Application of tribulus terrestris polysaccharide

Abstract

The invention belongs to the technical field of pharmaceutical chemistry and natural product separation, and provides application of tribulus terrestris polysaccharide. The tribulus terrestris polysaccharide can be used for preparing drugs for treating hypertension. The tribulus terrestris polysaccharide has the effects of reducing blood pressure, relieving vascular endothelium pathological injury, improving vascular endothelium dysfunction and protecting vascular endothelium on a hypertensive rat model. The treatment effect of the fried tribulus terrestris polysaccharide is obviously superior to that of the tribulus terrestris polysaccharide, and meanwhile, the blood pressure reducing effect of the fried tribulus terrestris polysaccharide is not obviously different from that of a positive medicine valsartan. A new material basis is provided for development of antihypertensive drugs, and meanwhile the medicinal value of the tribulus terrestris polysaccharide is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry and natural product separation, and particularly relates to a tribulus terrestris extract. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Tribulus terrestris Tribulus terrestris The dried mature fruit of L. is pungent, bitter, slightly warm, and slightly toxic. It enters the liver meridian and has the effects of calming the liver and relieving depression, promoting blood circulation and dispelling wind, improving eyesight, and relieving itching. It is often used to treat headaches and dizziness, chest and flank pain, breast closure and abscesses, conjunctivitis and cataracts, and urticaria and itching. Modern research shows that Tribulus terrestris mainly contains chemical components such as saponins, polysaccharides, flavonoids, and alkaloids, which have pharmacological activities such as lowering blood pressure and blood lipids, anti-tumor and antioxidant properties.

[0004] In recent years, research on the active ingredients of Tribulus terrestris has primarily focused on saponins and flavonoids. Specifically regarding the antihypertensive effects of Tribulus terrestris, it is widely believed that steroidal saponins, converted during stir-frying, are the underlying material basis for stir-fried Tribulus terrestris's enhanced therapeutic efficacy for essential hypertension. Polysaccharides are macromolecular polymers, typically composed of multiple monosaccharides linked by glycosidic bonds, with molecular weights reaching hundreds of thousands or even millions. A literature search revealed that polysaccharides isolated from traditional Chinese medicine (TCM) have become a research hotspot due to their diverse biological activities, including antioxidant, immunomodulatory, anti-tumor, and anti-inflammatory properties. While studies have examined the antihypertensive effects of Dendrobium and Polygonatum polysaccharides, there are no reports on the antihypertensive effects of Tribulus terrestris polysaccharides. Traditional Chinese medicines often exhibit synergistic effects after processing. The present invention demonstrates that stir-fried Tribulus terrestris polysaccharides exhibit superior antihypertensive effects compared to raw Tribulus terrestris polysaccharides, providing a new material basis for the development of antihypertensive drugs and expanding the medicinal value of Tribulus terrestris polysaccharides. Summary of the Invention

[0005] In view of the problems in the prior art, the present invention provides a Tribulus terrestris extract, which uses Tribulus terrestris polysaccharide as an active substance, has good pharmacological activity and a simple preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] Application of Tribulus terrestris polysaccharide in preparing medicine for treating hypertension.

[0008] The preparation method of the tribulus terrestris polysaccharide comprises the following steps: precipitating the water extract of tribulus terrestris with ethanol, and purifying the precipitation to obtain the polysaccharide.

[0009] The method of obtaining Tribulus terrestris polysaccharides only affects the extraction rate. Without affecting the composition, a variety of auxiliary extraction methods can be used to improve the extraction rate, such as microwave-assisted extraction and ultrasound-assisted extraction.

[0010] The tribulus terrestris is tribulus terrestris or fried tribulus terrestris.

[0011] The purification method is to remove proteins and dialysis to remove small molecules; the protein removal method is preferably the Sevage method; the dialysis membrane is preferably 3000 Da. Specifically, the above preparation method includes the following steps: The tribulus terrestris is crushed and then water is added for ultrasonic-assisted extraction. After solid-liquid separation, water is added again for ultrasonic-assisted extraction for 1-4 times. After the extracts are combined, ethanol is added to a volume fraction of ethanol of 75%-95% to obtain a precipitate, and the precipitate is purified and dried to obtain the product.

[0012] Preferably, the above preparation method comprises the following steps: After the Tribulus terrestris is crushed, water is added according to the liquid-to-solid ratio of (5-25):1 mL / g, and ultrasonic-assisted extraction is performed at a temperature of 40°C-80°C and a time of 30-70 min. After extraction, the solid and liquid are separated, and the solid is repeatedly extracted 1-4 times, and the extract is obtained after merging. After the extract is concentrated, ethanol is added to a volume fraction of 75%-95% to obtain a precipitate, which is then purified and dried.

[0013] More preferably, the above preparation method comprises the following steps: After the Tribulus terrestris is crushed, water is added according to the liquid-to-solid ratio of (15-25): 1 mL / g, and ultrasonic-assisted extraction is performed at a temperature of 50°C-70°C and a time of 40-60 min. After extraction, the solid and liquid are separated, and the solid is extracted again once. The combined extracts are concentrated, and then ethanol is added to the concentrate to a volume fraction of 85%-95% to obtain a precipitate. The precipitate is then purified and dried to obtain Tribulus terrestris polysaccharide.

[0014] Most preferably, the above preparation method comprises the following steps: After the Tribulus terrestris was crushed, water was added at a liquid-to-solid ratio of 20:1 mL / g, and ultrasonic-assisted extraction was performed at a temperature of 62°C and a time of 51 min. After extraction, the solid and liquid were separated, and the solid was extracted again once. The combined extracts were concentrated, and then ethanol was added to the concentrate to a volume fraction of 95%. A precipitate was obtained at 4°C, and the precipitate was purified and dried to obtain Tribulus terrestris polysaccharide.

[0015] The present invention has the following advantages: The present invention utilizes conventional water extraction, alcohol precipitation, protein removal, and dialysis to obtain Tribulus terrestris polysaccharides, eliminating the need for complex processes and achieving a high extraction yield. The obtained Tribulus terrestris polysaccharides have the effects of lowering blood pressure, alleviating vascular endothelial pathological damage, improving vascular endothelial dysfunction, and protecting the vascular endothelium in a hypertensive rat model. Furthermore, it was unexpectedly discovered that the therapeutic effect of stir-fried Tribulus terrestris polysaccharides was significantly superior to that of stir-fried Tribulus terrestris polysaccharides, and that the antihypertensive effect of stir-fried Tribulus terrestris polysaccharides was not significantly different from that of the active drug valsartan. This provides a new material basis for the development of antihypertensive drugs and expands the medicinal value of Tribulus terrestris polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the glucose standard curve; Figure 2 The effects of different extraction parameters on the extraction rate of Tribulus terrestris polysaccharides; A is different extraction times, B is different liquid-to-solid ratio, C is different ultrasonic time, D is different ultrasonic temperature, and E is different alcohol precipitation concentration; Figure 3 This is the HPSEC spectrum of Tribulus terrestris polysaccharide; wherein FP is Tribulus terrestris polysaccharide, and SFP is stir-fried Tribulus terrestris polysaccharide; Figure 4 is the infrared spectrum of Tribulus terrestris polysaccharide; wherein, FP is Tribulus terrestris polysaccharide, and SFP is stir-fried Tribulus terrestris polysaccharide; Figure 5 This is a scanning electron microscope image of Tribulus terrestris polysaccharide; FP is Tribulus terrestris polysaccharide, and SFP is stir-fried Tribulus terrestris polysaccharide; Figure 6 is the systolic blood pressure of rats in each group; compared with the blank group, # P <0.05, ## P <0.01; compared with the model group, P <0.05, P <0.01; compared with the positive drug group, P <0.05, P <0.01; compared with the Tribulus terrestris polysaccharide group, ▲ P <0.05, ▲▲ P <0.01; Figure 7 is the content of NO, eNOS, Ang Ⅱ and ET-1 in the serum of rats in each group; compared with the blank group, # P <0.05, ## P <0.01; compared with the model group, P<0.05, P <0.01; compared with the Tribulus terrestris polysaccharide group, ▲ P <0.05, ▲▲ P <0.01; Figure 8 The effect of Tribulus terrestris polysaccharide on vascular tissue morphology (A) and collagen deposition (B) in SHR rats; the red arrows indicate endothelial cell shedding, the black arrows indicate vascular endothelial damage and incompleteness, and the yellow arrows indicate disordered arrangement of smooth muscle cells; compared with the blank group, # P <0.05, ## P <0.01; compared with the model group, P <0.05, P <0.01; compared with the Tribulus terrestris polysaccharide group, ▲ P <0.05, ▲▲ P <0.01. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0018] Example 1 Extraction of Tribulus terrestris polysaccharide 1. Determination of polysaccharide content The phenol-sulfuric acid method was used to determine the polysaccharide content in Tribulus terrestris. Add an appropriate amount of glucose to a volumetric flask, dilute to volume, and shake well to prepare a glucose standard solution (0.1 mg / mL). Place six test tubes in a test tube rack. Accurately measure 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution and add appropriate amounts of distilled water. Add 6% phenol solution and concentrated sulfuric acid. Mix thoroughly and place in a boiling water bath. After cooling, measure the absorbance at 490 nm and plot a glucose standard curve.

[0019] Glucose standard curve Figure 1 As shown, the linear regression equation is: y=16.631x+0.0148, R 2 =0.9992, indicating that the linear relationship is good in the range of 0.01~0.05 mg / mL.

[0020] 2. Single-factor experiment Weigh the Tribulus terrestris powder into a beaker, add distilled water to a certain liquid-to-solid ratio, ultrasonically extract in an ultrasonic cleaner at a set temperature for a period of time, centrifuge and filter, and collect the filtrate; repeat the above operation, combine the filtrates and concentrate them by rotary evaporation to an appropriate volume, add anhydrous ethanol to a certain volume fraction (alcohol precipitation concentration), perform alcohol precipitation overnight at 4°C, centrifuge, discard the supernatant, take the precipitate, and dry to obtain Tribulus terrestris polysaccharide (crude polysaccharide).

[0021] Under the above conditions, the effects of five factors on the extraction efficiency of Tribulus terrestris polysaccharides were investigated: extraction times (1, 2, 3, 4, and 5), ultrasonic time (30, 40, 50, 60, and 70 min), ultrasonic temperature (40, 50, 60, 70, and 80°C), liquid-to-solid ratio (5:1, 10:1, 15:1, 20:1, and 25:1 mL / g), and alcohol precipitation concentration (75%, 80%, 85%, 90%, and 95%). Three parallel experiments were conducted at each level.

[0022] Take an appropriate amount of Tribulus terrestris polysaccharide and dilute it with distilled water to prepare a polysaccharide solution of a certain concentration. The polysaccharide content in the crude Tribulus terrestris polysaccharide is determined by the phenol-sulfuric acid method. The extraction rate of Tribulus terrestris polysaccharide is calculated according to the following formula: Polysaccharide extraction rate ( Y )= Where, ρ ——polysaccharide concentration (mg / mL); V ——volume (mL); n ——dilution multiple; M ——Mass of Tribulus terrestris powder (g).

[0023] (1) Effect of extraction times on the extraction rate of Tribulus terrestris polysaccharides Depend on Figure 2 A concluded that with increasing extraction times, the extraction rate of Tribulus terrestris polysaccharides initially increased and then slightly decreased. The extraction rate of Tribulus terrestris polysaccharides reached 1.88% during the second extraction and reached a maximum of 1.90% during the fourth extraction. However, increasing the number of extractions had little effect on the polysaccharide extraction rate. Considering practical conditions and economic costs, the response surface experiment eliminated the number of extractions and fixed the number of extractions at two.

[0024] (2) Effect of liquid-solid ratio on the extraction rate of Tribulus terrestris polysaccharides Depend on Figure 2 B knows that under this condition, Y The range is 1.48%~2.08%. Y In the range of 5:1 (mL / g) ~ 25:1 (mL / g), it shows a trend of first increasing and then decreasing. When the liquid-to-solid ratio is 20:1 (mL / g),Y The highest was 2.08%. Therefore, liquid-to-solid ratios of 15:1 (mL / g), 20:1 (mL / g), and 25:1 (mL / g) were selected for subsequent response surface optimization experiments.

[0025] (3) Effect of ultrasonic time on the extraction rate of Tribulus terrestris polysaccharides Depend on Figure 2 C shows that when the ultrasound time is 50 min, Y The maximum value reached 2.04%. In this study, ultrasound for 40 min, 50 min, and 60 min was selected for subsequent response surface optimization experiments.

[0026] (4) Effect of ultrasonic temperature on the extraction rate of Tribulus terrestris polysaccharides Depend on Figure 2 D The effect of ultrasonic temperature on the extraction rate of Tribulus terrestris polysaccharide Y The range is 1.67%~1.99%, Y In the range of 30℃~70℃, the ultrasonic temperature increases and then decreases. When the ultrasonic temperature is 60℃, Y Reach the maximum value. In the range of 60℃~70℃, although Y It showed a downward trend, but compared with other temperature conditions, it was still at a high level. When extracting Tribulus terrestris polysaccharide, the temperature was 50℃, 60℃, and 70℃. Y Therefore, temperatures of 50°C, 60°C, and 70°C were selected for subsequent response surface optimization experiments.

[0027] (5) Effect of alcohol precipitation concentration on the extraction rate of Tribulus terrestris polysaccharides Depend on Figure 2 E shows that as the concentration of alcohol precipitation increases Y It also showed an increasing trend and reached a maximum value of 2.17% when the alcohol precipitation concentration was 95%. Y exist D The growth rate is fastest when the alcohol precipitation concentration is 80%~85%, and the growth rate is slowest when the alcohol precipitation concentration is 75%~80%. Y Therefore, alcohol precipitation concentrations of 85%, 90%, and 95% were selected for subsequent experiments.

[0028] 3. Response Surface Experiment Based on the single factor experiment, a response surface experiment with four factors and three levels was conducted. Y Response values ​​were used, and three optimal levels (A, B, C, and D) were selected. A regression equation was fitted to optimize the extraction process for Tribulus terrestris polysaccharides. Each group was repeated three times to minimize error and determine an accurate extraction process for Tribulus terrestris polysaccharides. The response surface design factors and levels are shown in Table 1.

[0029] Table 1 Response surface experimental factor design (1) Response surface methodology experiment on the extraction rate of Tribulus terrestris polysaccharides As can be seen from Table 2, a total of 29 groups of experiments were carried out, including 5 central replicates to reduce errors and improve the reliability of the experiments; at the same time, the highest polysaccharide extraction rate was 2.16% and the lowest was 1.81%.

[0030] Table 2 Response surface Box-Behnken design and experimental results (2) Variance analysis of the response surface regression model for the extraction rate of Tribulus terrestris polysaccharides The extraction rate of Tribulus terrestris polysaccharides varies due to different experimental conditions, ranging from 1.81% to 2.16%. The quadratic polynomial regression equation for ultrasonic extraction of Tribulus terrestris polysaccharides was obtained by data analysis: Y (%) = 2.14 + 0.04 A +0.06 B +0.03 C +0.08 D -0.01 AB +0.02 AC -0.02 AD +0.02 BC -0.04 BD +0.02 CD -0.14 A ²-0.11 B ²-0.12 C ² Where, A is the liquid-to-solid ratio (mL / g); B is the ultrasound time (min); C is the ultrasonic temperature (°C); D is the alcohol precipitation concentration (%); Y Represents the extraction rate (%).

[0031] The response surface variance analysis results are shown in Table 3. The lack-of-fit term is 0.3308 (>0.05), which is not significant, indicating that the equation is significant. 2 =0.9760, indicating that the model fits well and the equation can predict the actual situation. 2 =0.9521, about 95.21% of the experimental data can be explained by this model, there is 4.79% of the experimental error that this model cannot explain, the signal-to-noise ratio of this model is 19.5735 (>4), indicating that the degree of interference is small. As can be seen from the table below, the response surface regression model under investigation is extremely significant ( P<0.01), indicating that these four variables have Y The impact is significant. BD right Y It shows a very significant effect, while the interaction of other items is not strong. According to the F value, it is inferred that the four experimental variables have a significant effect on Y The order of significance of the impact degree is: D>B>A>C.

[0032] Table 3 Analysis of variance table of response surface experiment Note: Indicates extremely significant ( P <0.01, Not means not significant ( P >0.05).

[0033] (3) Response surface analysis and verification of the optimal extraction process for crude polysaccharides from Tribulus terrestris Response surface analysis using Design Expert 13.0 software revealed the theoretically optimal process parameters for ultrasonic extraction as follows: liquid-to-solid ratio of 20.46:1 mL / g, ultrasonic time of 51.01 min, ultrasonic temperature of 62.32°C, and ethanol precipitation concentration of 95%. The predicted extraction yield under these conditions was 2.227%. Taking into account experimental conditions and actual operation, the optimal process parameters were adjusted to: liquid-to-solid ratio of 20:1 mL / g, ultrasonic time of 51 min, ultrasonic temperature of 62°C, and ethanol precipitation concentration of 95%. Three replicate experiments were conducted under these adjusted conditions to validate the model. The average extraction yield of Tribulus terrestris polysaccharides was 2.182%, with a relative error of 0.29% and a deviation of 0.045% from the optimal predicted value. This value is close to the model prediction, demonstrating the reliability of the polysaccharide extraction yield model established using the response surface analysis method.

[0034] Example 2 Preparation of Tribulus terrestris polysaccharide 1. Preparation of Tribulus terrestris polysaccharides Purchase Tribulus terrestris and stir-fried Tribulus terrestris that comply with the 2020 edition of the Chinese Pharmacopoeia, crush them to pass through a 40-mesh sieve, and extract Tribulus terrestris polysaccharides according to the following method: Two kinds of Tribulus terrestris powder were added with water at a liquid-to-solid ratio of 20:1 mL / g, and ultrasonic extraction was performed at an ultrasonic temperature of 62°C and a ultrasonic time of 51 min to obtain an extract. After filtration, the filter residue was ultrasonically extracted again according to the above conditions. The two filtrates were combined and concentrated to 1 / 4 of the volume of the original medicinal solution to obtain a concentrate. Anhydrous ethanol was added to the concentrate to a volume fraction of 95%, and the mixture was precipitated at 4°C for 12 hours. The mixture was centrifuged, the supernatant was discarded, and the precipitate was freeze-dried to obtain a dry precipitate. Sevage reagent was prepared in a 4:1 ratio of chloroform to n-butanol. The precipitate was dissolved in pure water to a 125 mg / mL solution. Sevage reagent was then added in a 4:1 ratio of solution to Sevage reagent. The mixture was shaken vigorously for 30 minutes, and the layers were allowed to stand. The upper layer was retained and the lower organic layer was discarded. This process was repeated until no flocs were left between the upper and lower organic layers. The resulting solution was dialyzed with pure water using a 3000 Da dialysis bag for 48 hours. The polysaccharide solution in the bag was collected, concentrated under reduced pressure, and freeze-dried to obtain the polysaccharides. These were designated as Tribulus terrestris polysaccharide (FP) and stir-fried Tribulus terrestris polysaccharide (SFP) according to their sources.

[0035] 2. Analysis of the physicochemical properties of Tribulus terrestris polysaccharides The sugar content of Tribulus terrestris polysaccharides and stir-fried Tribulus terrestris polysaccharides was determined by the phenol-sulfuric acid method, the protein content of Tribulus terrestris polysaccharides and stir-fried Tribulus terrestris polysaccharides was determined by the Coomassie Brilliant Blue method, and the uronic acid content of Tribulus terrestris polysaccharides and stir-fried Tribulus terrestris polysaccharides was determined by the m-hydroxybiphenyl method. The results showed that the polysaccharide content of Tribulus terrestris polysaccharides was 52.41% and the polysaccharide content of stir-fried Tribulus terrestris polysaccharides was 63.55%, and the polysaccharide content increased significantly after stir-frying. The protein content of Tribulus terrestris polysaccharides was 6.60% and 7.12% in stir-fried Tribulus terrestris polysaccharides, and the protein content in both cases was less than 10%. The uronic acid content of Tribulus terrestris polysaccharides was 4.22% and 11.75% in stir-fried Tribulus terrestris polysaccharides, and the uronic acid content increased significantly after stir-frying.

[0036] Example 3 Characterization of Tribulus terrestris polysaccharide 1. Determination of Molecular Weight of Tribulus Terrestris Polysaccharides The molecular weight of raw and fried Tribulus terrestris polysaccharides was determined by HPSEC-MALLS-RID method.

[0037] Chromatographic conditions: Gel chromatography-differential refractive index-multi-angle laser light scattering system was used, with gel exclusion chromatography columns Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) connected in series; column temperature: 45°C, injection volume: 100 μL; mobile phase A (0.02% NaN3, 0.1 M NaNO3) at a flow rate of 0.6 mL / min, and elution gradient: isocratic for 75 min.

[0038] Table 4 Molecular weight and content of Tribulus terrestris polysaccharides The molecular weight results are shown in Table 4. Figure 3As shown, the Tribulus terrestris polysaccharide (FP) has two peaks, with absolute molecular weights of 151 kDa for peak 1 and 39.86 kDa for peak 2, and PDIs of 1.456 and 1.096, respectively. The stir-fried Tribulus terrestris polysaccharide (SFP) has a single peak, with an absolute molecular weight of 89.41 kDa and a PDI of 1.588. These results suggest that processing reduces the molecular weight of Tribulus terrestris polysaccharides and makes their distribution more uniform.

[0039] 2. Fourier Transform Infrared Spectroscopy Analysis of Tribulus Terrestris Polysaccharides Figure 4 The infrared spectra of Tribulus terrestris and stir-fried Tribulus terrestris polysaccharides are shown: 3300 cm -1 and 2900 cm -1 The absorption bands at 1738 cm⁻¹ are typical absorption bands of polysaccharides, which are related to the OH stretching vibration and CH stretching vibration in the polysaccharide molecules. The absorption peak at 1738 cm⁻¹ is attributed to the stretching vibration of the C=O bond in the uronic acid of the stir-fried Tribulus terrestris polysaccharide. -1 The broad absorption peak at 1412 cm -1 The absorption peak at 1248 cm corresponds to the CH deformation vibration. -1 and 1072 cm -1 The absorption peak at 1027 cm is related to COC and COH groups. -1 The prominent signal appearing nearby corresponds to the CO stretching vibration of the glycosidic bond or the backbone mode of the pyranose ring, indicating the characteristics of polysaccharides. -1 The weak absorption peak at corresponds to the skeleton mode of the pyran ring.

[0040] 3. Scanning electron microscopy observation of the morphology and structure of Tribulus terrestris polysaccharides The scanning electron microscopy results of Tribulus terrestris polysaccharide at 100×, 300× and 1000× magnifications are as follows Figure 5 As shown in the results, the raw Tribulus terrestris polysaccharide has a flaky structure with a smooth surface and a complete and uniform structure, while the fried Tribulus terrestris polysaccharide has an irregular fragmented structure with a large number of rough and small spherical particles attached to the surface. The above results show that after the Tribulus terrestris is fried, the structure and morphology of the polysaccharide change significantly. The flaky structure of the raw Tribulus terrestris polysaccharide is destroyed after heating and frying, and the flaky structure of the fried Tribulus terrestris polysaccharide is fragmented.

[0041] Example 4 Effects of Tribulus terrestris polysaccharide on hypertensive rats Twenty-four 11-week-old SHR hypertensive rats and eight 11-week-old WKY rats were used for this experiment. All rats were SPF-grade, weighing 250 ± 10 g and purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a quality certificate number of SCXK (Beijing) 2021-0006. Animal husbandry and experiments were conducted in the SPF laboratory of Shandong University of Traditional Chinese Medicine. Temperature and humidity were maintained constant throughout the experiment. Rats in each group had free access to food and water, and a regular 12-h day / night cycle was maintained. All experimental procedures adhered to the relevant regulations of the Laboratory Animal Welfare Ethics Committee and were overseen by the Laboratory Animal Welfare Ethics Review Committee of Shandong University of Traditional Chinese Medicine, with an ethics approval number (SDUTCM202101005).

[0042] After one week of adaptive feeding, 24 11-week-old SHR rats were randomly divided into a model group (SHR), a positive drug group (VAL), a Tribulus terrestris polysaccharide group (FP), and a stir-fried Tribulus terrestris polysaccharide group (SFP), with 8 rats in each group. Eight WKY rats served as a blank group (WKY). The Tribulus terrestris polysaccharide and stir-fried Tribulus terrestris polysaccharide groups were administered a dose of 400 mg / kg (400 mg of Tribulus terrestris polysaccharide freeze-dried powder per 1 kg rat), which is within the safety range. The positive drug group received valsartan capsules (14.4 mg / kg, twice the adult standard dose) by oral gavage, while the model and blank groups received an equal volume of distilled water. Administration was continued daily between 8:00 and 10:00 AM for 5 weeks. Body weights were re-weighed every 7 days, and the dose was adjusted accordingly.

[0043] 1. Regulatory effect of Tribulus terrestris polysaccharide on systolic blood pressure in SHR rats At the same time every weekend after drug administration, the BP-2000 animal non-invasive blood pressure analysis system was used to measure the systolic blood pressure (SBP) of the rats' tail artery in a non-anesthetized state. The rats in each group were measured in a conscious and quiet state. The rats were fixed in a matching fixator, and a pressure sensor was placed at the base of the rat's tail. The instrument was inflated and deflated to simulate the pressure application and release of the rat's tail artery. The measurement was performed continuously for 10 times, and the average value was taken.

[0044] The systolic blood pressure of rats in each group before and after administration was Figure 6 As shown in the figure, before administration, there was no statistically significant difference in the systolic pressure of the rat tail artery among the model group, positive drug group, Tribulus terrestris polysaccharide group, and stir-fried Tribulus terrestris polysaccharide group ( P >0.05). The systolic blood pressure of the model rats in each group was higher than that in the blank group, and the difference was statistically significant ( P <0.01). The systolic blood pressure of the blank group and the model group remained at a relatively stable level throughout the experiment.

[0045] After administration, the systolic blood pressure of the positive drug group, raw Tribulus terrestris polysaccharide group, and stir-fried Tribulus terrestris polysaccharide group decreased compared with the model group ( P<0.05 or 0.01); after 4-5 weeks of administration, the systolic blood pressure of the stir-fried Tribulus terrestris polysaccharide group decreased significantly compared with the raw Tribulus terrestris polysaccharide group, and the difference was statistically significant ( P <0.05); Compared with the model group, the systolic blood pressure of the raw Tribulus terrestris polysaccharide group and the stir-fried Tribulus terrestris polysaccharide group decreased, among which the stir-fried Tribulus terrestris polysaccharide group had a better antihypertensive effect. There was no significant difference in the antihypertensive effect of the stir-fried Tribulus terrestris polysaccharide group compared with the positive drug group at week 1, 3, 4, and 5 ( P >0.05).

[0046] 2. Effects of Tribulus terrestris polysaccharide on serum NO, eNOS, Ang II, and ET-1 in SHR rats After the last blood pressure measurement, rats in each group were fasted but not watered for 12 h and anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg / kg). Blood was collected from the abdominal aorta using a vacuum blood collection tube, kept at room temperature for 30 min, and centrifuged at 4°C and 3000 rpm for 10 min. The upper serum was collected, quickly frozen in liquid nitrogen, and stored at -80°C for the determination of serum indicators.

[0047] The levels of eNOS, ET-1, and AngⅡ in rat serum were determined by enzyme-linked immunosorbent assay (ELISA), and the level of NO in rat serum was determined by (enzymatic) colorimetric method. The specific operations were carried out strictly in accordance with the kit instructions.

[0048] The levels of NO, eNOS, Ang Ⅱ and ET-1 in the serum of rats in each group were as follows: Figure 7 The results showed that compared with the blank group, the serum NO and eNOS levels in the model group were significantly reduced ( P <0.01), ET-1 and Ang Ⅱ levels were significantly increased ( P <0.01).

[0049] 3. Effects of Tribulus terrestris polysaccharides on vascular tissue morphology and collagen deposition in SHR rats After blood collection in each treatment group, the rats' thoracic cavities were opened, other tissues removed, and the thoracic aorta completely exposed. The entire aorta was carefully and quickly dissected, and the vessels from the ascending aortic arch to the bifurcation of the abdominal aorta were cut. Perivascular impurities such as fat and connective tissue were removed, and the vessels and residual blood in the vascular lumen were washed with normal saline (pre-chilled at 4°C). One-third of the thoracic aorta was fixed with 10% neutral formaldehyde and stained with hematoxylin and eosin to assess vascular damage.

[0050] Masson staining was used to evaluate the relative level of vascular collagen deposition: the blue area of ​​Masson staining was semi-quantitatively analyzed using Image ProPlus (IPP) 6.0 software, and the average optical density of the blue area [AOD (%) = IOD / Area × 100%] was calculated, which is the relative level of vascular collagen deposition.

[0051] The results of HE staining of blood vessels are as follows Figure 8 As shown in Figure A, the results showed that the thoracic aorta in the blank group was normal in morphology, with no thickening of the vascular wall, a smooth and intact intima, no endothelial cell shedding or blood cell adhesion, and regular and orderly arrangement of thin cells in the smooth muscle layer of the tunica media with no thickening. The structure between the intima, media, and adventitia was intact, with clear layers. Compared with the blank group, the thoracic aorta in the model group showed thickening of the vascular wall, loss of normal structure, disorganized cell arrangement, a rough and uneven intima, partial damage and shedding of endothelial cells, significant thickening of the media, disorganized arrangement of elastic fibers and smooth muscle cells, and unclear adventitia structure with a blurred boundary with the media. Compared with the model group, both the raw and stir-fried Tribulus terrestris polysaccharide groups improved the vascular tissue morphology of SHR rats to some extent. In the raw Tribulus terrestris polysaccharide group, occasional intima and cell shedding were observed, while the tunica media was slightly thickened. Hyperplasia of elastic fibers and smooth muscle cells in some tunica media was still observed, with disorganized cell arrangement. The improvement of vascular tissue morphology in the fried Tribulus terrestris polysaccharide group was better than that in the raw Tribulus terrestris polysaccharide group. The integrity of the intima and the shedding of endothelial cells were significantly improved. The elastic fibers and smooth muscle cells of the media were arranged more neatly without obvious thickening.

[0052] Masson staining results Figure 8 As shown in Figure B, the results showed that there was a small amount of vascular collagen deposition in the blank group. Compared with the blank group, the vascular collagen deposition in the model group was significantly increased. Compared with the model group, both raw and fried Tribulus terrestris polysaccharides could improve the vascular collagen deposition, and fried Tribulus terrestris polysaccharide was better than raw Tribulus terrestris in improving the vascular collagen deposition.

[0053] The above results show that both Tribulus terrestris polysaccharides and stir-fried Tribulus terrestris polysaccharides can reduce blood pressure in SHR rats, alleviate vascular endothelial pathological damage in rats, improve vascular endothelial dysfunction, and have a protective effect on the vascular endothelium. However, the therapeutic effect of stir-fried Tribulus terrestris polysaccharide on SHR rats is significantly better than that of Tribulus terrestris polysaccharide, and the antihypertensive effect of stir-fried Tribulus terrestris polysaccharide is not significantly different from that of the positive drug valsartan.

[0054] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. Application of Tribulus terrestris polysaccharide in the preparation of drugs for treating hypertension.

2. The use according to claim 1, characterized in that Tribulus terrestris is Tribulus terrestris or fried Tribulus terrestris.

3. The use according to claim 1, characterized in that The preparation method of the tribulus terrestris polysaccharide comprises the following steps: precipitating the water extract of the tribulus terrestris with ethanol, and purifying the polysaccharide through precipitation.

4. A method for extracting tribulus terrestris polysaccharide, characterized in that: The following steps are involved: The water extract of Tribulus terrestris is precipitated with ethanol, the precipitate is dried and then purified to obtain the product; the Tribulus terrestris is Tribulus terrestris or fried Tribulus terrestris.

5. The extraction method according to claim 4, characterized in that The water extract of Tribulus terrestris is extracted by microwave-assisted extraction or ultrasound-assisted extraction; the purification steps are protein removal and small molecule removal; the protein removal method is the Sevage method; the small molecule removal method is dialysis, and the dialysis membrane is 3000 Da.

6. The extraction method according to claim 4, characterized in that The following steps are involved: The tribulus terrestris is crushed and then water is added for ultrasonic-assisted extraction. After solid-liquid separation, water is added again for ultrasonic-assisted extraction for 1-4 times. After the extracts are combined, ethanol is added to a volume fraction of ethanol of 75%-95% to obtain a precipitate, and the precipitate is purified and dried to obtain the product.

7. The extraction method according to claim 4, characterized in that The following steps are involved: After the Tribulus terrestris is crushed, water is added according to the liquid-to-solid ratio of (5-25):1 mL / g, and ultrasonic-assisted extraction is performed at a temperature of 40°C-80°C and a time of 30-70 min. After extraction, the solid and liquid are separated, and the solid is repeatedly extracted 1-4 times, and the extract is obtained after merging. After the extract is concentrated, ethanol is added to a volume fraction of 75%-95% to obtain a precipitate, which is then purified and dried.

8. The extraction method according to claim 4, characterized in that The following steps are involved: After the Tribulus terrestris is crushed, water is added according to the liquid-to-solid ratio of (15-25): 1 mL / g, and ultrasonic-assisted extraction is performed at a temperature of 50°C-70°C and a time of 40-60 min. After extraction, the solid and liquid are separated, and the solid is extracted again once. The combined extracts are concentrated, and then ethanol is added to the concentrate to a volume fraction of 85%-95% to obtain a precipitate. The precipitate is then purified and dried to obtain Tribulus terrestris polysaccharide.

9. The extraction method according to claim 4, characterized in that The following steps are involved: After the Tribulus terrestris was crushed, water was added at a liquid-to-solid ratio of 20:1 mL / g, and ultrasonic-assisted extraction was performed at a temperature of 62°C and a time of 51 min. After extraction, the solid and liquid were separated, and the solid was extracted again once. The combined extracts were concentrated, and then ethanol was added to the concentrate to a volume fraction of 95%. A precipitate was obtained at 4°C, and the precipitate was purified and dried to obtain Tribulus terrestris polysaccharide.

10. Tribulus terrestris polysaccharide obtained according to the extraction method according to any one of claims 4 to 9.

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