CSRP-50, CSRP-70 and CSRP-90 was 0.0%. CSRP-50, CSRP-70 and CSRP-90 with higher total sugar content were selected as the subsequent research objects.

[0038] The monosaccharide composition, molecular weight and homogeneity, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis showed that CSRP-50, CSRP-70 and CSRP-90 were mainly composed of glucose. CSRP-50, CSRP-70 and CSRP-90 were homogeneous polysaccharide components with molecular weights of 5.7 kDa, 4.5 kDa and 2.0 kDa, respectively. CSRP-50, CSRP-70 and CSRP-90 had a molecular weight of 4000-400 cm -1 All of them have characteristic absorption peaks of polysaccharides. And the spectrum signals show that CSRP-50, CSRP-70 and CSRP-90 are all α-configuration pyranose glucans. It is concluded that the primary structure of CSRP-50 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain; while the primary structures of CSRP-70 and CSRP-90 are glucans with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming side chains at the O-6 position of α-(1,4)-D-Glcp.

[0039] Atomic force microscopy and scanning electron microscopy showed that the polysaccharide chains of CSRP-50, CSRP-70 and CSRP-90 all had different degrees of molecular aggregation and cross-linking. The overall appearance of CSRP-50 was honeycomb-like. The overall appearance of CSRP-70 was irregular. The overall appearance of CSRP-90 was an aggregated network structure. The connectivity of CSRP-70 was higher and tighter than that of CSRP-50 and CSRP-90. The structure of CSRP-70 was the tightest among the three, and CSRP-90 was the loosest.

[0040] All levels of the Cynomorium songaricum polysaccharide components of the present invention have good overall antioxidant capacity in vitro. The present invention uses the MC3T3-E1 oxidative stress model induced by homocysteine ​​to study the antioxidant activity of CSRP-50, CSRP-70 and CSRP-90 and the effect of treating postmenopausal osteoporosis. The results show that CSRP-50, CSRP-70 and CSRP-90 can increase the levels of GSH, SOD and T-AOC in MC3T3-E1 cells, reduce the level of MDA, and reduce the level of LDH released outside the cells, thereby achieving a protective effect. The Keap1 / Nrf2 signaling pathway can be activated to increase the levels of the regulatory factor Nrf2 and the antioxidant enzymes HO-1 and NQO1, and reduce the level of the regulatory factor Keap1, thereby achieving the effect of protecting cells.

[0041] Based on the results of antioxidant activity, primary structure and micromorphology observations, it was concluded that CSRP-90 with side chains, smaller molecular weight and loose micromorphology had a better protective effect.

[0042] In summary, the Cynomorium songaricum polysaccharide provided by the present invention can increase endogenous antioxidant enzymes and antioxidant substances in cells to remove oxides in cells by activating the Keap1-Nrf2 signaling pathway, thereby achieving the protective effect of oxidative damage to osteoblasts, and has broad application prospects in the preparation of prevention and / or treatment of osteoporosis caused by oxidative stress of osteoblasts caused by homocysteine.

[0043] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0044] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Technical roadmap of the present invention.

[0046] Figure 2 Na 2 CO 3 Determination of solution dosage.

[0047] Figure 3 The color development time is determined.

[0048] Figure 4 Flow chart of Cynomorium songaricum polysaccharide extraction.

[0049] Figure 5 Standard curve of total sugar content.

[0050] Figure 6 Protein content standard curve.

[0051] Figure 7 Standard curve of uronic acid content.

[0052] Figure 8 Standard curve of total phenol content.

[0053] Fig. 9 TSK-Ge1 G3000PWXL molecular weight standard curve.

[0054] Fig.10 HPGPC elution profile of Cynomorium songaricum polysaccharide on TSK-Gel G3000PWXL column.

[0055] Fig.11 HPGPC elution profile of Cynomorium songaricum polysaccharide on Sephacryl S-200HR column.

[0056] Fig.12 FT-IR spectrum of Cynomorium songaricum polysaccharide.

[0057] Fig.13 CSRP-50 1 H NMR spectrum.

[0058] Fig.14 CSRP-50 13 C NMR spectrum.

[0059] Fig.15 CSRP-70 1 H NMR spectrum.

[0060] Fig.16 CSRP-70 13 C NMR spectrum.

[0061] Fig.17 CSRP-90 1 H NMR spectrum.

[0062] Fig.18CSRP-90 13 C NMR spectrum.

[0063] Fig.19 HH COSY spectrum of CSRP-50.

[0064] Fig. 20 HSQC spectrum of CSRP-50.

[0065] Fig.21 HMBC spectrum of CSRP-50.

[0066] Fig. 22 NOESY spectrum of CSRP-50.

[0067] Fig.23 HH COSY spectrum of CSRP-70.

[0068] Fig.24 HSQC spectrum of CSRP-70.

[0069] Fig.25 HMBC spectrum of CSRP-70.

[0070] Fig.26 NOESY spectrum of CSRP-70.

[0071] Fig. 27 HH COSY spectrum of CSRP-90.

[0072] Fig.28 HSQC spectrum of CSRP-90.

[0073] Fig.29 HH HMBC spectrum of CSRP-90.

[0074] Fig.30 NOESY spectrum of CSRP-90.

[0075] Fig.31 Primary structures of CSRP-50, CSRP-70, and CSRP-90.

[0076] Fig.32 Atomic force microscopy images of Cynomorium songaricum polysaccharides. A: CSRP-30; B: CSRP-50; C: CSRP-90; 1: 2D; 2: 3D.

[0077] Fig.33 Scanning electron micrographs of Cynomorium songaricum polysaccharides. A: CSRP-30; B: CSRP-50; C: CSRP-90; 1: 10000×; 2: 50000×.

[0078] Fig.34Standard curve of total antioxidant capacity.

[0079] Fig.35 The inhibitory effect of Hcy on the proliferation of MC3T3-E1 cells.

[0080] Fig.36 Effects of CSRP-50, CSRP-70 and CSRP-90 on the survival rate of MC3T3-E1 cells. Note: *p<0.05 compared with 0μg / mL CSRP-50, CSRP-70 and CSRP-90 groups.

[0081] Fig.37 BZA promotes the proliferation of MC3T3-E1 cells damaged by Hcy.

[0082] Fig.38 Proliferation-promoting effect of CSRP-50 on Hcy-damaged MC3T3-E1 cells. Note: *p<0.05 compared with 0μg / mL CSRP-50 group; cell activity value is the ratio to the normal group.

[0083] Fig.39 Proliferation-promoting effect of CSRP-70 on Hcy-damaged MC3T3-E1 cells. Note: *p<0.05 compared with 0μg / mL CSRP-70 group; cell activity value is the ratio to the normal group.

[0084] Fig.40 Proliferation-promoting effect of CSRP-90 on Hcy-damaged MC3T3-E1 cells. Note: *p<0.05 compared with 0μg / mL CSRP-90 group; cell activity value is the ratio to the normal group.

[0085] Fig.41 Effects of Cynomorium songaricum polysaccharide on the levels of MDA, GSH, SOD, T-COA and LDH in MC3T3-E1 cells. Note: *p<0.05 compared with the blank control group; #p<0.05 compared with the model group.

[0086] Fig.42 Effect of Cynomorium songaricum polysaccharide on protein expression in Keap1 / Nrf2 signaling pathway. Note: *p<0.05 compared with the blank control group; #p<0.05 compared with the model group. DETAILED DESCRIPTION

[0087] Unless otherwise specified, the raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0088] The "alcohol content" and "ethanol concentration" in the present invention refer to the volume percentage of ethanol in the solution.

[0089] 1. Reagents

[0090] Anhydrous ethanol (Shanghai Sangon Biotechnology Co., Ltd.); DEAE cellulose (Shanghai Yuanye Biotechnology Co., Ltd.); sodium chloride (Sinopharm Chemical Reagent Co., Ltd.); phenol (Shanghai McLean Biochemical Technology Co., Ltd.); concentrated hydrochloric acid, hydrochloric acid (Xilong Science Co., Ltd.), sodium hydroxide (Tianjin Hedong District Hongyan Reagent Factory), chloroform (Baiyin Liangyou Chemical Reagent Co., Ltd.); 1-phenyl-3-methyl-5-pyrazolone (PMP), trifluoroacetic acid (TFA), aminosulfonic acid, m-hydroxybiphenyl (Shanghai Aladdin Biotechnology Co., Ltd.); Folin-Phenol reagent (Folin-Phenol 1N) (Beijing Bio-Toda Technology Co., Ltd.).

[0091] 2. Instruments

[0092] HH-6 digital constant temperature water bath (Bangxi Instrument Technology Co., Ltd.); UPH floor-standing ultrapure water machine (Sichuan Youpu Ultrapure Technology Co., Ltd.); FD-1A-50 vacuum freeze dryer (Beijing Boyikang Laboratory Instrument Co., Ltd.); ordinary dialysis bag (3500) MD-3544, cellulose dialysis bag (100-500) SP131057 (Shanghai Yuanye Biotechnology Co., Ltd.); TD-5A desktop low-speed large-capacity centrifuge (Changzhou Jintan Youlian Instrument Research Institute); chromatography column (Shanghai Xiamei Biochemical Technology Development Co., Ltd.); B T100-2J precision peristaltic pump (Lange Constant Flow Pump Co., Ltd.); HL-2 constant flow pump, BSZ-100 automatic fraction collector (Shanghai Huxi Analytical Instrument Factory Co., Ltd.); 722 spectrophotometer (Shanghai Lingguang Technology Co., Ltd.); DHG-9030A blast dryer (Shanghai Yiheng Scientific Instrument Co., Ltd.); high performance liquid chromatography system (Shimadzu Corporation, Japan); TGL-16M desktop high-speed refrigerated centrifuge (Shanghai Luxiang Centrifuge Instrument Co., Ltd.); MG alcohol meter (Hengshui Chuangji Instrument Co., Ltd.); Sephacryl S-200HR cross-linked dextran gel chromatography column, TSK-Gel G3000PW XL Chromatographic column (Shanghai Tosoh Biotechnology Co., Ltd.); Heizbad HB digit rotary evaporator (Heidolph Company).

[0093] Example 1: Preparation of Cynomorium songaricum polysaccharide

[0094] 1. Obtaining Cynomorium songaricum fleshy stems

[0095] Fresh Cynomorium songaricum samples were collected in Dulan County, Qinghai Province during the growing season between April and May, and the sampling points and habitat altitudes were recorded (2800m; 36°2′25″N, 97°40′26″E). After the samples were collected, the inflorescences and roots of the Cynomorium songaricum samples were removed in accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia, and only the fleshy stems were retained for frozen storage. The plant samples were identified as Cynomorium songaricum Rupr., Cynomorium genus, Cynomorium family (Cynomoriaceae).

[0096] 2. Extraction of Crude Polysaccharides

[0097] Take 1.0kg of frozen Cynomorium songaricum fleshy stem, slice it, add 15 times the amount of water (i.e., the material-liquid ratio is 1:15), boil and extract 4 times, 4 hours each time. Filter and combine the liquid, and concentrate to 1L. Add 99.8% ethanol to make the solution contain 75% alcohol, and let it stand at 4℃ for 24 hours to allow the polysaccharide to precipitate to the bottom. Centrifuge at 4000rmp for 30 minutes, discard the supernatant, and collect the precipitate. Freeze the obtained precipitate in a -80℃ refrigerator for 24 hours, then transfer it to a vacuum freeze dryer for freeze-drying to obtain Cynomorium songaricum crude polysaccharide (CSRP), with a yield of 4.7%.

[0098] The extraction process of Cynomorium songaricum crude polysaccharide (CSRP) is as follows Figure 2-3 shown.

[0099] 3. Anion Exchange Chromatography Fractionation Preparation

[0100] Dissolve 100.0 mg of Cynomorium songaricum crude polysaccharide CSRP in deionized water to prepare a 25.0 mg / mL polysaccharide solution. After centrifugation at 10000 rpm for 10 min to remove insoluble matter, load the supernatant solution onto a balanced DEAE-Cellulose cellulose chromatography column (100 mm×600 mm). Elute with pure water, set the elution flow rate to 5 mL / min, and the collection volume to 8.0 mL / tube. Use the phenol-sulfuric acid method to detect the polysaccharide content of each tube sample in turn until the polysaccharide content is 0, then stop collecting. Finally, the polysaccharide solutions are combined and collected, and after concentration and freeze-drying, Cynomorium songaricum neutral sugar (CSRP-N) can be obtained, with a yield of 13.89%.

[0101] The extraction process of neutral sugar from Cynomorium songaricum (CSRP-N) is as follows Figure 2-3 shown.

[0102] 4. Step-by-step precipitation preparation

[0103] CSRP-N was prepared into a polysaccharide solution with a concentration of 1%. Step-by-step precipitation was used. First, anhydrous ethanol was used to prepare the prepared polysaccharide solution to an ethanol concentration of 30%, and the solution was stored at 4°C for 24 hours, centrifuged at 12500 rpm at 4°C for 30 minutes, the supernatant was transferred to another centrifuge tube, and the precipitate was freeze-dried to obtain the Cynomorium songaricum polysaccharide component CSRP-30.

[0104] The ethanol concentration in the supernatant solution obtained in the previous step was adjusted to 50%, and the solution was kept at 4°C for 24 hours, and centrifuged at 12500 rpm for 30 minutes at 4°C. The supernatant was transferred to another centrifuge tube, and the precipitate was freeze-dried to obtain the Cynomorium songaricum polysaccharide component CSRP-50.

[0105] The ethanol concentration in the supernatant solution obtained in the previous step was adjusted to 70%, and the solution was kept at 4°C for 24 hours, and centrifuged at 12500 rpm for 30 minutes at 4°C. The supernatant was transferred to another centrifuge tube, and the precipitate was freeze-dried to obtain the Cynomorium songaricum polysaccharide component CSRP-70.

[0106] The ethanol concentration in the supernatant solution obtained in the previous step was adjusted to 90%, and the solution was kept at 4°C for 24 hours, and centrifuged at 12500 rpm for 30 minutes at 4°C. The supernatant was transferred to another centrifuge tube, and the precipitate was freeze-dried to obtain the Cynomorium songaricum polysaccharide component CSRP-90.

[0107] The supernatant solution obtained in the previous step was subjected to rotary evaporation, the precipitate was dissolved with pure water, and then collected and freeze-dried to obtain the last Cynomorium songaricum polysaccharide component CSRP-S.

[0108] The extraction process of Cynomorium songaricum polysaccharide CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S is as follows Figure 4 shown.

[0109] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0110] Experimental Example 1: Determination of chemical composition of polysaccharides

[0111] I. Experimental Methods

[0112] 1. Determination of total sugar content

[0113] The total sugar content of Cynomorium songaricum polysaccharide was determined by the phenol-sulfuric acid method (Dubois et al., 1951; Fangfang et al., 2022). The specific steps are as follows:

[0114] (1) Preparation of experimental solution

[0115] Prepare 60% phenol solution: weigh 600.0 g phenol and dissolve it in 400.0 mL ultrapure water. Store at 4°C away from light for later use. When using, dilute to 6% with distilled water.

[0116] Preparation of 0.1 mg / mL glucose solution: Weigh 5.0 mg of D-glucose and dissolve it in distilled water. Use 50 mL to make up to 50 mL and mix well for later use.

[0117] (2) Drawing of standard curve for total sugar content determination

[0118] According to Table 1, prepare 0.01mg / mL, 0.02mg / mL, 0.04mg / mL, 0.06mg / mL, 0.08mg / mL, and 0.10mg / mL D-glucose standard solutions. Take 1.0mL of the polysaccharide sample solution to be tested and the glucose standard solution in a test tube, and add 0.50mL of 6% phenol solution and 2.50mL of concentrated sulfuric acid to each test tube in turn, shake quickly, cool to room temperature, and measure the light absorption value at 490nm. Use the glucose concentration C (mg / mL) as the horizontal coordinate and the light absorption value A at 490nm as the vertical coordinate to draw a standard curve of total sugar content.

[0119] (3) Determination of total sugar content of Cynomorium songaricum polysaccharide samples

[0120] Prepare 0.1 mg / mL of the tested Cynomorium songaricum polysaccharide sample solution, and repeat three times for each sample. Take 1.0 mL of the tested polysaccharide sample solution in a test tube, and add 0.50 mL of 6% phenol solution and 2.50 mL of concentrated sulfuric acid to each test tube in turn, shake quickly, cool to room temperature, and measure the light absorption value at 490 nm. Calculate the total sugar content of each Cynomorium songaricum polysaccharide sample according to the total sugar content standard curve.

[0121] Table 1 Preparation method of glucose standard solution

[0122]

[0123] 2. Protein Content Determination

[0124] The protein content of Cynomorium songaricum polysaccharide was determined by Bradford method (Coomassie Brilliant Blue method) (Marion et al., 1976). The specific steps are as follows:

[0125] (1) Preparation of experimental solution

[0126] Preparation of Coomassie Brilliant Blue Solution: Weigh 10.0 mg of Coomassie Brilliant Blue G-250, dissolve in 5 mL of 95% ethanol, add 10 mL of 85% phosphoric acid, dilute to 100 mL with distilled water, filter with filter paper and set aside. The final reagent contains 0.01% Coomassie Brilliant Blue G-250 (w / v) and 4.7% ethanol (w / v).

[0127] Preparation of protein standard solution: Prepare a 5 mg / mL stock solution of bovine serum albumin. Dilute to 50 μg / mL with pure water before use.

[0128] (2) Plotting of standard curve for protein content determination

[0129] According to Table 2, prepare 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, and 50μg / mL protein standard solutions. Take 1.0mL of protein standard solution in a test tube, and add 4mL of 0.01% Coomassie Brilliant Blue reagent to each test tube in turn, shake quickly and evenly, let it stand for 5 minutes, and then measure its absorbance at 562nm. Draw a protein content standard curve with the concentration C (μg / mL) of BSA as the horizontal coordinate and its absorbance at 562nm as the vertical coordinate.

[0130] (3) Determination of protein content of Cynomorium songaricum polysaccharide samples

[0131] Take 1.0mL of 0.1mg / mL polysaccharide sample solution to be tested into a test tube, and add 4mL of 0.01% Coomassie Brilliant Blue reagent to each test tube in turn, shake quickly and evenly, let stand for 5 minutes, and then measure the absorbance at 562nm. The protein content of each Cynomorium songaricum polysaccharide sample is calculated according to the protein content standard curve.

[0132] Table 2 Preparation method of BSA standard solution

[0133]

[0134] 3. Determination of uronic acid content

[0135] The uronic acid content of Cynomorium songaricum polysaccharide was determined by the m-hydroxybiphenyl method (N. Blumenkrantz & G. Asboe-Hansen., 1973). The specific steps are as follows:

[0136] (1) Preparation of experimental solution

[0137] Preparation of 4M aminosulfonic acid solution (pH 2.5): weigh 3.90 g aminosulfonic acid and dissolve it in 8 mL ultrapure water. Add saturated potassium hydroxide solution dropwise to adjust the pH to 2.5, make up to 10 mL, and store at 4°C for later use.

[0138] Preparation of 0.3% m-hydroxybiphenyl solution: Weigh 30.0 mg of m-hydroxybiphenyl, dissolve it in 10 mL of 0.5% sodium hydroxide, and store it at 4°C in the dark for later use.

[0139] Preparation of 1mg / mL D-galacturonic acid solution: weigh 50.0mg D-galacturonic acid in 40mL ultrapure water, add ultrapure water to 50mL after it is completely dissolved, and store at -20℃ for later use. Before use, dilute to 100μg / mL with ultrapure water.

[0140] (2) Drawing of standard curve for determination of uronic acid content

[0141] According to Table 3, 12.5μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, and 100μg / mL uronic acid standard solutions were prepared. 1.0mL uronic acid standard solution was placed in a test tube, and 40μL 4M aminosulfonic acid (pH=2.5) and 2.5mL concentrated sulfuric acid were added to each test tube in turn, and then the mixture was placed in a boiling water bath for 20min. After being taken out, the mixture was immediately cooled to room temperature with running water, and then 40μL 0.30% m-hydroxybiphenyl / 0.50% sodium hydroxide was added to each test tube in turn, and the mixture was placed at room temperature for 30min, and the absorbance was measured at 525nm. The standard curve for the determination of uronic acid content was drawn with the D-galacturonic acid content C (μg / mL) as the abscissa and the absorbance at 525nm as the ordinate.

[0142] (3) Uric acid content of Cynomorium songaricum polysaccharide samples

[0143] Take 1.0mL 0.1mg / mL polysaccharide sample solution to be tested into a test tube, and add 40μL 4M aminosulfonic acid (pH=2.5) and 2.5mL concentrated sulfuric acid to each test tube in turn, shake well, boil in a boiling water bath for 20min, take out and immediately cool to room temperature with running water, then add 40μL 0.30% m-hydroxybiphenyl / 0.50% sodium hydroxide to each test tube in turn, shake well, leave at room temperature for 30min, and measure the absorbance at 525nm. And calculate the uronic acid content of each Cynomorium songaricum polysaccharide sample based on the uronic acid content standard curve.

[0144] Table 3 Preparation method of D-galacturonic acid standard solution

[0145]

[0146] 4. Determination of total phenolic content

[0147] The total content of Cynomorium songaricum polysaccharide samples was determined by the Folin phenol reagent method (Xiong Ruqin et al., 2019; Batool NR et al., 2023). The specific steps are as follows:

[0148] (1) Determine the amount of sodium carbonate content

[0149] Weigh 4.0 mg of Cynomorium songaricum polysaccharide sample, dissolve it in 60.0 mL of pure water, transfer it to a 100.00 mL volumetric flask, add 1.0 mL of Folin phenol reagent, shake well, let it stand for 2 to 3 minutes, and add different volumes (0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL) of 10% Na 2 CO 3 The solution was made up to volume with pure water, kept in a constant temperature water bath at 25°C for 120 min, and the absorbance was measured at 765 nm.

[0150] Depend on Figure 2 It can be seen that when 10% Na 2 CO 3 The volume of the solution is 2.5 mL, so the 10% Na 2 CO 3 The amount of solution used is 2.5 mL.

[0151] (2) Determine the reaction time

[0152] Take 4.00 mg of Cynomorium songaricum polysaccharide sample, dissolve it in 60.0 mL of pure water, transfer it to a 100.00 mL volumetric flask, add 1.0 mL of Folin phenol reagent, shake well, let it stand for 2 to 3 minutes, then add 2.5 mL of 10% Na 2 CO 3 The solution was made up to volume with pure water, and then kept in a constant temperature water bath at 25°C. The absorbance was measured at 765nm every 20 minutes.

[0153] Depend on Figure 3 It can be seen that the color development reaction tends to be stable after 100 minutes, so 100 minutes is selected as the color development reaction time.

[0154] (3) Draw a standard curve

[0155] Prepare 1.0 mg / mL gallic acid standard solution, pipette 0, 0.10, 0.20, 0.30, 0.40, 0.5 mL into a 100.00 mL volumetric flask, add 1.0 mL of Folin phenol reagent, shake well, let stand for 2-3 min, then add 2.5 mL of 10% Na 2 CO 3 The solution was made up to volume with pure water, kept in a constant temperature water bath at 25°C for 100 min, and the absorbance was measured at 765 nm.

[0156] (4) Determination of total phenol content in Cynomorium songaricum samples

[0157] Take 0.5mL of Cynomorium songaricum sample to be tested in a 100.00mL volumetric flask, add 1.0mL of Folin phenol reagent, shake well, let stand for 2-3min, then add 2.5mL of 10% Na 2 CO 3 The solution was diluted with pure water and kept in a water bath at 25°C for 100 min, and the absorbance was measured at 765 nm. The total phenol content of each Cynomorium songaricum polysaccharide sample was calculated based on the standard curve.

[0158] II. Experimental Results

[0159] 1. Total sugar content of Cynomorium songaricum polysaccharide

[0160] The standard curve of total sugar content was drawn with the glucose concentration C (mg / mL) as the horizontal axis and the absorbance value A at 490nm as the vertical axis. Figure 5 As shown, the regression equation is A 490nm =13.64C+0.020702, R 2 =0.9979. The absorbance value A of the Cynomorium songaricum polysaccharide sample at 490 nm was put into the standard curve, and it was calculated that the total sugar contents of CSRP, CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S were 61.35%, 47.57%, 33.62%, 93.54%, 96.79%, 92.82% and 1.75% respectively.

[0161] 2. Protein content of Cynomorium songaricum polysaccharide

[0162] The protein content standard curve was plotted with the concentration of bovine serum albumin (BSA) (μg / mL) as the horizontal axis and the absorbance at 562nm as the vertical axis. Figure 6 As shown, the regression equation is A 562nm =0.6443C+0.0203, R 2 =0.9974. The absorbance value A of the Cynomorium songaricum polysaccharide sample at 562 nm was put into the standard curve, and it was calculated that the protein content of CSRP and CSRP-S was 5.02% and 0.10% respectively, and the protein content of CSRP-N, CSRP-30, CSRP-50, CSRP-70, and CSRP-90 was 0.0%.

[0163] 3. Uronic acid content of Cynomorium songaricum polysaccharide

[0164] The standard curve of uronic acid content determination was drawn with D-galacturonic acid content C (μg / mL) as the horizontal axis and its absorbance value A at 525nm as the vertical axis. The obtained standard curve is as follows: Figure 7 As shown, the regression equation is A 525nm =5.7248C-0.0154, R 2 =0.9938. The absorbance value A of the Cynomorium songaricum polysaccharide sample at 525nm was put into the standard curve, and it was calculated that only CSRP contained uronic acid, with a content of 4.63%. CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S did not contain uronic acid.

[0165] 4. Total phenol content of Cynomorium songaricum polysaccharide

[0166] The standard curve of uronic acid content determination was drawn with gallic acid content C (μg / mL) as the horizontal axis and its absorbance value A at 765nm as the vertical axis. The obtained standard curve is as follows: Figure 8 As shown, the regression equation is A 765nm =0.0771C+0.0311, R 2 =0.9993. The absorbance value A of the Cynomorium songaricum polysaccharide sample at 765nm was brought into the standard curve, and the total phenol content per gram of polysaccharide sample was calculated. The total phenol content per gram of CSRP, CSRP-N, CSRP-30, CSRP-50, CSRP-70, CSRP-90 and CSRP-S was 14.43%, 2.50%, 1.36%, 0.00%, 0.34%, 2.09% and 0.75%, respectively.

[0167] The chemical compositions of Cynomorium songaricum polysaccharides are summarized in Table 4.

[0168] Table 4 Yield, total sugar content, protein content, uronic acid content and total phenol content of Cynomorium songaricum polysaccharide

[0169]

[0170]

[0171] Note: The total sugar content, protein content, uronic acid content and total phenol content in the table are mass contents.

[0172] The above experimental results show that the total sugar content of CSRP-30 and CSRP-S is low, 33.62% and 1.75% respectively, while the total sugar content of CSRP-50, CSRP-70 and CSRP-90 is high, 93.54%, 96.79% and 92.82% respectively. Therefore, CSRP-50, CSRP-70 and CSRP-90 are selected as the key research objects for subsequent research. CSRP-30 with low total sugar content and CSRP-S with extremely low total sugar content are not taken as the main research objects, but are treated as non-sugar compounds. The results of protein content, uronic acid content and total phenol content of Cynomorium songaricum polysaccharide components show that the protein content and uronic acid content of CSRP-50, CSRP-70 and CSRP-90 are all 0, and their total phenol content is 0.00%, 0.34% and 2.09% respectively. The total phenol content of CSRP-50, CSRP-70 and CSRP-90 is less than 5%, which can be ignored.

[0173] Experimental Example 2: Characterization of the primary structural characteristics of Cynomorium songaricum polysaccharide

[0174] It is well known to those skilled in the art that polysaccharides are carbohydrates composed of more than ten monosaccharides, and different structures have different biological activities (Liu He et al., 2018). The structure of polysaccharides can be divided into primary, secondary, tertiary and quaternary structures, among which the primary structure of polysaccharides includes molecular weight, monosaccharide composition, configuration of anomeric carbon (anomeric hydrogen proton), configuration of sugar ring, arrangement of monosaccharides, linkage of sugar residues, and presence or absence of side chains (Li Xueqin et al., 2021; Jing Yongshuai et al., 2022).

[0175] This experiment determined the monosaccharide composition and ratio, molecular weight and homogeneity of CSRP-50, CSRP-70 and CSRP-90. Fourier transform infrared spectroscopy was used to determine the sugar ring structure type and the configuration of the anomeric carbon of CSRP-50, CSRP-70 and CSRP-90. Further combined with nuclear magnetic resonance spectroscopy analysis, the possible sugar residue connection mode of CSRP-50, CSRP-70 and CSRP-90 was determined, and finally the primary structure of CSRP-50, CSRP-70 and CSRP-90 was characterized.

[0176] IReagents and Instruments

[0177] 1. Reagents

[0178] 1-Phenyl-3-methyl-5-pyrazolone (PMP), trifluoroacetic acid (TFA), aminosulfonic acid (Shanghai Aladdin Biotechnology Co., Ltd.); sodium hydroxide (Hongyan Reagent Factory, Hedong District, Tianjin); nine monosaccharide standard substances including mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose (Beijing Solebow Technology Co., Ltd.); acetonitrile (Shandong Yuwang Hetianxia New Materials Co., Ltd.); potassium bromide (KBr) (Sinopharm Chemical Reagent Co., Ltd.); D 2 O.

[0179] 2. Instruments

[0180] HH-6 digital constant temperature water bath (Bangxi Instrument Technology Co., Ltd.); UPH floor-standing ultrapure water machine (Sichuan Youpu Ultrapure Technology Co., Ltd.); Nicolet iN10 Fourier transform infrared spectrometer (Thermo Fisher scientific Thermo Fisher Scientific Co., Ltd.); AV600 nuclear magnetic resonance spectroscopy (Bruker); ESJ30-5A electronic balance (Shenyang Shenyu Longteng Balance Co., Ltd.); UItiMate 3000 two-dimensional liquid chromatography system (Thermo Fisher Scientific Co., Ltd.); TSK-Gel G3000PW XL Chromatographic column (Shanghai Tosoh Biotechnology Co., Ltd.); C18 chromatography column (2.6 μm 100×4.6mm)(Xiamen Guangshengda Instrument Equipment Co., Ltd.).

[0181] II. Experimental Methods

[0182] 1. Determination of monosaccharide composition

[0183] The monosaccharide composition was determined by acid hydrolysis, PMP pre-column derivatization and HPLC detection (Huang X et al., 2018; Lin R et al., 2021). The specific steps are as follows:

[0184] (1) Acid hydrolysis of Cynomorium songaricum polysaccharide

[0185] Weigh 5.0 mg of polysaccharide sample into a brown glass vial, add 1.0 mL of 2 M trifluoroacetic acid (TFA), place it in a 120 °C oven for hydrolysis for 2 h, transfer the reaction solution to an evaporating dish, evaporate it in a 60 °C water bath, and repeatedly add anhydrous ethanol slowly along the edge of the evaporating dish to accelerate the evaporation of TFA.

[0186] (2) PMP pre-column derivatization

[0187] After the TFA in the acid-hydrolyzed polysaccharide sample is completely evaporated, 0.50mL 0.5M PMP and 0.50mL 0.3M NaOH are added to the evaporating dish to fully dissolve the sample in the evaporating dish. 300.0μL of the dissolved solution is placed in a 1.5mL centrifuge tube, and after derivatization reaction at 70℃ water bath for 30min, it is cooled to room temperature, and 150.0μL 0.3M HCl and 1.0mL chloroform are added to each centrifuge tube, mixed and vortexed, the water layer is retained, the chloroform layer is discarded, and the extraction is repeated 3 times, and filtered through a 0.22μm filter membrane into a liquid phase loading bottle for use.

[0188] (3) Preparation of monosaccharide standard reference solution

[0189] Prepare 0.5 mg / mL nine monosaccharide standard solutions (mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, fucose), take 5.0 μL of each and mix them into a 1.5 mL centrifuge tube, add 50.0 μL 0.5MPMP and 50.0 μL 0.3 M NaOH, derivatize in a 70 °C water bath for 30 min, cool to room temperature, add 50.0 μL 0.3 M HCl and 100.0 μL HCl, and then add 100 μL HCl. 2 O, mix well; add 1.0 mL of chloroform and vortex extract, retain the water layer, discard the chloroform layer, repeat the extraction 3 times, and filter through a 0.22 μm filter membrane into a liquid phase sample bottle for later use.

[0190] (4) High performance liquid chromatography detection

[0191] The monosaccharide composition of the samples was determined on a Thermo Scientific UItiMate 3000 two-dimensional liquid chromatography system and a UV-VIS detector. The mobile phase used was 20% acetonitrile and 80% 0.1 M phosphate buffer solution (pH = 7.0), and the chromatographic column was 2.6μm C18 (LC Column 100×4.6nm), injection volume was 2.0μL, flow rate was 0.5mL / min, column temperature was 35℃, and detection wavelength was 245nm.

[0192] 2. Determination of molecular weight distribution and uniformity of Cynomorium songaricum polysaccharide

[0193] High performance gel filtration chromatography (HPGPC) was used to analyze the molecular weight distribution and homogeneity of Cynomorium songaricum polysaccharide (Huang X et al., 2018; Lin R et al., 2021). The specific steps are as follows:

[0194] (1) Preparation of experimental solution

[0195] Prepare 0.15M NaCl: weigh 8.7660g NaCl powder, dissolve it in 1.0L ultrapure water, filter it through a 0.22μm filter membrane to remove impurities, and store it at room temperature for later use.

[0196] (2) Drawing of molecular weight determination standard curve

[0197] 5.0 mg of each of the sugar molecular weight standards of 50,000, 25,000, 12,000, 5,000, 1,000, 666 (Dp4), 342 (lactose), and 180 (glucose) Da were weighed and dissolved in 1.0 mL of ultrapure water, filtered through a 0.22 μm filter membrane to remove impurities, and then loaded onto a Shimadzu HPLC system (LC-10ATvp pump, RID-10A detector and TSK-Gel G3000PW XL The mobile phase was 0.15 M NaCl solution, the flow rate was 0.6 mL / min, and the column temperature was 40.0°C.

[0198] (3) Molecular weight distribution and homogeneity determination of Cynomorium songaricum polysaccharide samples

[0199] Prepare a 5.0 mg / mL polysaccharide solution and load it into the same detection system. The liquid phase conditions are consistent with those for the standard determination. Substitute the measured retention time into the standard curve formula to calculate the molecular weight.

[0200] (4) Determination of elution peak properties

[0201] To determine TSK-Gel G3000PW XL Whether the elution peak in the chromatographic column is an impurity peak. Weigh 2.0 mg of CSRP-30, CSRP-50, CSRP-70 and CSRP-90 respectively and dissolve them in 1.0 mL of pure water. After filtering with a 0.22 μm filter membrane, load the sample into a Sephacryl S-200HR cross-linked dextran gel chromatography column and elute with pure water at a flow rate of 0.45 mL / min2.0 mL / tube.

[0202] 3. Fourier transform infrared spectroscopy (FT-IR) analysis

[0203] Take 2.0 mg of Cynomorium songaricum polysaccharide sample, press it into a KBr pellet, place it on the ATR accessory sample loading platform, and collect the sample at 4000-500 cm -1 Mid-infrared spectra within the wavenumber range. Each sample was measured three times in parallel, and the average spectrum was taken as the mid-infrared spectrum of the sample. The spectrum acquisition range is 4000~400cm -1 The number of scans for the sample and background is 32, and the resolution is 4 cm -1 . H is deducted in real time during scanning2 O and CO 2 OMNIC 9.2 software was used for automatic peak marking, and the sensitivity of absorption peak recognition was set to 51 (Fan Haiyan et al., 2020).

[0204] 4. Nuclear magnetic resonance (NMR) analysis

[0205] 20.0 mg of polysaccharide sample was dissolved in 0.5 mL of D 2 O (99.8% D), and the samples were measured using a Bruker AV600 NMR spectrometer. 1 H-NMR, 13 C-NMR, HSQC, COSY, HMBC and NOESY spectra, among which the 600MHz measurement of Cynomorium songaricum polysaccharide 1 H-NMR spectrum, measured at 150MHz 13 C-NMR spectrum. 1 The H-NMR spectrum was scanned 32 times. 13 The C-NMR spectrum was scanned 7000 times, the HSQC spectrum was scanned 32 times, the COSY spectrum was scanned 32 times, the HMBC spectrum was scanned 100 times and the NOESY spectrum was scanned 32 times (PW Needs & R. R. Selvendran, 1993; Ghosh K et al., 2008).

[0206] III. Experimental Results

[0207] 1. Monosaccharide composition of Cynomorium songaricum polysaccharide

[0208] The monosaccharide composition results are shown in Table 5. CSRP-30 is composed of mannose (5.14%), glucuronic acid (9.22%), rhamnose (2.05%), galacturonic acid (2.46%), glucose (78.23%), galactose (1.0.6%), arabinose (1.29%), and fucose (0.56%); CSRP-50 is composed of mannose (Man, 0.80%), glucuronic acid (GlcUA, 0.48%), rhamnose (Rha, 0.19%), galacturonic acid (GalUA, 0.58%), glucose (Glc, 96.58%), galactose (Gal, 0.45%), arabinose (Ara, 0.06%). .80%), and fucose (Fuc, 0.12%); CSRP-70 is composed of mannose (0.78%), glucuronic acid (0.24%), rhamnose (0.15%), galacturonic acid (0.69%), glucose (96.75%), galactose (0.61%), arabinose (0.66%), and fucose (0.11%); CSRP-90 is composed of mannose (0.86%), glucuronic acid (0.40%), rhamnose (0.06%), galacturonic acid (0.25%), glucose (96.01%), galactose (1.18%), arabinose (1.08%), and fucose (0.16%).

[0209] Table 5 Monosaccharide composition of Cynomorium songaricum polysaccharide

[0210]

[0211] Note: The monosaccharide composition in the table is in mass percentage.

[0212] 2. Study on the molecular weight and uniformity of Cynomorium songaricum polysaccharide

[0213] High performance gel permeation chromatography (HPGPC) is often used to determine the molecular weight and uniformity of polysaccharides. During the HPGPC elution process, substances with large molecular weights peak first and substances with small molecular weights peak later. The molecular weight MW, Ig MW and retention time RT of the standard are measured. A standard curve is drawn with the logarithm of molecular weight (MW) lg MW as the vertical coordinate and retention time (RT) as the horizontal coordinate. The standard curve is shown in the figure below. Fig. 9 The regression equation is Ig MW = -0.1897RT + 1.039, R 2 =0.9926.

[0214] The molecular measurements are shown in Fig.10The results showed that CSRP-30 in TSK-Gel G3000PW XL There are three elution peaks on the chromatographic column, CSRP-50, CSRP-70 and CSRP-90 each have two elution peaks, and one of the four peaks overlaps with the elution peak of glucose.

[0215] The elution curves of CSRP-30, CSRP-50, CSRP-70 and CSRP-90 in Sephacryl S-200HR cross-linked dextran gel chromatography column are shown in Fig.11 The results of the collected polysaccharide samples using the phenol-sulfuric acid method showed that CSRP-30, CSRP-50, CSRP-70 and CSRP-90 were detected in TSK-Gel G3000PW XL The tail peak of the chromatographic column (the elution peak that overlaps with glucose) is an impurity peak.

[0216] 3. Fourier Transform Infrared Spectroscopy Analysis of Cynomorium songaricum Polysaccharide

[0217] The FT-IR test results of Cynomorium songaricum polysaccharide are as follows Fig.12 As shown. Among them, 3306.86cm -1 (CSRP-50), 3300.03cm -1 (CSRP-70), 3335.14cm -1 The strong and broad absorption peak at (CSRP-90) represents the -OH stretching vibration. -1 (CSRP-50), 2924.08cm -1 (CSRP-70), 2928.83cm -1 The weaker absorption peak at (CSRP-90) represents the CH stretching vibration, at 1000-1150 cm -1 The presence of three absorption peaks indicates that it is pyranose. 853.36cm -1 (CSRP-50), 846.25cm -1 (CSRP-70), 853.27cm -1 The absorption peak at (CSRP-90) indicated the presence of α-configuration monosaccharide residues in the polysaccharide component.

[0218] 4. Nuclear Magnetic Resonance Spectroscopy Analysis of Cynomorium songaricum Polysaccharide

[0219] 4.1 One-dimensional NMR spectroscopy analysis

[0220] Known in 13 In C-NMR, the chemical shift of the anomeric carbon of α-D-glucose is distributed in δ97-101ppm, and the chemical shift of the anomeric carbon of β-D-glucose is distributed in δ103-106ppm.1 In H-NMR, the chemical shift of the anomeric hydrogen proton of α-D-glucose is generally δ>4.95 ppm, and the chemical shift of the anomeric hydrogen proton of β-D-glucose is generally δ<4.95 ppm.

[0221] According to CSRP-50 1 H NMR spectrum ( Fig.13 ) and CSRP-50 13 C NMR spectrum ( Fig.14 ) shows that CSRP-50 has two anomeric carbon signals and two anomeric hydrogen proton signals, indicating that there are two monosaccharide residue linking modes in CSRP-50. Combined with the monosaccharide composition analysis, it can be seen that the glucose content in CSRP-50 is 96.58%, and the content of the remaining monosaccharides is less than 5%. However, these monosaccharide signals with a content less than 5% cannot be actively detected in the NMR spectrum, which also shows that CSRP-50 has two glucose residue linking modes. At the same time, the anomeric carbon signal and anomeric hydrogen proton signal of CSRP-50 are located in the signal distribution area corresponding to α-D-glucose, indicating that the pyranose glucose residues in CSRP-50 are in α configuration.

[0222] According to CSRP-70 1 H NMR spectrum ( Fig.15 ) and CSRP-70 13 C NMR spectrum ( Fig.16 ) It can be seen that CSRP-70 has four anomeric carbon signals and four anomeric hydrogen proton signals, indicating that there are four sugar residue linking modes in CSRP-70. Combined with the monosaccharide composition analysis, it can be seen that the glucose content in CSRP-70 is 96.75%, and the contents of the remaining monosaccharides are all less than 5%. However, these monosaccharide signals with a content less than 5% cannot be collected in the NMR spectrum, which also shows that CSRP-70 has four glucose residue linking modes. At the same time, the anomeric carbon signal and anomeric hydrogen proton signal of CSRP-70 are located in the signal distribution area corresponding to α-D-glucose, indicating that the pyranose glucose residues in CSRP-70 are in α configuration.

[0223] According to CSRP-90 1 H NMR spectrum ( Fig.17 ) and CSRP-90 13 C NMR spectrum ( Fig.18) It can be seen that CSRP-90 has four anomeric carbon signals and four anomeric hydrogen proton signals, indicating that there are four sugar residue linking modes in CSRP-90. Combined with the monosaccharide composition analysis, it can be seen that the glucose content in CSRP-90 is 96.01%, and the contents of the remaining monosaccharides are all less than 5%. However, these monosaccharide signals with a content less than 5% cannot be collected in the nuclear magnetic resonance spectrum, which also shows that CSRP-90 has four glucose residue linking modes. At the same time, the anomeric carbon signal and anomeric hydrogen proton signal of CSRP-90 are located in the signal distribution area corresponding to α-D-glucose, indicating that the pyranose glucose residues in CSRP-90 are α-configuration. It can be concluded that the pyranose glucose residues of CSRP-50, CSRP-70 and CSRP-90 are all α-configuration, which is consistent with the analysis results of FT-IR.

[0224] Compared with the chemical shifts of known sugar residues, CSRP-50 has a signal peak of anomeric carbon (anomeric hydrogen proton) of 1,6-α-D-Glcp at 99.54 / 5.42ppm and a signal peak of anomeric carbon (anomeric hydrogen proton) of 1,4-α-D-Glcp at 99.53 / 5.41ppm. At the same time, its signal peaks at 76.65 / 3.68ppm and 69.29 / 3.95,3.44ppm indicate the presence of glycosides at the 4th and 6th positions.

[0225] 99.52 / 5.41ppm of CSRP-70 is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,4-α-D-Glcp, 99.71 / 5.40ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,6-α-D-Glcp, 99.58 / 5.41ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,4,6-α-D-Glcp, and 95.78 / 5.33ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of T-α-D-Glcp. The absorption peaks of CSRP-70 at 76.14 / 3.65ppm, 69.27 / 3.67,3.85ppm, 76.64 / 3.67ppm and 69.89 / 3.54,3.85ppm represent the glycoside at position 4 of 1,4-α-D-Glcp, the glycoside at position 6 of 1,6-α-D-Glcp and the glycosides at positions 4 and 6 of 1,4,6-α-D-Glcp, respectively.

[0226] 99.32 / 5.30ppm of CSRP-90 is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,4-α-D-Glcp, 99.5 / 5.38ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,6-α-D-Glcp, 99.69 / 5.38ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of 1,4,6-α-D-Glcp, and 95.72 / 5.2ppm is the signal peak of the anomeric carbon (anomeric hydrogen proton) of T-α-D-Glcp. The absorption peaks of CSRP-70 at 76.64 / 3.59ppm, 69.26 / 3.50,3.82ppm, 76.64 / 3.60ppm and 69.87 / 3.50,3.82ppm represent the glycoside at position 4 of 1,4-α-D-Glcp, the glycoside at position 6 of 1,6-α-D-Glcp and the glycosides at positions 4 and 6 of 1,4,6-α-D-Glcp, respectively.

[0227] 4.2 Two-dimensional NMR spectroscopy analysis

[0228] For the convenience of analysis, the 1,4-Glcp of CSRP-50 was named as residue A, and the 1,6-Glcp was named as residue B; the 1,4-Glcp of CSRP-70 and CSRP-90 were named as residue A, 1,4,6-Glcp was named as residue B, 1,6-Glcp was named as residue C, and 1-Glcp was named as residue D.

[0229] The HH COSY spectrum can provide cross-peak signals of mutually coupled hydrogen nuclei. The HSQC (CH COSY) spectrum is a spectrum directly related to C and H, which can provide signals of directly connected carbon-hydrogen relationships.

[0230] According to the HH COSY spectrum of CSRP-50 ( Fig.19 ) It can be inferred that the chemical shifts of H1, H2, H3, H4, H5 and H6 of its A residue are 5.41, 3.65, 3.98, 3.68, 3.64, 3.85 / 3.97 ppm respectively; and the chemical shifts of H1, H2, H3, H4, H5 and H6 of its B residue are 5.42, 3.6, 3.98, 3.85, 3.62, 3.42 / 3.95 ppm respectively.

[0231] According to the HSQC spectrum of CSRP-50 ( Fig. 20) It can be inferred that the chemical shifts of C1, C2, C3, C4, C5 and C6 of its A residue are 99.53, 71.49, 73.31, 76.65, 70.32 and 60.42 ppm respectively; and the chemical shifts of C1, C2, C3, C4, C5 and C6 of its B residue are 99.54, 72.84, 73.31, 71.14, 72.68 and 69.29 ppm respectively.

[0232] According to the HMBC spectrum of CSRP-50 ( Fig.21 ) shows that there is a cross peak between H-4 of A residue (3.68ppm) and C-1 of B residue (99.54ppm); there is a cross peak between H-6 of B residue (3.85ppm) and C-1 of A residue (99.53ppm); there is a cross peak between H-1 of A residue (5.41ppm) and C-6 of B residue (69.29ppm); there is a cross peak between H-1 of B residue (5.42ppm) and C-4 of A residue (76.65ppm); it can be inferred that the possible primary structure of CSRP-50 is a straight-chain α-glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected to form the main chain.

[0233] According to the NOESY spectrum of CSRP-50 ( Fig. 22 ) It can be seen that there are cross peaks between H-1 of A residue and its H-2, H-3 and H-6 of B residue, and there are cross peaks between -1 of B residue and its H-2, H-3 and H-4 of A residue. There is NOE reaction between them, which further proves that the main chain of CSRP-50 is formed by alternating connection of α-(1,4)-D-Glcp and α-(1,6)-D-Glcp.

[0234] According to the HH COSY spectrum of CSRP-70 ( Fig.23 ) It can be inferred that the chemical shifts of H1, H2, H3, H4, H5, and H6 of its A residue are 5.4, 3.63, 3.95, 3.65, 3.82, and 3.83 / 3.54 ppm, respectively; the chemical shifts of H1, H2, H3, H4, H5, and H6 of its B residue are 5.41, 3.63, 3.94, 3.67, 3.85, 3.85 / 3.54, respectively. ppm; the chemical shifts of H1, H2, H3, H4, H5 and H6 of its C residue are 5.41, 3.63, 3.75, 3.73, 3.82, 3.67 / 3.85 ppm respectively; the chemical shifts of H1, H2, H3, H4, H5 and H6 of its D residue are 5.33, 3.6, 3.67, 3.4, 3.95, 3.85 / 3.71 ppm respectively.

[0235] According to the HSQC spectrum of CSRP-70 ( Fig.24 ) It can be inferred that the chemical shifts of C1, C2, C3, C4, C5, and C6 of its A residue are 99.52, 71.69, 72.83, 76.14, 71.69, and 60.43 ppm, respectively; the chemical shifts of C1, C2, C3, C4, C5, and C6 of its B residue are 99.58, 71.48, 73.3, 76.64, 73.17, 69.89, respectively. ppm; the chemical shifts of C1, C2, C3, C4, C5 and C6 of its C residue are 99.71, 71.69, 74.49, 71.12, 71.62 and 69.27 ppm respectively; the chemical shifts of C1, C2, C3, C4, C5 and C6 of its D residue are 95.78, 74.8, 73.3, 72.83, 72.67 and 60.64 ppm respectively.

[0236] According to the HMBC spectrum of CSRP-70 ( Fig.25) shows that there are cross peaks between H-4 of A residue (3.65ppm) and C-1 of C residue (99.52ppm), as well as between H-1 of C residue (3.65ppm) and C-4 of A residue (99.71ppm); there are cross peaks between H-6 of C residue (3.67ppm) and C-1 of A residue (99.52ppm), as well as between H-1 of A residue (5.4ppm) and C-6 of C residue (69.27ppm), indicating that there is an alternating connection between A residue and C residue, that is, α-(1,4)-D-Glcp and α-(1,6)-D-Glcp are alternately connected to form the main chain. There are cross peaks between H-6 (3.67ppm) of C residue and C-1 (95.78ppm) of D residue, and between H-1 (5.33ppm) of D residue and C-6 (69.27ppm) of C residue, indicating that there is a connection between C residue and D residue. There are cross peaks between H-4 (3.67ppm) of B residue and C-1 (99.71ppm) of C residue, and between H-1 (5.41ppm) of C residue and C-4 (76.64ppm) of B residue, indicating that there is a mutual connection between position 4 of B residue and position 1 of C residue. There are cross peaks between H-6 (3.85ppm) of B residue and C-1 (99.71ppm) of C residue, and between H-1 (5.41ppm) of C residue and C-6 (69.89ppm) of B residue, indicating that there is a mutual connection between position 6 of B residue and position 1 of C residue. There are cross peaks between H-6 of C residue (3.85ppm) and C-1 of B residue (99.58ppm), and between H-1 of B residue (5.41ppm) and C-6 of C residue (69.27ppm), indicating that there is a mutual connection between position 6 of C residue and position 1 of B residue. It is inferred that the possible primary structure of CSRP-70 is that α-(1,4)-D-Glcp and α-(1,6)-D-Glcp are alternately connected to form the main chain, and there is a glucan with T-α-D-Glcp and α-(1,6)-D-Glcp forming the side chain at the O-6 position of α-(1,4)-D-Glcp.

[0237] According to the NOESY spectrum of CSRP-70 ( Fig.26 ) It can be seen that there are cross peaks between H-1 of residue A and its H-2, H-3 and H-5, indicating that there is a NOE effect between them.

[0238] According to the HH COSY spectrum of CSRP-90 ( Fig. 27) It can be inferred that the chemical shifts of H1, H2, H3, H4, H5, and H6 of its A residue are 5.3, 3.81, 3.93, 3.59, 3.63, and 3.97 / 3.45 ppm, respectively; the chemical shifts of H1, H2, H3, H4, H5, and H6 of its B residue are 5.38, 3.82, 3.94, 3.6, 3.69, 3.5 / 3.82, respectively. ppm; the chemical shifts of H1, H2, H3, H4, H5 and H6 of its C residue are 5.38, 3.83, 3.93, 3.6, 3.68 and 3.5 / 3.82 ppm respectively; the chemical shifts of H1, H2, H3, H4, H5 and H6 of its D residue are 5.2, 3.62, 3.67, 3.4, 3.61 and 3.95 / 3.81 ppm respectively.

[0239] According to the HSQC spectrum of CSRP-90 ( Fig.28 ) It can be inferred that the chemical shifts of C1, C2, C3, C4, C5, and C6 of its A residue are 99.32, 71.24, 73.15, 76.64, 71.15, and 60.43 ppm, respectively; the chemical shifts of C1, C2, C3, C4, C5, and C6 of its B residue are 99.69, 71.67, 73.15, 76.64, 72.81, 69.87, respectively. ppm; the chemical shifts of C1, C2, C3, C4, C5 and C6 of its C residue are 99.5, 71.47, 73.27, 71.67, 72.65 and 69.26 ppm respectively; the chemical shifts of C1, C2, C3, C4, C5 and C6 of its D residue are 95.72, 71.69, 73.3, 72.83, 72.65 and 60.63 ppm respectively.

[0240] According to the HMBC spectrum of CSRP-90 ( Fig.29) shows that there are cross peaks between H-4 (3.59ppm) of A residue and C-1 (99.5ppm) of C residue, and between H-1 (5.38ppm) of C residue and C-4 (76.64ppm) of A residue, indicating that there is mutual connection between A residue and C residue. There are cross peaks between H-4 (3.6ppm) of B residue and C-1 (99.5ppm) of C residue, and between H-4 (3.6ppm) of C residue and C-4 (76.64ppm) of B residue, indicating that position 4 of B residue and position 1 of C residue are mutually connected. There is a cross peak between H-1 (5.38ppm) of B residue and C-6 (69.26ppm) of C residue, indicating that position 1 of B residue and position 6 of C residue are mutually connected. There is a cross peak between H-1 (5.38 ppm) of C residue and C-6 (69.87 ppm) of B residue, indicating that there is a mutual connection between position 1 of C residue and position 6 of B residue. There is a cross peak between H-1 (5.2 ppm) of D residue and C-6 (99.5 ppm) of C residue, indicating that there is a mutual connection between position 1 of D residue and position 6 of C residue. It can be deduced that the primary structure of CSRP-90 is a main chain formed by alternating connection of α-(1,4)-D-Glcp and α-(1,6)-D-Glcp, and there is a glucan with T-α-D-Glcp and α-(1,6)-D-Glcp forming a side chain at the O-6 position of α-(1,4)-D-Glcp.

[0241] According to the NOESY spectrum of CSRP-90 ( Fig.30 ) It can be seen that there are cross peaks between H-1 of residue A and its H-2, H-3 and H-5, indicating that there is a NOE effect between them, but the signal is weak.

[0242] Based on the above analysis, CSRP-50 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp connected alternately as the main chain; while CSRP-70 and CSRP-90 are glucans with α-(1,4)-D-Glcp forming a branch at its O-6 position and connected to the side chains of T-α-D-Glcp and α-(1,6)-D-Glcp. The chemical shift assignments of the C and H signals of all sugar residues are summarized in Table 6.

[0243] Table 6 Cynomorium songaricum polysaccharide 13 C NMR and 1 Summary of H NMR signal assignments

[0244]

[0245] 5. Primary structure characterization of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSRP-90

[0246] Based on the results of monosaccharide composition analysis, Fourier transform infrared spectroscopy analysis, one-dimensional nuclear magnetic resonance spectroscopy analysis, and two-dimensional nuclear magnetic resonance spectroscopy analysis, a structure of CSRP-50, CSRP-70, and CSRP-90 was inferred, such as Fig.31 The results showed that CSRP-50, CSRP-70, and CSRP-90 were all α-glucans, and it was inferred that the primary structure of CSRP-50 was a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp connected alternately as the main chain; while the primary structures of CSRP-70 and CSRP-90 were glucans with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp connected alternately as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming side chains at the O-6 position of α-(1,4)–D–Glcp.

[0247] The above experimental results show that:

[0248] (1) CSRP-50, CSRP-70 and CSRP-90 are mainly composed of glucose and are all polysaccharide components with molecular weights of 5.7 kDa, 4.5 kDa and 2.0 kDa, respectively. Further analysis by Fourier transform infrared spectroscopy showed that CSRP-50, CSRP-70 and CSRP-90 were located at 4000-500 cm -1 The mid-infrared spectra within the wavenumber range contain characteristic absorption peaks of CH stretching vibration, -OH stretching vibration, pyranose and α-configuration monosaccharide residues. At the same time, the chemical shift signals of the anomeric carbon of CSRP-50, CSRP-70 and CSRP-90 in the nuclear magnetic resonance spectrum analysis are also within the range of the chemical shift signals of the anomeric carbon of glucose residues and α-configuration monosaccharide residues, which proves that CSRP-50, CSRP-70 and CSRP-90 have α-configuration monosaccharide residues and are glucans.

[0249] (2) CSRP-50, CSRP-70 and CSRP-90 are all glucans, wherein the primary structure of CSRP-50 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp connected alternately as the main chain; while the primary structures of CSRP-70 and CSRP-90 are glucans with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp connected alternately as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming the side chain at the O-6 position of α-(1,4)-D-Glcp. CSRP-50, CSRP-70 and CSRP-90 are the neutral sugar uniform polysaccharide components newly discovered by the present invention. The primary structures of CSRP-50, CSRP-70 and CSRP-90 are different from the primary structures of known Cynomorium songaricum polysaccharides. The reasons may be that the Cynomorium songaricum was collected from different habitats, at different times, and by different extraction, separation and purification methods.

[0250] Experimental Example 3: Microscopic morphology observation of Cynomorium songaricum polysaccharide

[0251] It is well known to those skilled in the art that the primary structure and spatial structure of polysaccharides have an impact on their biological activity. Therefore, the exploration of the microscopic morphology of polysaccharides has attracted the attention of more and more researchers. In this experiment, the microstructure of CSRP-50, CSRP-70 and CSRP-90 was observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM).

[0252] IReagents and Instruments

[0253] 1. Reagents

[0254] Fluorphlogopite mica sheet (for atomic force microscope only) (Hangzhou Hangdan Optoelectronics Technology Co., Ltd.)

[0255] 2. Instruments

[0256] Dimension ICON atomic force microscope (Bruker); SU8010 scanning electron microscope (Hitachi).

[0257] II. Experimental Methods

[0258] 1. Atomic force microscopy (AFM) observation

[0259] The microscopic morphology of Cynomorium songaricum polysaccharide was analyzed using Bruker's Dimension ICON atomic force microscope. The Cynomorium songaricum polysaccharide samples were dissolved in ultrapure water at a concentration of 5 μg / mL. 5 μL of the solution was dropped on the surface of a newly peeled mica sheet, dried at room temperature, and observed in the Tapping mode of the atomic force microscope with a scanning rate of 1.0 Hz and a scanning size of 3000 nm (Morris JV et al., 2001; Wang J&Nie S, 2019).

[0260] 2. Scanning electron microscopy (SEM) observation

[0261] The microscopic image of Cynomorium songaricum polysaccharide was observed using a Hitachi SU8010 scanning electron microscope with an observation voltage of 5.0 KV and observation magnifications of 10000× and 50000× (Qiang S et al., 2017).

[0262] III Results

[0263] 1. Atomic force microscopy (AFM) observation and analysis results

[0264] Depend on Fig.32 It can be seen that CSRP-50, CSRP-70, and CSRP-90 are all composed of cluster structures. Using Nano ScopeAnalysis 1.7 software to analyze, the average height of CSRP-50 is 4.379nm, the average height of CSRP-70 is 7.436nm, and the average height of CSRP-90 is 1.459nm. The height of a single polysaccharide chain is between 0.1 and 1.0nm. The average heights of CSRP-50, CSRP-70, and CSRP-90 are greater than 1.0nm, indicating that there is mutual entanglement between the sugar chains in these uniform polysaccharide components, and the degree of entanglement is from high to low in the order of CSRP-70, CSRP-50, and CSRP-90.

[0265] 2. Scanning electron microscope (SEM) observation and analysis results

[0266] Scanning electron microscopy can be used to observe the microscopic morphology and spatial structure of the polysaccharide sample surface. Fig.33As shown. The structure of CSRP-50 is relatively tight, with a high degree of connectivity. It is mainly composed of lamellar structures and a small amount of spherical structures, and the overall appearance is honeycomb-like. The structure of CSRP-70 is relatively tight, with a high degree of connectivity. It is mainly lamellar structure, and the overall appearance is irregular. The strip-like fiber structure runs through it, and the connectivity is higher and tighter than that of CSRP-50 and CSRP-90. The structure of CSRP-90 is relatively loose, with a low degree of connectivity. It is mainly composed of spherical structures and a small amount of lamellar structures, presenting a relatively uniform aggregate, and the overall appearance is an aggregated network structure. This aggregation may be related to the mutual entanglement of sugar chains. This is consistent with the results obtained by atomic force microscopy observation and analysis. Among them, the spatial structure of CSRP-70 is the tightest, and the spatial structure of CSRP-90 is the loosest.

[0267] The above experimental results show that:

[0268] (1) The results of SEM observation and analysis show that the overall appearance of CSRP-50 is honeycomb-like. The overall appearance of CSRP-70 is irregular. The overall appearance of CSRP-90 is a clustered network structure. The connectivity of CSRP-70 is higher and tighter than that of CSRP-50 and CSRP-90. The spatial structure of CSRP-70 is the tightest among the three, and CSRP-90 is the loosest.

[0269] (2) Combined with the AFM analysis results, it was shown that there were different degrees of molecular aggregation and cross-linking in the polysaccharide chains of CSRP-50, CSRP-70 and CSRP-90.

[0270] Experimental Example 4: Study on the antioxidant activity of Cynomorium songaricum polysaccharide, its protective effect on osteoblast oxidative damage and its effect on the treatment of postmenopausal osteoporosis

[0271] Studies have shown that homocysteine ​​(Hcy) is a new independent risk factor for osteoporosis (Jacobsen W Det al., 2000). Using Hcy to induce MC3T3-E1 cells is a known method for constructing a postmenopausal osteoporosis model (1. Ethanolic extract of Actaea racemosa (black cohosh) potentiates bone nodule formation in MC3T3-E1 preosteoblast cells, Bone, Volume 43, Issue 3, September 2008, Pages 567-573; 2. MiR-320a was highly expressed in postmenopausal osteoporosis and acts as a negative regulator in MC3T3E1 cells by reducing MAP9 and inhib iting PI3K / AKT signaling pathway, Experimental and Molecular Pathology, Volume 110, October 2019, 104282; 3. Characterization of steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line, Biology of Reproduction, 2007; 4. Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis and Accumulation of Advanced Glycation EndProducts by Reducing Oxidative Stress in MC3T3-E1 Cells, Calcif Tissue Int (2017) 100:286-297).

[0272] In this study, the antioxidant activity of Cynomorium songaricum polysaccharide was analyzed through the overall antioxidant capacity in vitro, and the MC3T3-E1 oxidative stress model induced by homocysteine ​​(Hcy) was established to simulate postmenopausal osteoporosis. The changes in the levels of cytokines such as MDA, SOD, GSH, T-COA, and LDH in MC3T3-E1 cells and the effects on the expression of Keap1 and Nrf2 regulatory factors and antioxidant enzymes (HO-1 and NQO1) in the Keap1 / Nrf2 signaling pathway were detected.

[0273] IReagents, consumables and instruments

[0274] 1. Reagents

[0275] 1.1 Antibodies

[0276] Monoclonal rabbit antibodies against Nrf2, Keap1, and NQO1 (Abcam, Shanghai Abcam Trading Co., Ltd.); monoclonal rabbit antibodies against HO-1 and β-actin (Cell Signaling Technology); PageRuler TM Prestained protein molecular weight standard Marker 10-180kDa (Thermo Fisher scientific Thermo Fisher Scientific Co., Ltd.); HRP-goat anti-rabbit IgG (Wuhan Boster Bioengineering Co., Ltd.).

[0277] 1.2 Kits and other reagents

[0278] 2,2-Azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) (Sigma); water-soluble Trolox (Sigma); horseradish peroxidase (HRP) (Shanghai MacLean Biochemical Technology Co., Ltd.); sodium dihydrogen phosphate, sodium hydrogen phosphate (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.); malondialdehyde (MDA) assay kit (TBA method), total superoxide dismutase (SOD) assay kit (WST-1 method), reduced glutathione (GSH) assay kit (microplate method), lactate dehydrogenase (LDH ) kit (microplate method) (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.); total antioxidant capacity (T-AOC) detection kit (ABTS method) (Jiangsu Biyuntian High-tech Co., Ltd.); BCA protein quantification kit (enhanced), RIPA (strong) lysis buffer, PMSF (100mM), primary and secondary antibody dilution buffer, RIPA (strong) lysis buffer, Western and IP cell lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.); 0.25% trypsin solution (containing EDTA, dissolved in PBS), MC3T3-E1Subclone 14 cell-specific culture medium (Wuhan Pronocell Life Science Co., Ltd.); homocysteine, bazedoxifene (Shanghai McLean Biochemical Technology Co., Ltd.); membrane regeneration solution (strong), 1× Tris-glycine electrophoresis buffer (powder), 1× electrophoresis transfer buffer (transfer solution) (powder), 1× TBST buffer (powder) (Beijing Solebold Technology Co., Ltd.); ammonium persulfate (Sigma); TEMED (Shanghai Guangnuo Chemical Technology Co., Ltd.); isopropanol (Tianjin Fuyu Fine Chemical Co., Ltd.); ultra-sensitive ECL chemiluminescence ready-to-use substrate, Cell Counting Kit-8 (CCK-8) kit, SDS-PAGE protein loading buffer (5×), sterile PBS (Wuhan Boster Bioengineering Co., Ltd.).

[0279] 1.3 Consumables

[0280] 0.45μm PVDF membrane (Millipore); Cell Scraper, 50mL centrifuge tube, 15mL centrifuge tube, 5mL centrifuge tube (Wuxi Nice Life Science Co., Ltd.); 0.22μm filter membrane (Millex-GP); 6-well plate, 96-well plate, 10cm 2 Sterile cell culture dishes (Corning).

[0281] 1.4 Instruments

[0282] HH-6 digital constant temperature water bath (Bangxi Instrument Technology Co., Ltd.); UPH floor-standing ultrapure water machine (Sichuan Youpu Ultrapure Technology Co., Ltd.); 722 spectrophotometer (Shanghai Lingguang Technology Co., Ltd.); TGL-16M desktop high-speed refrigerated centrifuge (Shanghai Luxiang Centrifuge Instrument Co., Ltd.), electrophoresis instrument (Bio-Rad); multifunctional microplate reader (PerkinElmer); HF90 carbon dioxide cell culture incubator (Likang Biomedical Technology Holdings Co., Ltd.); AUW220D electronic balance (Shimadzu Corporation, Japan); 3-18KS desktop refrigerated centrifuge (Sigma); pipette (Eppendoff China Limited / Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.); TGL-16M desktop high-speed refrigerated centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.); clean bench (Suzhou Sujie Medical Instrument Co., Ltd.); Tanon5200 fully automatic chemiluminescence image analysis system (Shanghai Tianneng Technology Co., Ltd.); IX73 advanced live cell imaging inverted microscope system (Olympus Corporation); ZD-9560 decolorization shaker (Jiangsu Shenglan Instrument Manufacturing Co., Ltd.); snowflake ice machine (Henan Huashang Brothers Technology Co., Ltd.), HB120-S constant temperature metal bath (Japan Sanbote Co., Ltd.).

[0283] II. Experimental Methods

[0284] 1. Determination of total antioxidant capacity of Cynomorium songaricum polysaccharide in vitro

[0285] 1.1 Preparation of experimental solution

[0286] (1) Prepare 50 mM pH 7.5 phosphate buffer solution (PBS): weigh 3.760 g Na 2 HPO 4 12H 2 O, 0.3120 g NaH 2 PO 4 ·2H 2 O was dissolved in 250 mL of pure water; 1.0 μM HRP solution was prepared by weighing 8.80 mg HRP and dissolving it in 20 mL of 50 mM pH 7.5 PBS; 5 mM ABTS solution was prepared by weighing 68.750 mg ABTS and dissolving it in 25 mL of 50 mM pH 7.5 PBS; 6 mM H 2 O 2 Solution: Take 12.0 μL of 30% HO 2 O 2 Solution was prepared by adding 50 mM pH 7.5 PBS to a volume of 20 mL (Floegel et al., 2011; A Biskup I et al., 2013).

[0287] 1.2 Preparation of Trolox standard solution

[0288] Take 500μL of 5mM Trolox solution and dilute it 1-fold with 500μL 50mM pH 7.5 phosphate buffer to obtain 1000μL 2.5mM Trolox solution (see Table 7). Add 400.0μL 5mM ABTS solution, 250.0μL 1.0μM HRP solution, 10.0μL 6.0mM H 2 O 2 Solution, 340.0 μL 50 mM pH 7.5 phosphate buffer solution, mixed volume 1.0 mL, containing 2 mM ABTS, 15 μM H 2 O 2 Solution, 0.25μM HRP; test temperature is 25℃, protected from light for 10min; after the 730nm light absorption value is stable, add 70μL Trolox solution, and measure the light absorption value A after 5min 730nm A standard curve was drawn with tocopherol C (μg) as the horizontal axis and its absorbance value A at 730 nm as the vertical axis.

[0289] 1.3 Determination of antioxidant activity of Cynomorium songaricum polysaccharide samples

[0290] Add 400.0 μL 5 mM ABTS solution, 250.0 μL 1.0 μM HRP solution, and 10.0 μL 1.5 mM H 2 O 2 Solution, 340.0 μL 50 mM pH 7.5 phosphate buffer solution, mixed volume 1.0 mL, containing 2 mM ABTS, 15 μM H 2 O 2 Solution, 0.25μM HRP; test temperature is 25℃, protected from light for 10min; after the 730nm light absorption value is stable, add 70μL of 0.10mg / mL Cynomorium songaricum polysaccharide sample solution to be tested, and measure the light absorption value A after 5min 730nm The antioxidant activity of each Cynomorium songaricum polysaccharide fraction was calculated based on the standard curve. The total antioxidant capacity of each Cynomorium songaricum polysaccharide fraction was expressed as Trolox equivalent (each gram of sample is equivalent to x milligrams of Trolox, TEAC (mg TE / g) and ABTS free radical scavenging rate.

[0291] Table 7 Preparation method of Trolox standard

[0292]

[0293] 2. Study on the protective effect of Cynomorium songaricum polysaccharide on oxidative stress induced by homocysteine ​​in MC3T3-E1 cells

[0294] 2.1 MC3T3-E1 cell recovery, culture and passaging

[0295] MC3T3-E1 Subclone 14 cell line was purchased from Wuhan Punosai Biotechnology Co., Ltd., with the catalog number CL-0378, and the STR identification was correct. The cell culture medium was MEMα basal medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) 100U / mL penicillin / streptomycin (P / S). The cells were cultured in a cell culture incubator at a culture temperature of 37°C and a carbon dioxide concentration of 5%. Cell subculture can be performed after 72 to 96 hours of cell culture, and cells in good growth state in the logarithmic growth phase were taken for subsequent experiments (Chen Y et al., 2019).

[0296] 2.2 Establishment of MC3T3-E1 oxidative stress model

[0297] Homocysteine ​​Hcy was used to damage MC3T3-E1 cells to put them in an oxidative stress state, simulating postmenopausal osteoporosis. In order to select the appropriate modeling concentration, the Hcy concentration was screened in the range of 300 μM to 20 mM Hcy-containing medium. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and cultured in a 37°C cell culture incubator for 24 hours. The original culture medium was discarded, and 300 μM to 20 mM Hcy-containing culture medium was added, and cultured in a 37°C cell culture incubator for 24 hours. The Hcy-containing culture medium was discarded, and the cells were washed three times with sterile PBS, and fresh culture medium was added. 10.0 μL CCK-8 solution was added to each well, and the cells were cultured in the incubator for 1 hour. The absorbance (OD value) was measured at 450 nm using a multifunctional microplate reader, and the measurement temperature was 37.0°C. The cell viability was calculated. Cell viability % = [(OD Hcy -OD 空白 ) / (OD 对照 -OD 空白 )]×100% (Floegel A et al., 2011).

[0298] 2.3 Detection method of the effect of Cynomorium songaricum polysaccharide on the survival rate of MC3T3-E1 cells

[0299] In order to detect the effect of Cynomorium songaricum polysaccharide on the survival rate of MC3T3-E1 cells, a culture medium containing CSRP-50, CSRP-70 and CSRP-90 with a gradient concentration of 0.05-5 mg / mL was selected. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×104 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and cultured in a 37°C cell culture incubator for 24 hours. The original culture medium was discarded, and the culture medium containing different concentrations of CSRP-50, CSRP-70 and CSRP-90 to be tested was added, and the culture was continued in a 37°C cell culture incubator for 24 hours. The culture medium containing CSRP-50, CSRP-70 and CSRP-90 was discarded, and the cells were washed three times with sterile PBS. While adding fresh culture medium, 10.0 μL CCK-8 solution was added to each well, and the cells were continued to be cultured in the incubator for 1 hour. The absorbance value (OD value) was measured at 450 nm using a multifunctional microplate reader, and the measurement temperature was 37.0°C. The cell viability was calculated. Cell viability % = [(OD 多糖 -OD 空白 ) / (OD 对照 -OD 空白 )]×100%.

[0300] 3. Screening of Cynomorium songaricum polysaccharide and bazedoxifene dosage concentration

[0301] 3.1 Screening of positive control drug bazedoxifene (BZA) concentration

[0302] Bazedoxifene (BZA) was selected as the positive control drug for the polysaccharide sample. In order to select the appropriate positive control drug concentration, the BZA concentration was screened in the range of 10 to 800 nM BZA-containing medium. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and cultured in a 37°C cell culture incubator for 24 hours. The original culture medium was discarded, and a medium containing Hcy at an appropriate concentration was added, and the culture was continued in a 37°C cell culture incubator for 24 hours. The Hcy-containing culture medium was discarded, and the cells were washed three times with sterile PBS. The BZA-containing culture medium with a concentration screening range of 10 to 800 nM was added, and the culture was continued in a 37°C cell culture incubator for 24 hours. The BZA-containing culture medium was discarded, and the cells were washed three times with sterile PBS. While adding fresh culture medium, 10.0 μL CCK-8 solution was added to each well, and the cells were continued to be cultured in the incubator for 1 hour. The absorbance value (OD value) was measured at 450 nm using a multifunctional microplate reader. The measuring temperature was 37.0°C, and the cell viability was calculated. Cell viability % = [(OD BZA –OD 空白 ) / (OD 对照 –OD 空白 )]×100%.

[0303] 3.2 Determine the concentration of Cynomorium songaricum polysaccharide

[0304] The concentration of Cynomorium songaricum polysaccharide was screened in the range of 6.25 to 400 μg / mL of polysaccharide sample culture medium. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate and cultured in a 37°C cell culture incubator for 24 hours. The original culture medium was discarded, and a medium containing Hcy at an appropriate concentration was added, and the culture was continued in a 37°C cell culture incubator for 24 hours. The Hcy-containing culture medium was discarded, and the culture was washed three times with sterile PBS. The culture medium containing polysaccharide samples with a concentration screening range of 10 to 800 nM was added, and the culture was continued in a 37°C cell culture incubator for 24 hours. The culture medium containing polysaccharide samples was discarded and washed three times, and fresh culture medium was added and 10.0 μL CCK-8 solution was added to each well. The culture was continued in the incubator for 1 hour. The absorbance value (OD value) was measured at 450 nm using a multifunctional microplate reader. The measuring temperature was 37.0°C, and the cell viability was calculated. Cell viability % = [(OD 多糖 –OD 空白 ) / (OD 对照 –OD 空白 )]×100%.

[0305] Experimental groups were designed according to the screened Cynomorium songaricum polysaccharide concentration range.

[0306] 4. Detection of cytokines such as MDA, SOD, GSH, T-COA, LDH, etc.

[0307] 4.1 Preparation of MDA, SOD, GSH, and T-COA detection solutions

[0308] Prepare cell lysis solution: 1mL RIPA (strong) lysis solution is mixed with 10μL 100mM PMSF and placed in an ice box for later use. Remove the culture medium in the 6-well plate and wash it three times with 4℃ sterile PBS. Add 70μL cell lysis solution to each well, place it on an ice box, react at 4℃ for 10min, and use a cell scraper to scrape the adherent cells from the culture dish. Collect the cell lysis solution containing cells in a 1.5mL ep tube and place it in crushed ice for 60min. After the lysis is completed, move the ep tube to a pre-cooled low-temperature high-speed centrifuge and centrifuge at 4℃12500rmp for 15min. After the centrifugation is completed, collect the supernatant and discard the precipitate. Store the collected supernatant solution in crushed ice for later use.

[0309] 4.2 Determine the protein concentration of the sample to be tested

[0310] Prepare BCA working solution: Prepare BCA working solution A: BCA working solution B = 50:1. Prepare a suitable volume of BCA working solution according to the actual number of samples, following the principle of "prepare before use".

[0311] Prepare protein standard solution: weigh 5.0 mg of protein standard, dissolve in 10 mL of sterile PBS, and store at -80°C for a long time. Prepare protein standard gradient solution according to Table 8. Calculate the total protein concentration of each sample to be tested.

[0312] Take 1 μL of sample solution and add it to a 96-well plate, add 19 μL of sterile PBS, and shake to mix. Quickly add 200 μL of BCA working solution to each well in turn, and place in a 60°C water bath for 30 minutes. Use a multifunctional microplate reader to measure its absorbance at 562 nm. Calculate the total protein concentration of the sample based on the standard curve and the sample dilution multiple.

[0313] (3) Preparation of LDH detection solution

[0314] Collect the culture medium of the sample to be tested into an EP tube, move the EP tube to a pre-cooled low-temperature high-speed centrifuge, and centrifuge at 4°C 12500rmp for 15 minutes. After the centrifugation, collect the supernatant and discard the precipitate. Store the collected supernatant solution in crushed ice for later use.

[0315] 5. Western Blotting (WB)

[0316] 5.1 Extraction of total protein

[0317] Prepare cell lysis solution: 1mL RIPA (strong) lysis solution is mixed with 10μL 100mM PMSF and placed in an ice box for later use. Remove the culture medium in the 6-well plate and wash it three times with 4℃ sterile PBS. Add 70μL cell lysis solution to each well, place it on an ice box, react at 4℃ for 10min, and use a cell scraper to scrape the adherent cells from the culture dish. Collect the cell lysis solution containing cells in a 1.5mL ep tube and place it in crushed ice for 60min. After the lysis is completed, move the ep tube to a pre-cooled low-temperature high-speed centrifuge and centrifuge at 4℃12500rmp for 15min. After the centrifugation is completed, collect the supernatant and discard the precipitate. Store the collected supernatant solution in crushed ice for later use.

[0318] 5.2 BCA protein quantification

[0319] Prepare BCA working solution: Prepare BCA working solution A: BCA working solution B = 50:1. Prepare a suitable volume of BCA working solution according to the actual number of samples, following the principle of "prepare before use".

[0320] Prepare protein standard solution: weigh 5.0 mg of protein standard, dissolve in 10 mL of sterile PBS, and store at -80°C for a long time. Prepare protein standard gradient solution according to Table 8. Calculate the total protein concentration of each sample to be tested.

[0321] Take 1 μL of the protein sample solution to be tested and add it to a 96-well plate, add 19 μL of sterile PBS, and shake to mix. Quickly add 200 μL of BCA working solution to each well in turn, and place in a 60°C water bath for 30 minutes. Use a multifunctional microplate reader to measure its absorbance at 562 nm. Calculate the total protein concentration of the sample based on the standard curve and the sample dilution multiple.

[0322] Table 8 Gradient concentration preparation of protein standards

[0323]

[0324] 5.3 Preparation of sample solution

[0325] According to the protein quantification results of BCA, a protein sample loading solution with consistent concentration was prepared. SDS-PAGE protein loading buffer (5×) was added at a ratio of 5:1 (v / v), and the sample was placed in an ice box for 10 minutes in a 100°C metal bath (to fully denature the protein into a chain, and to fully mix the protein sample with the loading buffer), and then cooled. After cooling, the sample was centrifuged at 2000 rpm in a 4°C high-speed low-temperature centrifuge for 5 minutes. The sample was stored at -80°C or directly loaded onto a separation gel for electrophoresis.

[0326] 5.4 Electrophoresis

[0327] Select the upper layer of 5% concentrated gel and the lower layer of 10% separation gel, MOPS electrophoresis system, 20V constant voltage electrophoresis for about 1.5h. The protein loading amount is 25μg.

[0328] 5.5 Transfer

[0329] Prepare 1× electrophoresis transfer buffer (transfer solution): add 200 mL of methanol solution to 1× electrophoresis transfer buffer (transfer solution) powder, dilute to 1 L with pure water, and store at 4°C. Precool the transfer solution at -20°C for 2 h before transfer.

[0330] Activate PVDF membrane: Soak a PVDF membrane of appropriate size in methanol for 10 minutes. The PVDF membrane is activated and positively charged after being soaked in methanol, which can better bind to negatively charged proteins. The activated PVDF is soaked in the transfer solution for equilibrium.

[0331] Place the entire electrophoresis tank in an ice box, add an appropriate amount of pre-cooled transfer solution, and transfer for 90 minutes at 250 mA. After the transfer is completed, use tweezers to remove the PVDF membrane from the transfer clip and soak it in 1× TBST for later use.

[0332] 5.6 Immune Response

[0333] Prepare 1×TBST solution: dissolve 1×TBS in 1L pure water and add 1mL Tween-20. Store at 4℃ until use.

[0334] Prepare protein blocking solution (5% skim milk powder solution): weigh 2.250 g skim milk powder, fully dissolve it in 45 mL 1× TBST solution, and store it at 4°C for later use.

[0335] Primary antibody dilution: β-actin and NQO1 are diluted at 1:10000; Keap1, Nrf2 and HO-1 are diluted at 1:1000. Dilute with primary antibody diluent.

[0336] Secondary antibody dilution: HRP-goat anti-rabbit IgG is diluted at 1:10000. Dilute with secondary antibody diluent.

[0337] Blocking: Place the PVDF membrane after transfer into 5% skimmed milk powder solution and incubate on a decolorizing shaker at room temperature for 60 minutes.

[0338] Primary antibody incubation: Incubate the PVDF membrane in the corresponding primary antibody solution and incubate on a decolorizing shaker at 4°C for 24 hours.

[0339] Secondary antibody incubation: First, wash the PVDF membrane that has been incubated with the primary antibody with 1×TBST solution for 3 times, 10 minutes each time. Then, place the washed PVDF membrane in the secondary antibody solution and incubate it on a decolorizing shaker at room temperature for 60 minutes. After the incubation, wash it again with 1×TBST solution for 3 times, 10 minutes each time. After washing, soak the PVDF membrane in 1×TBST solution and store it at 4°C for later use.

[0340] 5.7 Development

[0341] Prepare the developer: prepare the ultra-sensitive ECL chemiluminescent ready-to-use substrate working solution A and working solution B in a 1:1 ratio, store in a dark place, and follow the principle of "prepare before use".

[0342] An appropriate amount of developer was added to the PVDF membrane, reacted in a dark room for 1 min, and then exposed and imaged in a Tanon 5200 fully automatic chemiluminescence image analysis system.

[0343] 6. Statistics

[0344] All experimental data are presented in the form of mean ± standard deviation (SD). Experimental data were analyzed and graphs were prepared using data analysis software such as SPSS.25, Origin Pro 2021, GraphPad Prism 8.3.0 and Image J.

[0345] III Results

[0346] 1. Total antioxidant capacity of Cynomorium songaricum polysaccharide in vitro

[0347] The standard curve is plotted with the mass C (μg) of tocopherol (Trolox) as the horizontal axis and the absorbance value A at 730nm as the vertical axis. Fig.34 As shown, the regression equation is A = 0.0556C, R 2 =0.9964. The absorbance value A of the Cynomorium songaricum polysaccharide sample at 490 nm was put into the standard curve, and it was calculated that the TEAC of CSRP-30, CSRP-50, CSRP-70, CSRP-90 and SRP-S were 17.34, 11.93, 11.39, 14.74 and 11.07, respectively. The ABTS clearance rates of CSRP-30, CSRP-50, CSRP-70, CSRP-90 and SRP-S were 99.23%, 72.51%, 69.84%, 86.40% and 68.26%, respectively (see Table 9).

[0348] Table 9 Total antioxidant activity of Cynomorium songaricum polysaccharide in vitro

[0349]

[0350] 2. Study on the protective effect of Cynomorium songaricum polysaccharide on oxidative stress induced by homocysteine ​​in MC3T3-E1 cells

[0351] 2.1 Establishment of MC3T3-E1 oxidative stress model

[0352] In order to study the antioxidant activity and oxidative stress protection mechanism of Cynomorium songaricum polysaccharide, this experiment chose to use the oxidative stress model of MC3T3-E1 induced by homocysteine ​​to simulate postmenopausal osteoporosis.

[0353] The Hcy modeling concentration was screened in the range of 300 μM to 20 mM Hcy-containing medium. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×10 4 The cells were inoculated into 96-well plates at a density of 100 cells / mL and cultured in a 37°C cell culture incubator for 24 h. The IC of Hcy was calculated. 50 =13.008mM, and finally 10mM Hcy was selected as the optimal modeling concentration (see Fig.35 ).

[0354] 2.2 Effect of Cynomorium songaricum polysaccharide on the survival rate of MC3T3-E1 cells

[0355] The results of the effects of CSRP-50, CSRP-70 and CSRP-90 on the survival rate of MC3T3-E1 cells are as follows Fig.36 shown.

[0356] When the concentrations of CSRP-50, CSRP-70 and CSRP-90 were lower than 1.0 mg / mL, they had a certain proliferative effect on the activity of MC3T3-E1 cells, and did not show an inhibitory effect on the activity of MC3T3-E1 cells. The proliferative effects of CSRP-50, CSRP-70 and CSRP-90 were, in order, CSRP-90 was better than CSRP-70 and better than CSRP-50. When the concentrations of CSRP-50, CSRP-70 and CSRP-90 were higher than 1.0 mg / mL, they showed a significant inhibitory effect on the activity of MC3T3-E1 cells (p<0.05), and the inhibitory effects of CSRP-50, CSRP-70 and CSRP-90 were, CSRP-90 was lower than CSRP-70 and lower than CSRP-50. This indicates that when the concentration is lower than 1.0 mg / mL, CSRP-50, CSRP-70 and CSRP-90 have no cytotoxic effect on MC3T3-E1 cells. Therefore, a concentration range lower than 1.0 mg / mL was selected for the screening of Cynomorium songaricum polysaccharide administration concentration.

[0357] 2.3 Screening of dosing concentrations of bazedoxifene (BZA) and Cynomorium songaricum polysaccharide

[0358] In order to select the appropriate positive control drug concentration, the BZA concentration was screened in the range of 10 to 800 nM in BZA-containing medium. The EC of BZA was calculated. 50 =99.94nM, and finally 200nM BZA was selected as the optimal positive control drug concentration (see Fig.37 ).

[0359] The appropriate dosing concentration of Cynomorium songaricum polysaccharide was screened, and the concentration screening range was 6.25-400 μg / mL of polysaccharide sample culture medium, and the polysaccharide-free sample culture medium (0 μg / mL) was used as a control. MC3T3-E1 cells in good growth state in the logarithmic growth phase were cultured at 2×10 4 The cells were seeded at a density of 100 cells / mL in a 96-well plate and cultured in a 37°C cell culture incubator for 24 hours. Fig.38 , Fig.39 and Fig.40As shown in the figure, in the process of screening the concentrations of CSRP-50, CSRP-70 and CSRP-90 for the proliferation promotion effect on Hcy-damaged MC3T3-E1 cells, the maximum proliferation promotion effect concentration of CSRP-50, CSRP-70 and CSRP-90 appeared at 25 μg / mL. Except for 6.25 μg / mL, the other concentrations still had significant proliferation promotion effects. Therefore, 12.5 μg / mL, 25 μg / mL and 50 μg / mL were selected as the low concentration group, medium concentration group and high concentration group of Cynomorium songaricum polysaccharide, respectively.

[0360] 2.4 Experimental Grouping Description

[0361] Based on the above experimental results, 10mM Hcy was finally selected as the optimal modeling concentration, and 200nM BZA was selected as the positive control drug concentration. The experiment was designed as follows: (1) blank control group (Normal): a group cultured in culture medium; (2) model group (Model): cultured in culture medium containing Hcy for 24 hours, followed by fresh culture for 24 hours; (3) positive control group (BZA): cultured in culture medium containing Hcy for 24 hours, followed by culture medium containing BZA for 24 hours; (4) polysaccharide group: cultured in culture medium containing Hcy for 24 hours, followed by culture medium containing polysaccharide samples for 24 hours, where low concentration (12.5μg / mL), medium concentration (25μg / mL), and high concentration (50μg / mL) of C SRP-50 was named CSRP-50L, CSRP-50M and CSRP-50H; low (12.5 μg / mL), medium (25 μg / mL) and high (50 μg / mL) concentrations of CSRP-70 were named CSRP-70L, CSRP-70M and CSRP-70H; low (12.5 μg / mL), medium (25 μg / mL) and high (50 μg / mL) concentrations of CSRP-90 were named CSRP-90L, CSRP-90M and CSRP-90H.

[0362] 2.5 Effects of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSRP-90 on the production of MDA, GSH, SOD, T-COA, LDH and other cytokines in MC3T3-E1 cells

[0363] MDA is a recognized marker of oxidative stress (Cui X et al., 2018). SOD, GSH and T-AOC are common antioxidants widely distributed in cells (Tan Y et al., 2010). LDH release is regarded as an important indicator of cell membrane integrity and is widely used in cytotoxicity detection (Li L et al., 2019).

[0364] In order to study the protective effect of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSEP-90 on oxidatively damaged MC3T3-E1 cells, the effects of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSRP-90 on the levels of cytokines such as MDA, SOD, GSH, T-COA and LDH produced by oxidatively damaged MC3T3-E1 cells were studied. Fig.41 shown.

[0365] (1) Changes in MDA levels in MC3T3-E1 cells: Compared with the blank control group, the model group significantly increased the MDA level in MC3T3-E1 cells (p<0.05). Compared with the model group, the BZA group, CSRP-50L, CSRP-50M, CSRP-50H, CSRP-70L, CSRP-70M, CSRP-70H, CSRP-90L, CSRP-90M and CSRP-90H all significantly decreased the MDA level in MC3T3-E1 cells (p<0.05).

[0366] (2) Changes in GSH levels in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the level of GSH in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). Compared with the model group, the BZA group, CSRP-70M, CSRP-70H, CSRP-90L, CSRP-90M and CSRP-90H all significantly increased the level of GSH in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). CSRP-50M significantly increased the level of GSH in MC3T3-E1 cells, reaching an extremely significant level (p<0.01). CSRP-50L, CSRP-50H and CSRP-70L significantly increased the level of GSH in MC3T3-E1 cells (p<0.05).

[0367] (3) Changes in SOD levels in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the SOD level in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). Compared with the model group, the BZA group, CSRP-50M, CSRP-50H and CSRP-70M all significantly increased the SOD level in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). CSRP-70L significantly increased the SOD level in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). CSRP-90M significantly increased the SOD level in MC3T3-E1 cells, reaching a significant level (p<0.05). However, CSRP-50L, CSRP-70H, CSRP-90L and CSRP-90H increased the SOD level in MC3T3-E1 cells, with no significant difference.

[0368] (4) Changes in T-AOC levels in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the level of SOD in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); compared with the model group, the BZA group significantly increased the level of T-AOC in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); CSRP-70M, CSRP-90L and CSRP-90M significantly increased the level of T-AOC in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); CSRP-50L, CSRP-50M, CSRP-50H, CSRP-70L, CSRP-70H and CSRP-90H all significantly increased the level of T-AOC in MC3T3-E1 cells (p<0.05).

[0369] (5) Changes in LDH levels in the culture medium of MC3T3-E1 cells: Compared with the blank control group, the model group significantly increased the level of LDH in the culture medium of MC3T3-E1 cells, reaching an extremely significant level (p<0.001). Compared with the model group, the BZA group, CSRP-50L, CSRP-50M, CSRP-70L, CSRP-70M, CSRP-90L, CSRP-90M and CSRP-90H all significantly decreased the level of LDH in the culture medium of MC3T3-E1 cells, reaching an extremely significant level (p<0.001). CSRP-50H significantly decreased the level of LDH in the culture medium of MC3T3-E1 cells, reaching an extremely significant level (p<0.01). CSRP-70H decreased the level of LDH in the culture medium of MC3T3-E1 cells, but there was no significant difference.

[0370] The above results showed that compared with the blank control group, the model group could significantly increase the level of MDA in MC3T3-E1 cells (p<0.05), significantly decrease the levels of SOD, GSH and T-AOC in MDA of MC3T3-E1 cells (p<0.05), and significantly increase the level of LDH released into the culture medium by MC3T3-E1 cells (p<0.05), indicating that after Hcy induction, the oxidative substances in MC3T3-E1 cells increased, the antioxidant active substances decreased, the cytotoxicity was significant, and the cells were broken and died.

[0371] Compared with the model group, the polysaccharide group can significantly reduce the MDA level of MC3T3-E1 cells (p<0.05), significantly increase the levels of SOD, GSH and T-AOC of MC3T3-E1 cells (p<0.05), and significantly reduce the level of LDH released into the culture medium by MC3T3-E1 cells (p<0.05), indicating that the damaged MC3T3-E1 cells have improved their antioxidant capacity under the stimulation of CSRP-50, CSRP-70 and CSRP-90, reduced the oxidants in the cells, reduced the cytotoxicity of Hcy, and reduced cell damage. Among the experimental concentrations of CSRP-50, CSRP-70 and CSRP-90, the optimal administration concentration was 25μg / mL, and the optimal polysaccharide was CSRP-90.

[0372] 2.6 Effects of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSEP-90 on protein expression in the Keap1 / Nrf2 signaling pathway of MC3T3-E1 cells

[0373] There is increasing evidence that Nrf2, as a negative regulator of ROS, is involved in the regulation of oxidative stress at the cellular and tissue levels (BHKA et al., 2016). In general, Keap1 negatively regulates the expression of Nrf2-dependent antioxidant enzymes (such as HO-1, GCS, NQO1, and G6PD) by inhibiting the nuclear translocation of Nrf2, which can counteract the generation of ROS and alleviate oxidative stress (Kaneko YS et al., 2016).

[0374] In order to study the protective effect mechanism of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSEP-90 on oxidatively damaged MC3T3-E1 cells, the effects of Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSRP-90 on the expression of Nef2 and Keap1 cell regulatory factors and antioxidant enzymes (HO-1, NQO1) in oxidatively damaged MC3T3-E1 cells were studied. Fig.42 shown.

[0375] (1) Changes in Keap1 expression in MC3T3-E1 cells: Compared with the blank control group, the model group significantly increased the expression of Keap1 in MC3T3-E1 cells (p<0.05). Compared with the model group, CSRP-70M significantly decreased the expression of Keap1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); BZA group significantly decreased the expression of Keap1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); CSRP-50M and CSRP-90M significantly decreased the expression of Keap1 in MC3T3-E1 cells (p<0.05); while CSRP-50L, CSRP-50H, CSRP-70L, CSRP-70H, CSRP-90L and CSRP-90H all decreased the expression of Keap1 in MC3T3-E1 cells, but there was no significant difference.

[0376] (2) Changes in Nrf2 expression in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the expression of Nrf2 in MC3T3-E1 cells, reaching a highly significant level (p<0.01). Compared with the model group, the BZA group significantly increased the expression of Nrf2 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); CSRP-90H and CSRP-90L significantly increased the expression of Nrf2 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); CSRP-50L, CSRP-50M, CSRP-50H, CSRP-70M and CSRP-90M significantly increased the expression of Nrf2 in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); CSRP-70L significantly increased the expression of Nrf2 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); CSRP-70H significantly increased the expression of Nrf2 in MC3T3-E1 cells (p<0.05).

[0377] (3) Changes in NQO1 expression in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the expression of NQO1 in MC3T3-E1 cells. Compared with the model group, CSRP-70M and CSRP-90M significantly increased the expression of NQO1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); CSRP-50L, CSRP-50M, CSRP-70L, CSRP-90L and CSRP-90H all significantly increased the expression of NQO1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); BZA group and CSRP-70H significantly increased the expression of NQO1 in MC3T3-E1 cells, reaching a significant level (p<0.05), while CSRP-50H increased the expression of NQO1 in MC3T3-E1 cells, but there was no significant difference.

[0378] (4) Changes in HO-1 expression in MC3T3-E1 cells: Compared with the blank control group, the model group significantly reduced the HO-1 expression in MC3T3-E1 cells, reaching an extremely significant level (p<0.001). Compared with the model group, CSRP-90M and CSRP-90H significantly increased the expression of HO-1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); CSRP-90L significantly increased the expression of HO-1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.001); CSRP-70M significantly increased the expression of HO-1 in MC3T3-E1 cells, reaching an extremely significant level (p<0.01); BZA group, CSRP-50M and CSRP-70H all significantly increased the expression of HO-1 in MC3T3-E1 cells (p<0.05), while CSEP-50L, CSRP-50H and CSRP-70L increased the expression of HO-1 in MC3T3-E1 cells, but there was no significant difference.

[0379] The above results show that the protein expression of Nrf2, HO-1, and NQO1 in MC3T3-E1 cells induced by Hcy significantly decreased (p<0.05), and the expression of Keap1 significantly increased (p<0.05), indicating that the cells are in a state of oxidative stress. This is consistent with the phenomenon found by Wan G et al. (2022) in the bone tissue of SAMP6 mice that the expression of Nrf2 decreased while the expression of Keap1 increased, and its ability to resist oxidative stress decreased. This shows that the modeling of this experiment was successful.

[0380] Compared with the model group, CSRP-50M, CSRP-70M, and CSRP-90M in the BZA group and the polysaccharide group could significantly reduce the expression of Keap1 in MC3T3-E1 cells (p<0.05). In terms of the ability to inhibit Keap1 protein expression, only CSRP-70M had a higher inhibitory ability than the BZA group, indicating that the inhibitory ability of CSRP-70 was superior to that of CSRP-50 and CSRP-90. Both the BZA group and the polysaccharide group could significantly increase the protein expression of Nrf2 in MC3T3-E1 cells (p<0.05). In terms of the ability to stimulate the protein expression of Nrf2, the polysaccharide group was superior to the BZA group, among which CSRP-90H had the best stimulating ability, and the stimulating abilities of CSRP-50M, CSRP-70M, and CSRP-90M were second only to CSRP-90H. There was no significant difference between CSRP-50H and CSRP-70H in the polysaccharide group in increasing the expression of NQO1 in MC3T3-E1 cells; there was no significant difference between CSRP-50L, CSRP-50H and CSRP-70L in the polysaccharide group in increasing the expression of HO-1 in MC3T3-E1 cells. In terms of the ability to stimulate the expression of the antioxidant enzyme NQO1, CSRP-90M and CSRP-70M were the strongest, followed by CSRP-50M. In terms of the ability to stimulate the expression of the antioxidant enzyme HO-1, the order from strong to weak was CSRP-90H, CSRP-90M, CSRP-70M, and CSRP-50M.

[0381] This indicates that CSRP-50, CSRP-70 and CSRP-90 can all activate the Keap1 / Nrf2 signaling pathway, inhibit the expression of Keap1, promote the expression of Nrf2 and antioxidant enzymes (HO-1, NQO1), and thus reduce the oxidative stress level of cells. Among the experimental concentrations of CSRP-50, CSRP-70 and CSRP-90, the optimal administration concentration was 25 μg / mL, and the best polysaccharide was CSRP-90.

[0382] 2.7 Comprehensive analysis of the primary structure and micromorphology of Cynomorium songaricum polysaccharide

[0383] The structures of CSRP-50, CSRP-70 and CSRP-90 were combined for analysis. CSRP-50 is superior to CSRP-70 and CSRP-90 in increasing SOD levels and reducing LDH levels. The primary structure of CSRP-50 has a larger molecular weight than that of CSRP-70 and CSRP-90, and its sugar chain does not have a side chain. It can be inferred that polysaccharides with larger molecular weight and unbranched structure may have more advantages in increasing the SOD level of cells and reducing the LDH level.

[0384] CSRP-90 is superior to CSRP-50 and CSRP-70 in increasing the levels of GSH and T-AOC, activating the Keap1 / Nrf2 signaling pathway, inhibiting the expression of Keap1, and promoting the expression of Nrf2 and antioxidant enzymes (HO-1, NQO1). CSRP-90 and CSRP-70 have side chains in their structures, while CSRP-50 has no side chains. At the same time, the effect of CSRP-90 is better than that of CSRP-70. The primary structure of CSRP-90 and CSRP-70 is similar, but the molecular weight of CSRP-90 is smaller and the structure is looser than that of CSRP-70. Therefore, it is inferred that polysaccharides with side chains, small molecular weight and loose structure may have more advantages in stimulating GSH secretion and increasing T-AOC levels.

[0385] In general, CSRP-90 is more effective than CSRP-50 and CSRP-70 in increasing the levels of cellular antioxidant enzymes (HO-1 and NQO1) and the regulatory factor Nrf2. Combined with the results of the primary structural feature analysis, the molecular weight of CSRP-90 is smaller than that of CSRP-50 and CSRP-70. The structure of CSRP-90 is similar to that of CSRP-70, but it differs from CSRP-50 in that it has a side chain. Considering the microscopic morphological observation results of CSRP-50, CSRP-70 and CSRP-90 at the same time, the structure of CSRP-90 presents an aggregated network structure and is loose. Its loose structural characteristics increase its specific surface area, which means that it increases the probability of contact with cell signal receptors. Based on the above analysis, it is concluded that the polysaccharide component CSRP-90, which has a small molecular weight, a branched chain and a loose microscopic morphological structure, has better antioxidant activity.

[0386] In this study, the total antioxidant capacity in vitro (ABTS scavenging method) and the homocysteine-induced MC3T3-E1 oxidative stress model were used to study the antioxidant activity of CSRP-50, CSRP-70, and CSRP-90 and their effects on the treatment of postmenopausal osteoporosis. The results showed that:

[0387] (1) All levels of the Cynomorium songaricum polysaccharide components of the present invention have good antioxidant activity.

[0388] (2) CSRP-50, CSRP-70 and CSRP-90 can significantly increase the levels of cellular GSH, SOD and T-AOC, and significantly reduce the level of MDA, thereby reducing the level of LDH released into the cells and achieving a protective effect.

[0389] (3) CSRP-50, CSRP-70, and CSRP-90 can increase the levels of the regulatory factor Nrf2 and antioxidant enzymes HO-1 and NQO1 and reduce the level of the regulatory factor Keap1 by activating the Keap1-Nrf2 signaling pathway, thereby achieving the effect of protecting cells.

[0390] (4) CSRP-90, which has side chains, smaller molecular weight, and loose microstructure, has better protection effect.

[0391] (5) The optimal protective concentration of CSRP-50, CSRP-70 and CSRP-90 was 25 μg / mL.

[0392] (6) CSRP-50, CSRP-70 and CSRP-90 can be used to prepare drugs for treating postmenopausal osteoporosis.

[0393] In summary, the Cynomorium songaricum polysaccharides CSRP-50, CSRP-70 and CSRP-90 provided by the present invention can increase endogenous antioxidant enzymes and antioxidant substances in cells to remove oxides in cells by activating the Keap1-Nrf2 signaling pathway, thereby achieving the protective effect of oxidative damage to osteoblasts, and have broad application prospects in the preparation of prevention and / or treatment of osteoporosis caused by oxidative stress of osteoblasts caused by homocysteine.

Claims

1. A Cynomorium songaricum polysaccharide, characterized in that: The polysaccharide is extracted from the fleshy stem of Cynomorium songaricum. The total sugar content of the Cynomorium songaricum polysaccharide is greater than 90wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%-3wt%.

2. The Cynomorium songaricum polysaccharide according to claim 1, characterized in that The total sugar content of the Cynomorium songaricum polysaccharide is 91wt%-97wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%-3wt%; the composition of monosaccharides in the total sugar includes: more than 96.00wt% of glucose.

3. The Cynomorium songaricum polysaccharide according to claim 2, characterized in that The Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-50, wherein the total sugar content is 93.45wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0wt%; the composition of the monosaccharides in the total sugar includes: 96.58wt% of glucose, 0.80wt% of mannose, 0.48wt% of glucuronic acid, 0.19wt% of rhamnose, 0.58wt% of galacturonic acid, 0.45wt% of galactose, 0.80wt% of arabinose and 0.12wt% of fucose; Or, the Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-70, wherein the total sugar content is 96.79wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 0.34wt%; the composition of monosaccharides in the total sugar includes: 96.75wt% of glucose, 0.78wt% of mannose, 0.24wt% of glucuronic acid, 0.15wt% of rhamnose, 0.69wt% of galacturonic acid, 0.61wt% of galactose, 0.66wt% of arabinose and 0.11wt% of fucose; Or, the Cynomorium songaricum polysaccharide is Cynomorium songaricum polysaccharide CSRP-90, wherein the total sugar content is 92.82wt%, the protein content is 0wt%, the uronic acid content is 0wt%, and the total phenol content is 2.09wt%; the composition of monosaccharides in the total sugar includes: 96.01wt% of glucose, 0.86wt% of mannose, 0.40wt% of glucuronic acid, 0.06wt% of rhamnose, 0.25wt% of galacturonic acid, 1.18wt% of galactose, 1.08wt% of arabinose and 0.16wt% of fucose.

4. The Cynomorium songaricum polysaccharide according to claim 3, characterized in that The molecular weight of the Cynomorium songaricum polysaccharide CSRP-50 is 5.7 kDa; Or, the molecular weight of the Cynomorium songaricum polysaccharide CSRP-70 is 4.5 kDa; Or, the molecular weight of the Cynomorium songaricum polysaccharide CSRP-90 is 2.0 kDa.

5. The Cynomorium songaricum polysaccharide according to claim 3, characterized in that The primary structure of the Cynomorium songaricum polysaccharide CSRP-50 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain; Or, the primary structure of the Cynomorium songaricum polysaccharide CSRP-70 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming the side chain at the O-6 position of α-(1,4)-D-Glcp; Alternatively, the primary structure of the Cynomorium songaricum polysaccharide CSRP-90 is a glucan with α-(1,4)-D-Glcp and α-(1,6)-D-Glcp alternately connected as the main chain, and T-α-D-Glcp and α-(1,6)-D-Glcp forming side chains at the O-6 position of α-(1,4)-D-Glcp.

6. The Cynomorium songaricum polysaccharide according to any one of claims 3 to 5, characterized in that The microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-50 is honeycomb-shaped; Or, the microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-70 is irregular in shape; Or, the microscopic morphology of the Cynomorium songaricum polysaccharide CSRP-90 is an aggregated network structure.

7. A method for preparing the Cynomorium songaricum polysaccharide according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) taking a fleshy stem of Cynomorium songaricum, adding water to extract, collecting the extract, concentrating it, adding ethanol to precipitate it, collecting the precipitate, and obtaining a crude polysaccharide CSRP of Cynomorium songaricum; (2) Separate and purify the crude polysaccharide CSRP from Cynomorium songaricum by using a DEAE-Cellulose cellulose chromatography column, collect the effluent containing the polysaccharide, concentrate it, and add ethanol for precipitation to obtain the product.

8. The method according to claim 7, characterized in that In step (1), the fleshy stem of Cynomorium songaricum is from fresh Cynomorium songaricum; the solid-liquid ratio of the extraction is 1:(12-18), the extraction temperature is 90-100° C., the number of extractions is 1-6 times, and the time of each extraction is 2-6 hours; the amount of ethanol added is added until the alcohol content of the solution reaches more than 70%; In step (2), the eluent used for separation and purification is water, and the method of adding ethanol precipitation is: first add ethanol to an ethanol concentration of 28%-32%, precipitate, centrifuge, collect the supernatant, adjust the ethanol concentration in the supernatant to 48%-52%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-50, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 68%-72%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-70, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 88%-92%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-90; Preferably, in step (1), the fleshy stem of Cynomorium songaricum is from fresh Cynomorium songaricum; the solid-liquid ratio of the extraction is 1:15, the extraction temperature is 95-100° C., the number of extractions is 4 times, and the time of each extraction is 4 hours; the amount of ethanol added is added until the alcohol content of the solution reaches 75%; In step (2), the method of adding ethanol precipitation is: first add ethanol to an ethanol concentration of 30%, precipitate, centrifuge, collect the supernatant, adjust the ethanol concentration in the supernatant to 50%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-50, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 70%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-70, and further adjust the ethanol concentration in the supernatant obtained in the previous step to 90%, precipitate, centrifuge, collect the solid, and obtain Cynomorium songaricum polysaccharide CSRP-90.

9. Use of the Cynomorium songaricum polysaccharide according to any one of claims 1 to 6 in the preparation of an antioxidant drug, wherein the antioxidant drug is preferably an osteoblast oxidative damage protective drug.

10. Use of the Cynomorium songaricum polysaccharide according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating osteoporosis, wherein the osteoporosis is preferably postmenopausal osteoporosis.

Images