High-purity preparation method of Mongolian astragaloside capable of resisting exercise-induced fatigue

Abstract

The invention relates to a high-purity preparation method of astragalus mongholicus saponin for resisting exercise-induced fatigue, which comprises the following steps: decocting astragalus mongholicus root with water, centrifuging the water decoction, and carrying out macroporous resin column chromatography to obtain astragalosides I, II, III and IV and soyasaponin I. The invention further discloses a preparation method of the astragalus mongholicus saponin for resisting exercise-induced fatigue. According to the invention, saponin is used as a surfactant and has a hydrotropy effect, and water is used as an extraction solvent for decoction and extraction; compared with the prior art, the method disclosed by the invention is simple and convenient to operate, and only needs two-step operation of water extraction and macroporous resin chromatography; the consumed time is short, and the preparation can be completed within 12 hours; the obtained product is high in purity, and the saponin content exceeds 80%.

Description

Technical Field

[0001] This invention relates to a technology in the field of biomedicine, specifically a method for preparing anti-exercise fatigue astragalus saponins with a purity exceeding 80%. Background Technology

[0002] Astragalus membranaceus var. mongholicus (Bunge) PKHsiao is a plant belonging to the genus Astragalus in the legume family. Modern research has found that the root of Astragalus membranaceus has a significant anti-exercise fatigue effect. However, the technical shortcomings of the existing extraction process are: there are no reported methods for preparing active saponins with anti-exercise fatigue effects from Astragalus membranaceus; and the saponins obtained by the existing extraction technology have low purity and low yield. Summary of the Invention

[0003] This invention addresses the shortcomings of existing extraction techniques, such as low purity, low yield, difficulty in obtaining single components, and high cost of saponins. It proposes a high-purity preparation method for Mongolian astragalus saponins that combat exercise fatigue. This method prepares high-purity active saponins with significant anti-exercise fatigue effects from Mongolian astragalus, and has the advantages of being simple, rapid, high-purity, and low-cost.

[0004] This invention is achieved through the following technical solution:

[0005] This invention relates to a class of mongholic astragaloside compounds, including astragaloside I, II, III, and IV, and soyasaponin I, with the following structural formulas:

[0006] Astragalus saponins: Among them: when the group R1 is 2,3-O-Ac2-Xyl and R2 is Glu, it is astragaloside I; when the group R1 is 2-O-Ac-Xyl and R2 is Glu, it is astragaloside II; when the group R1 is Glu-(1,2)-Xyl and R2 is H, it is astragaloside III; when the group R1 is Xyl and R2 is Glu, it is astragaloside IV.

[0007] Soy saponin I:

[0008] This invention relates to the application of the above-mentioned Mongolian astragalus saponins, using them in the preparation of drugs, health products and / or beverages for combating exercise-induced fatigue.

[0009] This invention relates to a method for extracting the above-mentioned Mongolian astragalus saponins. By decocting Mongolian astragalus root in water, centrifuging the decoction, and then performing macroporous resin column chromatography, astragalus saponins I, II, III, IV, and daidzein I are obtained.

[0010] The Mongolian Astragalus mentioned is the dried root of Astragalus membranaceus var. mongholicus (Bunge) PKHsiao, a plant belonging to the Fabaceae family.

[0011] The macroporous resin column chromatography used AB-8 type macroporous resin and ethanol solution as eluent.

[0012] The extraction method specifically includes: crushing the root of Astragalus membranaceus into coarse powder and then decocting it in water; chromatographically analyzing the decoction using an AB-8 macroporous resin column; washing away impurities with water and then eluting with 60% ethanol; collecting the 60% ethanol eluent; and recovering the solvent under reduced pressure to obtain Astragalus membranaceus saponins.

[0013] The water decoction method described above uses a water-to-medicinal-material ratio of 7:1 to 13:1, a decoction time of 0.5 to 1 hour, and is performed 1 to 2 times.

[0014] The macroporous resin column chromatography used is AB-8 macroporous resin, and the amount used is 1 to 2 times the mass of the drug material. Water is used as the eluent to remove impurities; then 60% ethanol is used as the eluent, with a volume of 3 to 5 times the column volume (BV). Technical effect

[0015] This invention utilizes saponins as surfactants, which have a solubilizing effect, and uses water as the extraction solvent for decoction extraction. Compared with the prior art, this invention is simple to operate, requiring only two steps: water extraction and macroporous resin chromatography. It is also time-efficient, with the preparation completed within 12 hours. The resulting product has high purity, with a saponin content exceeding 80%. Detailed Implementation Example 1

[0016] This embodiment includes the following steps:

[0017] Step 1: Add 1.0 kg of Astragalus membranaceus root to 10 L of water and decoct for 1 hour. Centrifuge the decoction at 5000 rpm for 20 minutes to obtain saponin extract.

[0018] Step 2: The extract was subjected to macroporous resin (1.0 kg) column chromatography with dynamic loading at a flow rate of 0.25 BV / h, i.e., dynamic adsorption for 4 h. Then, 3 BV of water was used to elute and remove impurities; 3 BV of 60% ethanol was used to elute and the ethanol was recovered to obtain a total mass of 113.7 g of astragaloside I, II, III, IV and daidzein I.

[0019] The above-mentioned Mongolian astragaloside sample was used, and astragaloside A was used as a control. The total astragaloside content was determined by the vanillin-concentrated sulfuric acid method, specifically including:

[0020] ① Preparation of reference solution: Weigh 1.0 mg of astragaloside A into a 10 mL volumetric flask, and dilute to volume with 80% ethanol to obtain a 1.0 mg / mL astragaloside A reference solution;

[0021] ② Preparation of the test solution: Weigh 100g of mongholic astragaloside sample into a 10mL volumetric flask, dissolve it in 80% ethanol, and dilute to the mark for later use;

[0022] ③ Construction of the standard curve: Accurately pipette 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, and 600 μL of astragaloside A reference solution with a mass concentration of 1.0 mg / mL into stoppered test tubes. Add 80% ethanol to each test tube to a volume of 600 μL. Then, add 600 μL of 8% vanillin-sulfuric acid solution and 6 mL of 72% sulfuric acid, respectively. Shake well and immediately place in a 62℃ constant temperature water bath for 20 min, then cool in an ice-water bath for 10 min. Shake well again and measure the absorbance at 562 nm. Plot the standard curve with the measured absorbance as the ordinate and the concentration of astragaloside A reference solution as the abscissa.

[0023] ④ Sample content determination: Pipette 80 μL of the test solution, add 80% ethanol to make up the volume to 600 μL, and then proceed as described above. Calculate the total saponin content of Astragalus membranaceus based on the standard curve.

[0024] The results showed that the total saponin content of the obtained Mongolian astragaloside sample, calculated as astragaloside A, was 80.6%. Example 2

[0025] This embodiment includes the following steps:

[0026] Step 1: Add 1.0 kg of Astragalus membranaceus root to 7 L of water and decoct twice, 0.5 h each time. Combine the two decoctions and centrifuge at 5000 rpm for 20 min to obtain saponin extract.

[0027] Step 2: The saponin extract was subjected to macroporous resin (2.0 kg) column chromatography with dynamic loading at a flow rate of 0.25 BV / h, i.e., dynamic adsorption for 4 h. 5 BV of water was used to elute and remove impurities; 5 BV of 60% ethanol was used to elute and the ethanol was recovered to obtain 141.0 g of mongholic saponins.

[0028] Step 3: Take the above-mentioned Mongolian astragaloside sample and, using astragaloside A as a control, determine the total astragaloside content using the vanillin-concentrated sulfuric acid method, following the same method as in Example 1. The results showed that the obtained Mongolian astragaloside sample contained 82.3% total astragaloside A. Example 3

[0029] This embodiment includes the following steps:

[0030] Step 1: Take 1 kg of the roots of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia, add 13 L of water and decoct for 0.75 h. Centrifuge the decoction at 5000 rpm for 20 min to obtain the saponin extract.

[0031] Step 2: Subject the saponin extract to macroporous resin (1.5 kg) column chromatography, elute with water for 4 BV to remove impurities, and then elute with 60% ethanol for 4 BV. Recover the ethanol to obtain the Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia saponin extract (129.2 g).

[0032] Step 3: Take the above-mentioned Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia saponin sample, use astragaloside IV as the control, and adopt the vanillin-sulfuric acid method to determine the content of total saponins in Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia. The specific determination method is the same as that in Example 1. It is found that the obtained Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia saponin sample contains 81.7% of total saponins calculated as astragaloside IV. Example 4

[0033] This example relates to the application of the above-mentioned Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao & K. T. Hsia saponins in anti-exercise fatigue. The specific detection indexes and detection methods are as follows:

[0034] 1. Animal grouping: Take 40 SPF-grade healthy male ICR mice, 6 weeks old, all purchased from Shanghai Lingchang Biotechnology Co., Ltd. The license number for the experimental animals used is: SYXK (Shanghai) 2018 - 0028. After 3 days of adaptive feeding of the mice, randomly divide the 40 mice into 4 groups, with 10 mice in each group: The first group is the blank control group (intragastric administration of normal saline), the second group is the low-dose astragalus saponin group (5 mg / kg), the third group is the high-dose astragalus saponin group (20 mg / kg), and the fourth group is the positive control group (800 mg / kg of taurine).

[0035] 2. Animal model establishment: Starting from the third day of intragastric administration, conduct 10 min of swimming training for the mice every day. Start the swimming training 30 min after the end of daily intragastric administration. Place the mice in a 5 L beaker containing 3 L of water, take them out and dry them after swimming for 10 min to construct a mouse fatigue model.

[0036] 3. Determination indexes: After continuous intragastric administration for one week, conduct a negative gravity exhaustion swimming experiment half an hour after intragastric administration on the morning of the eighth day. Wrap a tin foil block weighing 5% of the body weight of the mouse around the tail end and then let the mouse swim. When the mouse is exhausted, fish it out and record the swimming time. The criteria for judging mouse exhaustion: When the mouse swims, its movements are significantly disordered until it sinks into the water and still cannot swim to the water surface after 5 s; it is slow to react after getting out of the water, slowly escapes from strong stimuli, and its limbs droop when lifting the mouse's tail without struggling signs. Immediately after the mouse is exhausted, remove the eyeballs to take blood, dissect the mouse to obtain the liver, rinse it with normal saline, dry it with filter paper, and weigh it. Determine the activities of lactic acid (LD), liver glycogen, and creatine kinase (CK) according to the operation instructions on the purchased kit.

[0037] 4. Measurement Results

[0038] 4.1 Effects of Astragaloside on Swimming Time in Mice

[0039] The swimming time of mice under load is shown in Table 1. There were significant differences between the low-dose group, the high-dose group and the blank control group of astragaloside (P < 0.05), indicating that astragaloside can significantly increase the exercise time of mice.

[0040] Table 1. Effects of astragaloside on swimming time in mice (mean ± SD, n = 10)

[0041] Compared with the control group, *P<0.05; **P<0.01

[0042] 4.2 Effects of Astragalus saponins on liver glycogen content in mice

[0043] The results are shown in Table 2. There were statistically significant differences between the treatment groups (low-dose astragaloside group, high-dose group, and positive control group) and the blank control group (P<0.01). This indicates that astragaloside can significantly increase the liver glycogen reserves in mice. Since liver glycogenolysis provides energy for physiological activities, the results suggest that astragaloside solution can significantly delay exercise-induced fatigue in mice by increasing liver glycogen reserves.

[0044] Table 2. Effects of astragaloside on liver glycogen content in mice (mean ± SD, n = 10)

[0045] Compared with the control group, **P<0.01

[0046] 4.3 Effect of Astragalus saponins on lactic acid accumulation in mice

[0047] The results are shown in Table 3. Astragalus saponin solution significantly reduced lactic acid accumulation in mice after exercise (P<0.01). Lactic acid is a major metabolic product produced during anaerobic glycolysis in the body during short-term high-energy-consuming exercise. Reducing lactic acid accumulation can delay the onset of fatigue. These results suggest that astragalus saponins can delay exercise-induced fatigue in mice by reducing lactic acid accumulation in their blood.

[0048] Table 3. Effects of astragaloside on lactic acid levels in mouse blood (mean ± SD, n = 10)

[0049] Compared with the control group, **P<0.01

[0050] 4.4 Effects of Astragalus saponins on creatine kinase activity in mice

[0051] Both astragaloside solution and taurine solution significantly reduced serum creatine kinase activity in mice after exercise. Statistically highly significant differences were observed between the treated groups (low-dose and high-dose astragaloside groups) and the blank control group (as shown in Table 4). Serum creatine kinase is considered to indirectly indicate the degree of muscle damage and is related to hypoxia and cell damage in the body. It is one of the common biochemical indicators of fatigue. The significantly lower serum creatine kinase levels in the treated groups compared to the blank control group indicate that astragaloside can significantly combat exercise-induced fatigue by reducing the activity of CK enzymes in mouse blood and inhibiting the slowing of ATP production.

[0052] Table 4. Effects of astragaloside on creatine kinase activity in mice (mean ± SD, n = 10)

[0053] Compared with the control group, **P<0.01

[0054] Compared with existing technologies, this method improves the purity of mongholic saponins through water decoction extraction combined with macroporous resin column purification. The obtained mongholic saponins have good anti-fatigue effects, can increase the swimming time of mice, increase the liver glycogen reserves in mice, reduce the accumulation of lactic acid in mice after exercise, and reduce the activity of creatine kinase in mice, thereby making the energy supply of mice more sufficient during exercise. The amount of metabolic waste generated after exercise is reduced. It exerts its anti-exercise fatigue effect through multiple mechanisms and can be used in the preparation of anti-fatigue drugs and health products.

[0055] The above-described specific implementations can be partially adjusted by those skilled in the art in different ways without departing from the principles and purpose of the present invention. The scope of protection of the present invention is defined by the claims and is not limited to the above-described specific implementations. All implementation schemes within the scope of the claims are bound by the present invention.

Claims

1. A class of mongholic acid saponins, characterized in that, include: The structural formulas of astragaloside I, II, III, and IV, and soyasaponin I are as follows: Astragalus saponins: Wherein: when group R1 is 2,3-O-Ac2-Xyl and R2 is Glu, it is astragaloside I; when group R1 is 2-O-Ac-Xyl and R2 is Glu, it is astragaloside II; when group R1 is Glu-(1,2)-Xyl and R2 is H, it is astragaloside III; when group R1 is Xyl and R2 is Glu, it is astragaloside IV. Soy saponin I:

2. An application of the mongholic astragaloside compound according to claim 1, characterized in that, It can be used to prepare drugs, health products and / or beverages that combat exercise-induced fatigue.

3. A method for extracting saponins from mongholic astragalus, characterized in that, Astragalus membranaceus root was decocted in water, and the decoction was centrifuged and subjected to macroporous resin column chromatography to obtain astragaloside I, II, III, IV and soybean saponin I.

4. The extraction method according to claim 3, characterized in that, The Mongolian Astragalus mentioned is the dried root of Astragalus membranaceus var. mongholicus (Bunge) PKHsiao, a plant belonging to the Fabaceae family.

5. The extraction method according to claim 3, characterized in that, The macroporous resin column chromatography used AB-8 type macroporous resin and ethanol solution as eluent.

6. The extraction method according to claim 3, characterized in that, The extraction method specifically includes: crushing the root of Astragalus membranaceus into coarse powder and then decocting it in water; chromatographically analyzing the decoction using an AB-8 macroporous resin column; washing away impurities with water and then eluting with 60% ethanol; collecting the 60% ethanol eluent; and recovering the solvent under reduced pressure to obtain Astragalus membranaceus saponins.

7. The extraction method according to claim 3 or 6, characterized in that, The water decoction method described above uses a water-to-medicinal-material ratio of 7:1 to 13:1, a decoction time of 0.5 to 1 hour, and is performed 1 to 2 times.

8. The extraction method according to claim 5 or 6, characterized in that, In the macroporous resin column chromatography, the amount of macroporous resin used is 1 to 2 times the mass of the drug material, and the eluent for removing impurities is water; the ethanol solution is 60% ethanol, and the amount of eluent used is 3 to 5 times the BV (column volume).

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