Application of salvia miltiorrhiza crude polysaccharide, salvia miltiorrhiza oligosaccharide or salvia miltiorrhiza polysaccharide in identification of salvia miltiorrhiza from different producing areas

Abstract

The invention belongs to the field of natural product chemistry and phytochemistry, and particularly relates to application of salvia miltiorrhiza crude polysaccharide, salvia miltiorrhiza oligosaccharide or salvia miltiorrhiza polysaccharide to identification of salvia miltiorrhiza from different producing areas. The production places of different salvia miltiorrhiza bunge are judged according to the different saccharide fragments, the molecular weights of SMP and SMO, monosaccharide components and the proportion of the monosaccharide components, and subsequent research on the salvia miltiorrhiza bunge saccharide structure and function is facilitated. The adopted identification method is simple and easy to operate, low in cost and beneficial to being widely used in production. Meanwhile, in-depth study is carried out on different sugar fragments of salvia miltiorrhiza crude polysaccharides from different producing areas and monosaccharide components and structures of SMO and SMP, and the structure of sugar has a certain influence on the physiological function of the salvia miltiorrhiza, so that a foundation is laid for subsequent study of the salvia miltiorrhiza.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and phytochemistry, specifically relating to the application of crude polysaccharide, oligosaccharide or polysaccharide of Salvia miltiorrhiza in the identification of Salvia miltiorrhiza from different origins. Background Technology

[0002] Salvia miltiorrhiza Salvia miltiorrhiza (SM) is a plant of the genus Salvia in the family Lamiaceae. Salvia miltiorrhiza Bunge The dried roots and rhizomes of Salvia miltiorrhiza were first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). Salvia miltiorrhiza is bitter and slightly cold in nature, and has the effects of relieving pain and promoting blood circulation, regulating menstruation, and calming the mind. The medicinal parts of Salvia miltiorrhiza recorded in the *Chinese Pharmacopoeia* are the roots and rhizomes. Currently, about 22 species, 5 varieties, and 5 forms of Salvia miltiorrhiza in my country have their underground parts used as medicinal Salvia miltiorrhiza.

[0003] To date, over 100 chemical components have been isolated from *Salvia miltiorrhiza*, mainly including rosmarinic acid, tanshinone I, tanshinone IIA, tanshinone IIB, propargyl aldehyde, polysaccharides, and salvianolic acid. Salvia miltiorrhiza glycosides are one of the main active ingredients of this medicinal herb. Salvia miltiorrhiza polysaccharides, extracted and isolated from *Salvia miltiorrhiza*, possess various biological functions such as antioxidant, hepatoprotective, anti-inflammatory, hypoglycemic, antitumor, immunomodulatory, and cardioprotective effects. However, previous research on *Salvia miltiorrhiza* has primarily focused on salvianolic acid and tanshinone compounds, while a systematic and comprehensive study of the traditional efficacy and mechanisms of action of salvianolic acid glycosides has been lacking.

[0004] Patent CN101225422A discloses a method for extracting polysaccharides from Salvia miltiorrhiza. This patent utilizes ultrafine grinding technology to improve the dissolution rate of active ingredients in Salvia miltiorrhiza after ultrafine grinding. However, this patent does not provide a comparative analysis of the structural differences of polysaccharides in Salvia miltiorrhiza from different regions, thus focusing on a single research subject. (The paper "The Comparison of Effective In Radix") Salviae Miltiorrhizae Wang Daoping et al. compared the content of active ingredients in Salvia miltiorrhiza from different origins, but their research focused on several acidic and ketone substances in Salvia miltiorrhiza, and their research on sugars was not comprehensive.

[0005] This invention analyzes the differential sugar fragments of crude polysaccharides from different origins using UPLC LTQ-Orbitrap MS / MS. The characteristic sugar peaks, molecular weight, and monosaccharide composition of SMO and SMP from different origins are studied using infrared spectroscopy, high-performance gel permeation chromatography, and ion chromatography, providing a preliminary analysis of their sugar spectrum characteristics. This lays the foundation for further exploration of the relationship between the sugar structure and bioactivity of *Salvia miltiorrhiza*. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the purpose of this invention is to design and provide the application of crude polysaccharide, oligosaccharide or polysaccharide of tanshinone in the identification of tanshinone from different origins.

[0007] The present invention is implemented using the following technical solutions: In a first aspect, the present invention provides the application of crude polysaccharide, oligosaccharide or polysaccharide of tanshinone in the identification of tanshinone from different origins.

[0008] Furthermore, the crude polysaccharide from Salvia miltiorrhiza is a water-extracted and alcohol-precipitated crude polysaccharide from Salvia miltiorrhiza. The extraction process for the water-extracted and alcohol-precipitated crude polysaccharide from Salvia miltiorrhiza is as follows: extraction at 90 ℃, extraction time of 2.5 h, and extraction twice at a material-to-liquid ratio of 1:20 g / mL.

[0009] Furthermore, the tanshinone oligosaccharide is a low molecular weight sugar obtained by eluting crude tanshinone polysaccharide with water using a DEAE-52 cellulose column, and the tanshinone polysaccharide is a polysaccharide obtained by eluting crude tanshinone polysaccharide with NaCl solution using a DEAE-52 cellulose column.

[0010] Furthermore, mass spectrometry was used to characterize the glycosides from different origins of Salvia miltiorrhiza. The differentially characterized components of the different structures were six. Differentially characterized components 1-3 were mainly 1,6-linked β-D-glucan oligosaccharides, and differentially characterized components 4-6 were mainly 1,6-linked β-D-linear glucan oligosaccharides and galactooligosaccharides.

[0011] Furthermore, the molecular weight of SMP in Salvia miltiorrhiza from different origins was detected by high-performance gel permeation chromatography, and the origin of Salvia miltiorrhiza was determined based on the difference in molecular weight.

[0012] Furthermore, the molecular weight of SMO in Salvia miltiorrhiza from different origins was determined using high-performance gel permeation chromatography, and the origin of Salvia miltiorrhiza was determined based on the differences in molecular weight.

[0013] Furthermore, ion chromatography was used to detect the SMO monosaccharide components of Salvia miltiorrhiza from different origins, and the origin was determined based on the different monosaccharide components.

[0014] Furthermore, ion chromatography was used to detect the SMP monosaccharide components in Salvia miltiorrhiza from different origins, and the origin of Salvia miltiorrhiza was determined based on the proportion of SMP monosaccharide content in Salvia miltiorrhiza from different origins.

[0015] The present invention has the following beneficial effects: (1) This invention determines the origin of different Danshen by the molecular weight, monosaccharide components and their ratio of sugar differential fragments, SMP and SMO, which is beneficial to our subsequent research on the structure and function of Danshen sugars.

[0016] (2) The identification method used in this invention is simple and easy to operate, and has low cost, which is conducive to its widespread use in production.

[0017] (3) This invention has conducted in-depth research on the differential sugar fragments, SMO and SMP monosaccharide components and structures of crude polysaccharides from different origins of Salvia miltiorrhiza. The structure of sugars has a certain influence on their physiological functions, thus laying the foundation for subsequent research on Salvia miltiorrhiza. Attached Figure Description

[0018] Figure 1 Images of Salvia miltiorrhiza samples from different origins: Sichuan (a), Shandong (b), Hebei (c), Henan (d), Shaanxi (e), and Shanxi (f). Figure 2 The graph shows the effect of different material-to-liquid ratios on the yield of crude polysaccharides from Salvia miltiorrhiza. Figure 3 Figure showing the effect of different extraction times on the yield of crude polysaccharides from Salvia miltiorrhiza. Figure 4 Figure showing the effect of different extraction temperatures on the yield of crude polysaccharides from Salvia miltiorrhiza; Figure 5 The effect of different extraction times on the yield of crude polysaccharides from Salvia miltiorrhiza; Figure 6 Line graphs of DEAE-52 cellulose column elution of crude polysaccharide from Salvia miltiorrhiza from different origins. Note: Sichuan (a), Shandong (b), Hebei (c), Henan (d), Shaanxi (e), and Shanxi (f). Figure 7 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Sichuan; Figure 8 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Shandong; Figure 9 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Hebei Province; Figure 10 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Henan Province; Figure 11 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Shaanxi Province; Figure 12 Total ion chromatogram of crude polysaccharides from Salvia miltiorrhiza produced in Shanxi Province; Figure 13 Figure 1: β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal, showing the differential sugar fragment 1 of crude polysaccharides from different origins. Figure 14 Figure 2: Differential sugar fragment 2 of crude polysaccharides from different origins: β-D-Gal-1-6-β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal; Figure 15 Figure 3 shows the differential sugar fragment 3 of crude polysaccharides from different origins: β-D-Gal-1-6-β-D-Gal-1-6-β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal. Figure 16 Differential sugar fragment 4 of crude polysaccharides from different origins: β-D-Glc-1-6-β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal; Figure 17 Differential sugar fragments of crude polysaccharides from different origins: β-D-Glc-1-6-β-D-Gal-1-6-β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal; Figure 18 Differential sugar fragment 6 of crude polysaccharides from different origins: β-D-Glc-1-6-β-D-Glc-1-6-β-D-Gal-1-6-β-D-Gal-1-6-a / β-D-Gal; Figure 19 UV spectra of SMO from different origins: SMO-Sichuan (a), SMO-Shandong (b), SMO-Hebei (c), SMO-Henan (d), SMO-Shaanxi (e) and SMO-Shanxi (f); Figure 20 UV spectra of SMP from different origins: SMP-Sichuan (a), SMP-Shandong (b), SMP-Hebei (c), SMP-Henan (d), SMP-Shaanxi (e) and SMP-Shanxi (f). Figure 21 Infrared spectra of SMPs from different origins: SMP-1 Sichuan, SMP-2 Shandong, SMP-3 Hebei, SMP-4 Henan, SMP-5 Shaanxi, and SMP-6 Shanxi. Figure 22 Infrared spectra of SMO from different origins: SMO-1 from Sichuan, SMO-2 from Shandong, SMO-3 from Hebei, SMO-4 from Henan, SMO-5 from Shaanxi, and SMO-6 from Shanxi. Figure 23 HPGPC images of SMO from different origins, note: SMO-Sichuan (a), SMO-Shandong (b), SMO-Hebei (c), SMO-Henan (d), SMO-Shaanxi (e) and SMO-Shanxi (f); Figure 24 HPGPC images of SMP from different origins, note: SMP-Sichuan (a), SMP-Shandong (b), SMP-Hebei (c), SMP-Henan (d), SMP-Shaanxi (e) and SMP-Shanxi (f). Figure 25 The ion chromatogram is for a mixed standard of 11 sugars. Figure 26Ion chromatograms of SMO from different origins are shown. Note: SMO-Sichuan (a), SMO-Shandong (b), SMO-Hebei (c), SMO-Henan (d), SMO-Shaanxi (e), and SMO-Shanxi (f). Figure 27 Ion chromatograms of SMP from different origins are shown. Note: SMP-Sichuan (a), SMP-Shandong (b), SMP-Hebei (c), SMP-Henan (d), SMP-Shaanxi (e), and SMP-Shanxi (f). Detailed Implementation

[0019] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0020] The reagents used in the experiment are shown in Table 1.

[0021] Table 1 Experimental Reagents

[0022] The instruments and equipment used in the experiment are shown in Table 2.

[0023] Table 2 Instruments and Equipment

[0024] Example 1: Extraction, separation and purification of oligosaccharides and polysaccharides from Tanshinone from different origins I. Experimental Methods: (1) Collection of Salvia miltiorrhiza from different producing areas Previous studies have compiled information on Salvia miltiorrhiza sample plots based on traditional Chinese medicine textual research, and have collected information on altitude, topography, soil type, seedling type and planting history of 85 production areas, and selected six representative authentic Salvia miltiorrhiza varieties: white-flowered Salvia miltiorrhiza from Laiwu, Shandong; Salvia miltiorrhiza from Mianchi, Henan; Salvia miltiorrhiza from Changzhi, Shanxi; Salvia miltiorrhiza from Shangluo, Shaanxi; Salvia miltiorrhiza from Zhongjiang, Sichuan; and Salvia miltiorrhiza from Anguo, Hebei (Table 3).

[0025] Table 3. Source of Salvia miltiorrhiza samples

[0026] (2) Drying, pulverizing and defatting of Salvia miltiorrhiza. Drying and pulverizing: Fresh Salvia miltiorrhiza from different origins are sun-dried after removing the heads and fibrous roots to remove ash, and then dried at 60 ℃. After drying, the herbs are sliced, pulverized using a pulverizer, and sieved.

[0027] Degreasing: Weigh 500 g of dried Salvia miltiorrhiza powder, add 5 L of anhydrous ethanol, set the temperature to 85 ℃, reflux for 2 h, filter, and place the filter residue on a tray to dry for later use.

[0028] (3) Optimization of the extraction process of crude polysaccharide from Salvia miltiorrhiza by single-factor combined orthogonal experiment 1. Material-to-liquid ratio Accurately weigh 10 g of defatted Salvia miltiorrhiza and place it in a 500 mL round-bottom flask. Set the temperature to 85 ℃, the time to 2 h, and the rotation speed to 500 rpm to investigate the effect of different material-liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30) on the yield of crude polysaccharides from Salvia miltiorrhiza.

[0029] 2. Extraction time Accurately weigh 10 g of defatted Salvia miltiorrhiza and place it in a 500 mL round-bottom flask. Set the temperature to 85 ℃, the material-to-liquid ratio to 1:20, and the rotation speed to 500 rpm. Investigate the effect of different time periods (60 min, 90 min, 120 min, 150 min, and 180 min) on the yield of crude polysaccharides from Salvia miltiorrhiza.

[0030] 3. Extraction temperature Accurately weigh 10 g of defatted Salvia miltiorrhiza and place it in a 500 mL round-bottom flask. Set the time to 2 h, the material-to-liquid ratio to 1:20, and the rotation speed to 500 rpm. Investigate the effect of different temperatures (75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃) on the yield of crude polysaccharides from Salvia miltiorrhiza.

[0031] 4. Number of extractions Accurately weigh 10 g of defatted Salvia miltiorrhiza and place it in a 500 mL round-bottom flask. Set the extraction time to 2 h, the material-to-liquid ratio to 1:20, the temperature to 90 ℃, and the rotation speed to 500 rpm. Investigate the effect of different extraction times (1, 2, and 3 times) on the yield of crude Salvia miltiorrhiza polysaccharides.

[0032] 5. Orthogonal experimental design Based on the results of the single-factor experiments, factors that significantly influenced the extraction process (extraction temperature, extraction time, and solid-liquid ratio) were selected as independent variables for the orthogonal experiment. Three levels were selected for each factor, and a three-factor, three-level orthogonal experiment was conducted. The crude polysaccharide yield was used as the evaluation index to optimize the three extraction conditions. The orthogonal experiment factor and level design is shown in Table 4.

[0033] Table 4 L9 (3) 4 Orthogonal Experiment Factor Levels and Coding

[0034] (4) Extraction and purification of crude polysaccharides from Salvia miltiorrhiza Hot water extraction: Accurately weigh the Salvia miltiorrhiza powder and add it to a 5000 mL round-bottom flask. In a constant-temperature water bath with magnetic stirring, extract for 2.5 h at a material-to-liquid ratio of 1:20 and 90 ℃. Filter and collect the filtrate. Centrifuge the filtrate at 7000 rpm and 4 ℃ for 10 min. Pour off the supernatant and concentrate under reduced pressure in a rotary evaporator.

[0035] Alcohol precipitation: The extract concentrate and anhydrous ethanol were mixed at a ratio of 1:4 for precipitation, and the mixture was placed at 4 °C for 12 h. The supernatant was removed by centrifugation at 7000 rpm for 10 min at 4 °C. The precipitate was collected, excess ethanol was evaporated, and finally, the unpurified crude polysaccharide was obtained by freeze drying.

[0036] Decolorization: Dissolve the unpurified crude polysaccharide in purified water and decolorize it using activated carbon.

[0037] Protein removal: Dissolve 50 mg of papain in 100 mL of purified water to obtain a papain solution. Add the papain solution to the polysaccharide solution, adding 3-5 mL of papain solution per 100 mL of polysaccharide. Heat at 55 °C for 1.5 h, then at 95 °C for 1.5 h. Centrifuge to remove the protein layer. Prepare Sevage reagent at a ratio of n-butanol to chloroform of 1:4 to remove protein from the crude polysaccharide. Shake and stir for 25 min, then separate the protein layer using a separatory funnel. Repeat this process three times until no obvious layering occurs. Collect the supernatant and centrifuge at 7000 rpm for 10 min at 4 °C. Concentrate the supernatant. Freeze-dry to obtain crude polysaccharide. W0 and W1 are the weights of Salvia miltiorrhiza extract powder and crude Salvia miltiorrhiza polysaccharide, respectively.

[0038] Crude polysaccharide extraction rate (%) = .

[0039] (5) Separation and purification of tanshinone oligosaccharides and polysaccharides DEAE-52 column separation of crude polysaccharides: Pretreatment of the packing material: Before use, the packing material is regenerated and swollen by soaking it in 3-5 times its volume of purified water for 1 hour. After swelling, discard the liquid and fine fiber fragments on top. Repeat this process 3 times, then place it in purified water for 24 hours to allow it to completely dissolve. Filter the purified water and set aside for later use.

[0040] Column packing equilibration: Add purified water to DEAE-52 packing material and sonicate for 20 min. Use a glass rod to guide the packing solution into a 50 cm × 1.6 cm chromatography column. Wait for the packing material to settle naturally and the upper layer solution to become clear. Turn on the peristaltic pump to press the column. After the volume of the packing material in the column no longer changes, let it stand overnight to reach equilibrium.

[0041] Column separation: Weigh 1 g of crude Salvia miltiorrhiza polysaccharide and dissolve it thoroughly in 50 mL of purified water. Centrifuge and filter using a 0.45 μm microporous membrane. Load the sample slowly using a pipette. Prepare a gradient elution solution of 0-1 mol / L sodium chloride (NaCl). Adjust the constant flow pump to 1 mL / min, collect 100 mL for each gradient concentration, and collect 5 mL of eluent into a collection bottle. Detect the absorbance using the phenol-sulfuric acid method and plot the elution curve. Collect the eluent of each fraction, concentrate it using a rotary evaporator, and centrifuge. Analyze fraction 1 using a 300 Da molecular weight dialysis bag for 24 h, and analyze the remaining fractions using a 3500 Da molecular weight dialysis bag for 24 h. Finally, freeze-dry to obtain oligosaccharide and polysaccharide fractions from different origins.

[0042] (6) Determination of crude polysaccharide, oligosaccharide and polysaccharide content by phenol-sulfuric acid method Standard curve preparation: Weigh 20 mg of standard and dissolve it in purified water to obtain a standard stock solution (1 mg / mL). Prepare standard solutions of 0, 20, 40, 60, 80, and 100 μg / mL, and dilute to volume in a 25 mL volumetric flask. Quickly add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid solution to 1 mL of the standard solution, shake well, and react for 20 min. Measure the absorbance of crude polysaccharide, oligosaccharide, and polysaccharide at 490 nm, using purified water as a blank for zeroing, and plot the standard curve. Determine the total sugar content of the samples: Weigh 20 mg of each different sugar sample and dissolve it in purified water. Determine the sugar content according to the above steps.

[0043] (7) Protein content detection Standard curve construction: Following the BCA kit procedure, BSA standard solutions were diluted to obtain concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0 μg / μL. In a 96-well plate, 25 μL of the standard solution was pipetted into each well, with three replicates for each concentration. 200 μL of BCA working solution was added to each well and mixed thoroughly. The plate was incubated at 37 °C for 30 min. After cooling to room temperature, the absorbance at 562 nm was measured within 5 min, and a standard curve was plotted based on the absorbance values. Protein content determination: Crude polysaccharide, oligosaccharide, and polysaccharide solutions were prepared. The protein content was determined according to the steps described above.

[0044] (8) Data statistics and analysis SPSS software was used to conduct an orthogonal multivariate design, and one-way ANOVA was performed on the experimental data.

[0045] Experimental results were plotted using GraphPad Prism 8.0, and differences between groups were analyzed using one-way ANOVA. *P<0.05 ; **P<0.01 ;***P<0.001 ; ****P<0.0001 .

[0046] II. Experimental Results (1) Collection of Salvia miltiorrhiza from different producing areas Based on textual research in herbal medicine and studies on the authenticity of products, six were selected ( Figure 1 Representative Salvia miltiorrhiza from different origins. Thick red roots are a distinguishing feature of Salvia miltiorrhiza; the roots are thick, and Salvia miltiorrhiza from the six origins has red root bark and purple interior. Salvia miltiorrhiza without root heads or fibrous roots is often of higher quality.

[0047] (2) Results of single-factor experiments 1. Effect of different material-to-liquid ratios on the yield of crude polysaccharides from Salvia miltiorrhiza Line graphs showing the yield of crude polysaccharides extracted from Salvia miltiorrhiza under different material-to-liquid ratios are shown below. Figure 2 As shown, the solid-liquid ratio is a crucial factor affecting the extraction rate of crude polysaccharides from *Salvia miltiorrhiza*. Changes in the solid-liquid ratio directly influence the contact degree between the solvent and the raw material, thus affecting the extraction rate of crude polysaccharides. The extraction rate of crude polysaccharides from *Salvia miltiorrhiza* initially increases and then decreases with increasing solid-liquid ratio. When the solid-liquid ratio is too small, the contact between the solvent and the crude polysaccharides is insufficient, resulting in incomplete dissolution and a low extraction rate. As the solid-liquid ratio increases, the polysaccharide concentration in the solvent becomes moderate, which is conducive to the continuous transfer of polysaccharides from the raw material to the solvent. When the solid-liquid ratio reaches a certain value, all the crude polysaccharides from *Salvia miltiorrhiza* are dissolved. Afterward, if the solid-liquid ratio is too large, excessive solvent will create a dilution effect, preventing further dissolution of polysaccharides and instead dissolving other water-soluble substances besides polysaccharides. Single-factor experiments show that the optimal solid-liquid ratio for crude polysaccharides from *Salvia miltiorrhiza* is 1:20 (g / mL).

[0048] 2. Effect of different extraction times on the yield of crude polysaccharides from Salvia miltiorrhiza Line graphs showing the yield of crude polysaccharides from Salvia miltiorrhiza at different extraction times are shown below. Figure 3 As shown, with increasing extraction time, the yield of crude polysaccharides from *Salvia miltiorrhiza* initially increased and then stabilized, with a slight decrease. Between 1 and 2 hours of extraction, the yield of crude polysaccharides continuously increased, but decreased slowly after 2 hours. In the initial stage of extraction, the crude polysaccharides dissolved as the solvent penetrated, increasing the extraction rate. When the extraction time reached 2 hours, the polysaccharide concentration reached a dynamic equilibrium, and the extraction rate no longer increased significantly. However, excessively long extraction times led to the degradation of crude polysaccharides in a high-temperature environment, potentially damaging the glycosidic bonds and resulting in a slight decrease in yield. Single-factor experiments indicated that the optimal extraction time for crude polysaccharides from *Salvia miltiorrhiza* was 2 hours.

[0049] 3. Effect of different extraction temperatures on the yield of crude polysaccharides from Salvia miltiorrhiza Line graphs showing the yield of crude polysaccharides from Salvia miltiorrhiza at different extraction temperatures are shown below. Figure 4As shown, extraction temperature significantly affects the yield of crude polysaccharides from *Salvia miltiorrhiza*. The extraction rate of crude polysaccharides from *Salvia miltiorrhiza* initially increases and then decreases with increasing temperature. When the temperature is in the range of 75-90 °C, the yield of crude polysaccharides from *Salvia miltiorrhiza* increases with increasing extraction temperature. Higher temperatures accelerate the movement of solvent molecules, enhance solvent permeability, and promote the dissolution of crude polysaccharides. When the temperature exceeds 90 °C, the crude polysaccharide molecules may undergo thermal degradation, breaking glycosidic bonds and molecular chains, leading to a decrease in the yield of crude polysaccharides from *Salvia miltiorrhiza* and a decrease in the extraction rate. Therefore, the single-factor experimental results indicate that the optimal extraction temperature for crude polysaccharides from *Salvia miltiorrhiza* is 90 °C.

[0050] 4. Effect of different extraction times on the yield of crude polysaccharides from Salvia miltiorrhiza Line graphs showing the content of crude polysaccharides in Salvia miltiorrhiza obtained under different extraction times are shown below. Figure 5 As shown, the crude polysaccharide content of Salvia miltiorrhiza increases with the number of extractions. The average yield of crude polysaccharides after one extraction is 18.69%. After two and three extractions, the yield of crude polysaccharides of Salvia miltiorrhiza is greater than 20%, but the difference in yield is small. Multiple extractions are time-consuming and energy-intensive, so two extractions were ultimately chosen.

[0051] (3) Results of orthogonal experiment Based on the results of single-factor experiments, temperature (A), time (B), and material-to-liquid ratio (C) were determined as the orthogonal experimental factor levels. The extraction rate of crude polysaccharides from *Salvia miltiorrhiza* obtained by analyzing sugar content using the phenol-sulfuric acid colorimetric method was used as the evaluation index, and experiments were conducted according to the orthogonal experimental factor table. The orthogonal experimental data were processed using an intuitive analysis method, and each group of experiments was repeated a certain number of times to reduce error. The extraction rates of crude polysaccharides from *Salvia miltiorrhiza* under different factor levels were statistically analyzed, and the range R was compared. The larger the range, the more significant the effect of the factor on the extraction rate. The orthogonal experimental results are shown in Table 5. The results show that the degree of influence of each factor on the extraction rate is A > C > B, that is, the influencing factors of water extraction and alcohol precipitation of crude polysaccharides from *Salvia miltiorrhiza* are, in order: temperature, material-to-liquid ratio, and time. A2B3C2 represents the optimal extraction process: extraction twice at a temperature of 90 ℃, an extraction time of 2.5 h, and a material-to-liquid ratio of 1:20 (g / mL).

[0052] Table 5 L9 (3) 4 Orthogonal Experiment Results Table

[0053] Table 6 Analysis of Variance Table

[0054] Note: * indicates a significant difference ( P<0.05 ) The experiment was repeated three times under these optimal extraction conditions to verify the results. The extraction rate of unpurified crude polysaccharides from Salvia miltiorrhiza was 22.15 ± 0.12%, confirming it as the optimal combination.

[0055] (4) Separation, purification and analysis of oligosaccharides and polysaccharides from different origins of Tanshinone The separation and purification of crude polysaccharides from different origins were performed using a DEAE-52 cellulose column. (See the NaCl gradient elution diagrams for crude polysaccharides from different origins.) Figure 6 .

[0056] The DEAE-52 column separation spectra of *Salvia miltiorrhiza* from different origins were similar, with five basic peak concentrations: water, 0.2 M, 0.3 M, 0.4 M, and 0.5 M. Some origins showed a very small peak at 0.1 M. Peak shapes from Sichuan, Shandong, Hebei, and Henan were highly similar, while the peak at 0.5 M from Shaanxi was extremely small, and a large sugar component peak appeared at 0.2 M from Shanxi. This indicates that *Salvia miltiorrhiza* from different origins has similar elution peaks, unlike other traditional Chinese medicine species. However, the content of sugar components at different molecular weights varies.

[0057] The sugars eluted from water at each origin were subsequently identified as low molecular weight sugars and named oligosaccharides (SMO). The sugars eluted from the 0.2 M, 0.3 M, 0.4 M, and 0.5 M fractions were high molecular weight polysaccharides. Among them, the polysaccharide eluted with 0.3 M NaCl showed the highest extraction rate and sugar content, and was named polysaccharide (SMP). Physicochemical and pharmacological effects of the four polysaccharide fractions from the six origins were compared, and the polysaccharide eluted with 0.3 M NaCl exhibited better pharmacological activity. Therefore, the sugars eluted with water and 0.3 M NaCl were selected for more detailed research.

[0058] Example 2: UPLC LTQ-Orbitrap MS / MS analysis of crude polysaccharide components in Salvia miltiorrhiza from different origins I. Experimental Methods: Sample preparation: After centrifuging and lyophilizing 1.5 mL of liquid sample, add 1.5 mL of methanol and swirl until completely dissolved. Add 3 mL of dichloromethane and incubate at room temperature for 1 h. Add 1.25 mL of water and let stand at room temperature for 10 min. After drying, add 200 µL of a mixture (dichloromethane:methanol:water = 60:30:4.5). Take 250 µL of sample and add 750 µL of acetonitrile. Mix well and centrifuge at 13000 rpm for 10 min at 4 ℃. Take the supernatant for liquid chromatography-mass spectrometry (LC-MS).

[0059] Chromatographic conditions: Waters UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm); mobile phase: 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B); flow rate: 0.3 mL / min; column temperature: 40 ℃; injection volume: 5 µL; data acquisition in positive ion mode.

[0060] The elution method is as follows: Table 7 Elution methods for UPLC LTQ-Orbitrap MS / MS

[0061] Progenesis QI software was used to perform peak alignment and extraction on the RAW format data from the instrumentation process, with deconvolution performed using QI's default settings. The data exported from QI was further analyzed and filtered using R software, and the peaks were identified using R packages and the HMDB database.

[0062] II. Data Analysis UPLC LTQ-Orbitrap MS / MS (Ultra-High Performance Liquid Chromatography with Linear Ion Trap) is a novel analytical technique that combines ultra-high performance liquid chromatography (UPLC) and high-resolution mass spectrometry (LTQ-Orbitrap MS / MS). It is widely used for the qualitative and quantitative analysis of complex samples. Based on rapid and efficient chromatographic separation, it provides analytes with accurate molecular weights through multi-stage mass spectrometry, overcoming the limitations of traditional LC-MS and demonstrating significant advantages in screening unknown components in complex matrices. The Orbitrap mass analyzer provides extremely high mass accuracy (typically less than 1 ppm), enabling accurate determination of compound molecular weights. The linear ion trap (LTQ) enables multi-stage mass spectrometry analysis, providing structural information about the compounds. Therefore, this method is particularly suitable for analyzing multiple components in complex matrices of natural products.

[0063] Crude polysaccharides from *Salvia miltiorrhiza* are complex biomacromolecules, and their composition and structure vary depending on their source, origin, and extraction method. The components of crude polysaccharides from different origins were qualitatively and quantitatively analyzed using UPLC LTQ-Orbitrap MS / MS. High-resolution mass spectrometry data, combined with the R package and HMDB database, were used to identify peaks and determine the types of monosaccharides and oligosaccharides. Fragment ion information was used to infer the linkage patterns and structures of the sugars. The target sugar components were quantified using the internal standard method. The differences in the content of various sugar components in crude polysaccharides from different origins were compared. The results are as follows: the sugar components in crude polysaccharides from different origins are similar; the total ion chromatogram is shown below. Figure 7-12 .

[0064] Six sugar fragments with significantly different contents were detected (Table 8). The molecular weight and degree of polymerization of the polysaccharides can be determined by measuring the molecular ion peaks in primary mass spectrometry (PMS). Multi-stage mass spectrometry (MS / MS) utilizes characteristic fragment ions to resolve the monosaccharide composition, sequence structure, glycosidic bond configuration, and other structural features of the polysaccharides. Each monosaccharide residue has a different molecular weight, and specific fragment ions are generated during mass spectrometric fragmentation. Analysis showed that fragments 1-3, representing differences in origin, are mainly 1,6-linked β-D-glucan oligosaccharides, while fragments 4-6 are mainly 1,6-linked β-D-linear glucan oligosaccharides and galactooligosaccharides, possibly containing an α-configuration at the end. β-configured sugars often exhibit higher activity, with higher branching complexity indicating stronger activity. Oligosaccharide fragments have complex structures and numerous isomers; only a preliminary analysis was performed here, and only one possible structural diagram of each differential fragment was drawn. The specific structures should be further analyzed after separation and purification. The structural diagrams of the differential fragments and the primary and secondary mass spectra are shown below. Figure 13-18 .

[0065] Table 8. Information on Differential Glycofragments

[0066] Six differentially expressed sugar fragments were screened from crude polysaccharides of Salvia miltiorrhiza from different origins using UPLC LTQ-Orbitrap MS / MS. The types and contents of these differentially expressed sugar fragments varied across origins. Fragments 1-3 were mainly 1,6-linked β-D-glucan oligosaccharides, while fragments 4-6 were mainly 1,6-linked β-D-linear glucan oligosaccharides and galactooligosaccharides. The contents of differentially expressed sugar fragments 1, 2, and 3 were low in those from Shandong and Sichuan origins. β-configured sugars and complexly branched sugars often exhibited better activity.

[0067] Example 3: UV-Vis spectral detection of oligosaccharides and polysaccharides from different origins of Tanshinone. I. Experimental Methods: SMO and SMP solutions of Salvia miltiorrhiza from different origins were prepared with purified water at a concentration of 0.5 mg / mL, and the ultraviolet absorption spectra of the sample solutions were detected in the wavelength range of 200 nm to 800 nm.

[0068] II. Data Analysis Results of UV spectra of SMO and SMP from different origins are as follows Figure 19 , Figure 20 As shown, the absorption peaks of nucleic acids and proteins are generally at 260 nm and 280 nm. SMO and SMP from different origins do not have obvious absorption peaks at 260 nm and 280 nm, indicating that SMO and SMP contain almost no nucleic acids and proteins, but may contain trace amounts of glycoproteins.

[0069] Crude polysaccharides from Salvia miltiorrhiza from different origins were separated into SMO and SMP by column chromatography. Ultraviolet spectrophotometry analysis of SMO and SMP showed that they did not contain nucleic acids or proteins.

[0070] Example 4: Fourier transform infrared spectroscopy detection of oligosaccharides and polysaccharides from different origins of Tanshinone. I. Experimental Methods SMO and SMP samples from different origins were ground and compressed into thin sections using potassium bromide (KBr). The samples were then scanned at 4000-500 cm⁻¹. -1 Fourier transform infrared (FT-IR) spectra were obtained for SMO and SMP from different origins within the specified wavelength range.

[0071] II. Data Analysis Functional groups were analyzed using FT-IR, such as Figure 21 , Figure 22 As shown, SMO and SMP of tanshinone from different origins have characteristic absorption peaks of sugar.

[0072] SMP infrared absorption peaks from different origins are similar, around 3300 cm⁻¹. -1 The broad peak at 2937 cm⁻¹ is the stretching vibration peak of the hydroxyl group (OH). The presence of numerous intramolecular hydrogen bonds causes the stretching vibration peak of the hydroxyl group to appear at a lower wavenumber, resulting in a broader peak shape and higher intensity. -1 The peak at 1736 cm⁻¹ corresponds to the stretching vibration of the methyl methylene group (CH₂O), indicating the presence of numerous methylene groups within the glucose ring. -1 The sharp peak is the stretching vibration peak of the carbonyl C=O group, indicating that the sample contains uronic acid. (1596 cm⁻¹) -1 The peak at 1417 cm⁻¹ corresponds to the bending vibration of the hydroxyl group (OH). -1 The peak at 1143 cm⁻¹ represents the CH bending vibration. -1 and 1049 cm -1 This peak represents the stretching vibration of the numerous cyclic ether bonds and hydroxyl groups (CO) in glucose. (891 cm⁻¹) -1 This indicates the presence of a β configuration.

[0073] SMO from different origins exhibits similar infrared absorption peaks, but differs from SMP. At 3310 cm⁻¹... -1 The peak at 3236 cm⁻¹ represents the stretching vibration of the hydroxyl group (OH). -1 This is another characteristic peak for hydroxyl groups. 2965 cm⁻¹ -1 The peak at 1647 cm⁻¹ represents the stretching vibration of the methyl methylene group (CH₂O). Glucose contains a relatively large number of methylene groups, indicating the presence of glucose in the SMO sample. The absence of carbonyl characteristic peaks in the SMO sample's infrared spectrum indicates the absence of uronic acid in the sample. -1 The peak at 1418 cm⁻¹ corresponds to the bending vibration of the hydroxyl group (OH). -1The peak at 1144 cm⁻¹ represents the CH bending vibration, and its intensity is lower compared to the characteristic peak of the SMP. -1 This is the stretching vibration peak of the COC cyclic ether bond. 1042 cm⁻¹ -1 The peak at 984 cm⁻¹ represents the stretching vibration of hydroxyl CO. -1 The peak at 878 cm⁻¹ represents the stretching vibration of another hydroxyl group (CO). -1 The structure of the glucose ring undergoes CC deformation and vibration.

[0074] Example 5: Molecular weight determination of oligosaccharides and polysaccharides from different origins of Tanshinone. I. Experimental Methods The molecular weights of oligosaccharides and polysaccharides were determined by high-performance gel permeation chromatography (HPGPC).

[0075] Preparation of mobile phase solution: Prepare 0.05 M NaCl solution by accurately weighing 5.844 g NaCl, adding ultrapure water to a final volume of 2 L, sonicating, passing through a membrane, and transferring to a container.

[0076] Sample and standard solution preparation: Prepare 5 mg / mL standard (pullulan) and sample solutions, respectively, vortex to dissolve, and centrifuge at 12000 rpm for 10 min. Filter the supernatant through a 0.22 µm aqueous microporous membrane, and then transfer the sample to a 1.8 mL vial.

[0077] Chromatographic conditions: Mobile phase: 0.05 M NaCl solution; Column: BRT105-103-101 (8×300 mm); Flow rate: 0.7 mL / min; Column temperature: 40 ℃; Injection volume: 25 µL; Detector: Differential detector RID-20A; Analyze the standards and plot standard curves based on retention times: lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight-average molecular weight), and lgMn-RT (Mn number-average molecular weight). Analyze the samples, obtain chromatograms, and substitute the retention times into the standard curves to obtain the molecular weights Mp, Mw, and Mn.

[0078] Data Analysis Molecular weight was determined by HPGPC, and calibration curves were calculated based on the standard curves. The equations for the lgMp-RT calibration curves are: y = -0.1951x + 11.661, R² = 0.9956; the equations for the lgMw-RT calibration curves are: y = -0.1973x + 11.738, R² = 0.9956; and the equations for the lgMn-RT calibration curves are: y = -0.1964x + 11.7, R² = 0.9955. The molecular weight of each sample was calculated based on the standard curves. As shown in Table 9, the molecular weights of SMO from different origins range from 800 to 1200 Da, indicating they are low molecular weight oligosaccharides. Figure 23 As shown, SMO from Salvia miltiorrhiza from different origins exhibited a single peak with similar peak shapes when measured by HPGPC.

[0079] Table 9. Molecular weight determination results of SMO from different origins

[0080] Note: Mn is the number-average molecular weight, Mp is the peak molecular weight, and Mw is the weight-average molecular weight. The unit of molecular weight is Daltons.

[0081] As shown in Table 10, the molecular weight of SMP in Salvia miltiorrhiza from different origins is around 30,000 Da, which is a high molecular weight polysaccharide. Figure 24 As shown, SMP from different origins of Salvia miltiorrhiza showed two peaks by HPGPC analysis. The peak shapes were similar, and the molecular weight was concentrated in two fragments. The retention times were 30.279 min and 36.51 min, respectively (taking Sichuan as an example). The retention time of 36.51 min accounted for a very high proportion, and the peak area ratio was above 90%.

[0082] Table 10. Molecular weight determination results of SMP from different origins

[0083] Note: Mn is the number-average molecular weight, Mp is the peak molecular weight, and Mw is the weight-average molecular weight. The unit of molecular weight is Daltons.

[0084] The molecular weight of SMP from different origins is around 30,000 Da, indicating it is a polysaccharide. Monosaccharide composition analysis shows that SMO from different origins consists of five monosaccharides: galactose, glucose, mannose, arabinose, and xylose. Specifically, SMO from Shaanxi province consists of three monosaccharides: galactose, glucose, and arabinose. SMP from different origins consists of seven monosaccharides: galacturonic acid, galactose, arabinose, rhamnose, glucose, xylose, and fucose. While the types of monosaccharides in Tanshinone from different origins are similar, the proportions differ, which has a certain impact on its biological activity. Furthermore, this chapter only provides a preliminary structural analysis of SMO; unidentified oligosaccharide components may be present in Tanshinone SMO for further research.

[0085] Example 6: Monosaccharide composition analysis of oligosaccharides and polysaccharides from different origins of Tanshinone. I. Experimental Methods Monosaccharide composition was determined using ion chromatography. Carbohydrates are weak acids with pKa > 11, exhibiting electrochemical activity and existing as anions in strongly alkaline solutions. Different carbohydrates have different pKas, resulting in variations in ion exchange activity and hydrophobic interactions between the carbohydrates and anion exchange resins. The hydroxyl groups in the carbohydrate molecule can undergo oxidation reactions on the electrode surface, generating an electric current. Ion chromatography utilizes these principles for detection. The elution process is shown in Table 11.

[0086] Table 11 Elution Procedure for Ion Chromatography

[0087] Reagent preparation: Prepare 500 mM NaOH and 50 mM NaOAc solutions by accurately weighing 20.5 g of NaOAc and adding 1.3 mL of 50% NaOH solution to ultrapure water to a final volume of 500 mL; prepare 20 mM NaOH solution by adding 1.04 mL of 50% NaOH solution to ultrapure water to a final volume of 1 L.

[0088] Preparation of standard solutions: Accurately weigh 5 mg of each of the 11 monosaccharide standards (Table 12) and place them in ampoules. Add 5 mL of purified water and vortex to mix. Accurately prepare a mixed standard of a certain concentration. Determine the mass of different monosaccharides using an absolute quantitative method, and then calculate their molar ratio based on the molar mass of the monosaccharides.

[0089] Sample preparation: Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3 M trifluoroacetic acid, and hydrolyze at 120 °C for 3 h. Dry the hydrolysate in a nitrogen evaporator, add 5 mL of purified water and vortex to mix. Pipette 100 µL of the solution into 900 µL of deionized water and centrifuge at 12000 rpm for 5 min. Take the supernatant for analysis.

[0090] Chromatographic conditions: Column: Dionex Carbopac™ PA10 (4×250 mm); Mobile phase: A: H2O; B: 500 mM NaOH & 50 mM NaOAc; C: 20 mM NaOH; Flow rate: 1 mL / min; Injection volume: 25 µL; Column temperature: 30 ℃; Detector: Electrochemical detector.

[0091] Table 12 Standard Sample Sequence

[0092] Note: In the formula, C1 and A1 are samples, and C0 and A0 are standards.

[0093] II. Data Analysis Monosaccharide composition was determined using ion chromatography. Figure 25 The chromatogram of mixed standard 11 sugar ions shows, from left to right, fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, ribose, galacturonic acid, and glucuronic acid.

[0094] The monosaccharide composition results of SMO from different origins are shown in the figure. Figure 26 Table 13 shows that SMO from different origins consists of five sugars: galactose, glucose, mannose, arabinose, and xylose. SMO from Shaanxi province does not contain mannose or xylose. The monosaccharide composition results for SMP from different origins are shown in [Table 13]. Figure 27 Table 14 shows that SMP from different origins consists of seven components: galacturonic acid, galactose, arabinose, rhamnose, glucose, xylose, and fucose. While the monosaccharide composition is similar across different origins, their content varies.

[0095] Table 13 Analysis of SMO monosaccharide composition from different origins

[0096] Table 14. Analysis of SMP monosaccharide composition from different origins

[0097] By analyzing the differentially expressed sugar fragments of crude polysaccharides from Salvia miltiorrhiza from different origins, as well as the composition and structure of SMO and SMP monosaccharides from Salvia miltiorrhiza from different origins, it was found that the structure of sugars has a certain influence on their physiological functions. Therefore, in order to determine the relationship between structure and biological activity, it is necessary to study the structure of sugars to understand their properties. This lays the foundation for subsequent research on Salvia miltiorrhiza.

Claims

1. Application of crude polysaccharide, oligosaccharide or polysaccharide of tanshinone in the identification of tanshinone from different origins.

2. The application as described in claim 1, characterized in that, The crude polysaccharide from Salvia miltiorrhiza is obtained by water extraction and alcohol precipitation. The extraction process for the crude polysaccharide from Salvia miltiorrhiza is as follows: extraction temperature 90 ℃, extraction time 2.5 h, and extraction twice at a material-to-liquid ratio of 1:20 g / mL.

3. The application as described in claim 1, characterized in that, The oligosaccharides of tanshinone are low molecular weight sugars obtained by eluting crude tanshinone with water using a DEAE-52 cellulose column, while the polysaccharides of tanshinone are polysaccharides obtained by eluting crude tanshinone with NaCl solution using a DEAE-52 cellulose column.

4. The application as described in claim 1, characterized in that, Mass spectrometry was used to characterize the glycosides from different origins of Salvia miltiorrhiza. There were six differentially expressed glycoside fragments. Differentially expressed glycoside fragments 1-3 were mainly 1,6-linked β-D-glucan oligosaccharides, and differentially expressed glycoside fragments 4-6 were mainly 1,6-linked β-D-linear glucan oligosaccharides and galacto-oligosaccharides.

5. The application as described in claim 1, characterized in that, The molecular weight of SMP in Salvia miltiorrhiza from different origins was determined by high-performance gel permeation chromatography (HPLC). The origin of Salvia miltiorrhiza was determined based on the differences in molecular weight.

6. The application as described in claim 1, characterized in that, The molecular weight of SMO in Salvia miltiorrhiza from different origins was determined by high-performance gel permeation chromatography, and the origin of Salvia miltiorrhiza was determined based on the differences in molecular weight.

7. The application as described in claim 1, characterized in that, Ion chromatography was used to detect the SMO monosaccharide components of Salvia miltiorrhiza from different origins, and the origin was determined based on the different monosaccharide components.

8. The application as described in claim 1, characterized in that, Ion chromatography was used to detect the SMP monosaccharide components in Salvia miltiorrhiza from different origins, and the origin of Salvia miltiorrhiza was determined based on the proportion of SMP monosaccharide content in Salvia miltiorrhiza from different origins.

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