CN1361111A - Furost saponine analogue and its separation process and use - Google Patents

Abstract

The invention provides a class of five furostanol saponins isolated from Tribulus terrestris, which are proved to have obvious preventive and therapeutic effects on animal experiments against ischemic cerebral infarction and myocardial infarction through pharmacological tests and drug efficacy tests.

Description

Analogues, isolation methods and uses of furostanol saponins

The invention relates to the extraction of effective components in plants, more specifically the extraction of effective components of Tribulus terrestris and its application in cardiovascular disease medicine.

Tribulus terrestris L. is a plant of the family Zygophyllaceae. The medicine has the effects of calming the liver and suppressing yang, promoting blood circulation and dredging collaterals, regulating qi and resolving phlegm, dispersing numbness and unblocking yang, etc. It is used to treat cerebrovascular disorders, hypertension, coronary heart disease, etc. <this classic> main evil blood breaks the crux accumulation, <medicine property theory> controls all wind ulcers, breaks the place blood. <Materia Medica Zaixin>: calms liver wind, cools liver fire, disperses dampness and breaks diarrhea, nourishes qi and resolves phlegm, disperses dampness and breaks blood; <Compendium of Materia Medica>: good deeds and good detoxification, specially enters the stagnation of the lung, liver, and ventilating the lung, and soothes the stasis of the liver. <Materia Medica Huiyan>: It is also a medicine for dispelling wind and lowering qi, promoting water and removing blood stasis. <Compendium of Materia Medica>: Tribulus terrestris is used in ancient prescriptions to invigorate the kidney and control wind.

According to the above medical records, Tribulus terrestris is a highly effective traditional Chinese medicine and is recommended by physicians of all ages.

In recent years, scholars at home and abroad have also deepened their research work on the whole herb of Tribulus terrestris (hereinafter referred to as Tribulus terrestris). Various chemical extracts have been studied in pharmacology of traditional Chinese medicine, and there are continuous research reports that the extract of Tribulus terrestris has anti-atherosclerotic properties. It can significantly reduce experimental high cholesterol levels, prevent lipid precipitation in arteries, myocardium, and liver, reduce the scope of experimental myocardial infarction, and have obvious anti-myocardial ischemia capabilities. Although a lot of work has been done on the chemical structure and chemical extraction of Tribulus terrestris, an ideal drug for treating cardiovascular and cerebrovascular diseases has not yet been determined in Tribulus terrestris.

The object of the present invention is to chemically separate the Tribulus terrestris plant, find out effective parts or monomers for pharmacological clinical trials, and find out an effective drug for cardiovascular and cerebrovascular diseases.

Another object of the present invention is to find a preparation method of effective fractions or monomers for cardiovascular diseases.

Extraction of active ingredients from Tribulus terrestris:

Tribulus terrestris has been proved by a large number of experiments that the effective components of anti-cerebrovascular disorders are saponins with great water solubility, and the main active components are Hecogenin (sea cogenin) aglycone and Tigogenin (extract glycosides) after hydrolysis. Yuan) aglycone furostanol saponins with 5 or 6 sugars. To crude extraction method, at first studied 75% ethanol extraction method, water extract ethanol precipitation method and water extract liquid through macroporous resin method, three kinds of methods are compared, contain a lot of fat-soluble matter in 75% ethanol extract, concentrated extract It is necessary to use a large amount of CHCl to treat and remove the fat-soluble matter _first , and then to separate the macroporous resin. This method is costly and requires a large amount of ethanol and chloroform. In the water extraction and ethanol precipitation method, the water extract must be concentrated first, and the concentrated solution is then used for ethanol precipitation to remove ineffective impurities. Because it contains a large amount of saponin, it is easy to generate a large amount of foam during concentration, which makes it difficult. The third method is that the water extract directly passes through the macroporous resin to enrich the saponin. This process avoids the difficulty of flushing the material due to the foam generated by the concentration of a large amount of aqueous solution. The macroporous resin can enrich the saponin, remove a large amount of invalid impurities, and can It can be used repeatedly and eluted with 60% ethanol after enriching the saponin. This process is suitable for large-scale production and is relatively economical.

The crude product obtained by the above method was subjected to column chromatography, eluting with methanol: water, each fraction was divided into 50ml, 24-26 fractions were collected, combined and solvent removed to obtain 1b, other fractions were combined, and then column chromatography was performed several times , the mobile phase was eluted with chloroform: formic acid: water, and 2a, 2b, 3a, 3b were obtained successively.

MCI Gel column can also be used to elute with H ₂ O first, then H ₂ O: ethanol gradient elution, 100ml as a fraction, preliminary separation, controlled by THL, merge the fractions of the same spot, and then conduct multiple fractions respectively. Secondary normal phase silica gel column chromatography, before the same flow, yielded 1b, 2a, 2b, 3a, 3b.

Fig. 1 is the extraction process of effective components of Tribulus terrestris

Firstly, the whole herb of Tribulus terrestris is ground into a coarse powder, then boiled in water and filtered twice to separate the water extract and medicinal residues. The dregs are boiled and extracted with water, filtered, the separated dregs are discarded, and the water extract is used as the water volume of the next material. The water extract for the first two times is placed at 5-10°C for 12 hours, filtered, and the filtrate passes through 1400 type resin. After the liquid medicine passes through, continue to wash with water until the outflow is colorless, separate the water eluent, and after passing through the liquid medicine, elute with 60%-70% ethanol, and the eluent is distilled under reduced pressure to recover ethanol , continue to concentrate to a specific gravity between 1.37-1.40, add several times the amount of ethanol to the concentrated solution, make it a solution and place it at 5-10°C for 12 hours, separate the supernatant, filter the sediment slurry, discard the filter residue, and separate the supernatant from the filtrate The liquids were combined, concentrated under reduced pressure to obtain an extract, and put on a silica gel column, according to extract: silica gel = 1:10. Rinse with chloroform, then chloroform:methanol gradient elution to combine active components, and then concentrate to dryness under reduced pressure to obtain the product.

The product obtained by the above-mentioned active ingredient extraction process, 620mg, passed through the Fuji gel (silica gel for broken type transfer, ODSG 3 type) column, with methanol: water = 6: 4 elution, each fraction 50ml, with 24-26 streams Separated to obtain 1b, the other fractions were combined, and then several times of silica gel column chromatography (chromatographic silica gel 100-200 mesh produced by Qingdao Ocean Chemical Factory) was eluted with chloroform:methanol:water=7:3:0.5, and successively separated 1a, 2a, 2b, 3a, 3b were obtained. After repeated normal-phase and reverse-phase column chromatography, the above-mentioned active ingredients were mainly composed of mid-polar saponins, and six furostanol saponins were separated, which were JL-saponin. la. Their chemical structures are:

R ₁ is -β-D-Glu

_R2 is

_R3 is H; O

R' is CH ₃ , H

1a R ₁ =-β-D-Glu

      

R ³ =O

R'=CH ₃ 1b R ₁ =-β-D-Glu

 

R ₃ =O

R'=H2a R ₁ =-β-D-Glu

 

R ₃ =H

R'=CH ₃ 2b R ₁ =β-D-Glu

 

R ₃ =H

R'=H3a R ₁ =-β-D-Glu

 

R ₃ =H

R'=CH ₃ 3b R ₁ =β-D-Glu

 

R ₃ =H

R'＝H See Table 1-3 ¹ H NMR and ¹³ C NMR The data in Table 1-3 are all calibrated by pyridine solvent peak ( ¹ H NMR 7.190, ¹³ C NMR 123.55)

Table 1 NMR data of furostanol saponin JL-saponin 1a and 1b No.(c) ¹³ C(δ) ¹ H(δ) 1a 1b 1a 1b 1 36.54 36.53 2 29.65 29.64 3 77.48 77.47 4 34.64 34.61 5 44.40 44.38 6 28.52 28.50 7 31.61 31.61 8 34.22 34.21 9 55.47 55.45 10 36.18 36.16 11 37.85 37.88 12 212.75 212.84 13 55.75 55.72 14 55.63 55.62 15 31.29 31.61 16 79.82 79.57 17 55.47 54.98 2.65 (dd, J=8.2, 6.3) 2.87 (dd, J=83, 67) 18 15.95 16.13 1.02 1.10 19 11.61 11.60 0.62 0.63 20 41.03 41.14 twenty one 14.94 15.13 1.38 (J=6.8) 1.53 (J=6.7) twenty two 112.65 110.65 -OCH ₃ 47.29 3.23 twenty three 30.64 37.01 twenty four 28.11 28.29 25 34.11 34.14 26 75.10 75.09 27 17.04 17.33 0.96 (J=6.6) 0.96 (J=6.6) Sugar-1 (β-D-galactose) 1 105.23 105.22 5.44 (J=7.7) 5.45 (J=7.7) 2 73.69 73.69 3 73.94 73.89 4 70.32 70.34 5 77.24 77.25 6 60.47 60.42 Sugar-2 (β-D-glucose) 1 105.46 105.47 5.14 (J=7.8) 5.16 (J=7.9) 2 81.01 81.01 3 85.54 85.69 4 70.53 70.52 5 77.29 77.25 6 62.99 62.98 Sugar-3 (β-D-xylose) 1 104.73 104.72 5.01 (J=7.0) 5.05 (J=7.0) 2 74.99 74.49 3 78.41 78.37 4 70.65 70.62 5 67.17 67.16 Sugar-4 (β-D-glucose) 1 102.24 102.20 4.86 (J=7.6) 4.87 (J=7.6) 2 72.99 72.96 3 75.67 75.70 4 79.53 79.50 5 75.32 75.32 6 62.45 62.51 Sugar-5 (β-D-glucose) 1 104.89 104.84 4.81 (J=7.8) 4.79 (J=7.7) 2 75.13 75.17 3 78.55 78.51 4 71.68 71.62 5 78.34 78.33 6 62.82 62.74

Table 2 NMR data of furostanol saponin JL-saponin 2a and 2b No(c) ¹³ C(δ) ¹ H(δ) 2a 2b 2a 2b 1 37.08 37.15 2 29.87 29.93 3 77.60 77.64 4 34.81 34.83 5 44.64 44.64 6 28.84 28.89 7 32.32 32.38 8 35.13 35.17 9 54.39 54.37 10 35.72 35.75 11 21.12 21.23 12 39.92 40.15 13 41.02 41.06 14 56.27 56.31 15 31.99 32.33 16 81.24 81.08 17 64.24 63.88 18 16.36 16.70 0.75 0.82 19 12.20 12.25 0.60 0.58 20 40.41 40.62 twenty one 16.20 16.45 1.15 (J=6.6) 1.29 (J=6.6) twenty two 112.53 110.62 -OCH ₃ 47.20 3.23 twenty three 30.72 37.15 twenty four 28.11 28.34 25 34.09 34.22 26 75.03 75.13 27 17.01 17.43 0.96 (J=6.6) 0.93 (J=6.6) Sugar-1 (β-D-galactose) 1 105.11 105.29 5.41 (J=7.8) 5.43 (J=7.6) 2 73.59 73.72 3 73.94 73.92 4 70.19 70.26 5 77.11 77.26 6 60.43 60.50 Sugar-2(-D-glucose) 1 105.34 105.51 5.11 (J=8.4) 5.14 (J=7.7) 2 81.92 81.08 3 85.67 85.43 4 70.46 70.56 5 77.39 77.49 6 62.93 63.03 Sugar-3 (β-D-xylose) 1 104.68 104.72 5.01 (J=7.8) 5.05 (J＝6.9) 2 74.83 74.90 3 ^{78.26b 78.34c} 4 70.59 70.67 5 67.10 67.17 Sugar-4 (β-D-glucose) 1 102.24 102.28 4.85 (J=7.2) 4.88 (J=7.7) 2 72.99 73.04 3 75.23 75.32 ^d 4 79.54 79.57 5 75.36 75.66 ^d 6 62.28 62.34 Sugar-5 (β-D-glucose) 1 104.79 104.85 4 78 (J=7.8) 4.77 (J=7.7) 2 75.11 75.26 3 78.45 78.52 4 71.63 71.60 5 ^{78.30b 78.42c} 6 62.77 62.72

Table 3 NMR data of furostanol saponin JL-saponin 3a and 3b No(c) ¹³ C(δ) ¹ H(δ) 3a 3b 3a 3b 1 37.24 37.22 2 29.94 29.91 3 77.01 76.91 4 34.21 34.32 5 44.67 44.60 6 28.98 28.95 7 32.42 32.43 8 35.24 35.22 9 54.43 54.40 10 35.92 35.89 11 21.21 21.25 12 40.00 40.17 13 41.11 41.07 14 55.36 56.35 15 32.11 32.43 16 81.33 81.10 17 64.35 63.97 18 16.49 16.70 0.76 0.84 19 12.41 12.40 0.82 0.84 20 40.51 40.67 twenty one 16.32 16.47 1.17 (J=6.9) 1.32 (J=6.5) twenty two 112.61 110.58 -OCH3 47.27 3.24 twenty three 30.83 37.22 twenty four 28.20 28.37 25 34.21 34.27 26 75.20 75.28 27 17.15 17.47 0.97 (J=6.5) 0.96 (J=6.4) sugar-4 (β-D-galactose) 1 100.15 100.09 4.84 (J=7.7) 4.83 (J=8.0) 2 76.64 76.51 3 76.64 76.66 4 81.33 81.30 5 75.80 75.83 6 60.42 60.40 Sugar-2 (β-D-glucose) 1 105.34 105.33 4.99 (J=7.8) 4.99 (J=8.0) 2 81.49 81.53 3 87.61 87.55 4 70.43 70.41 5 77.77 77.77 6 62.91 62.90 Sugar-l (β-D-xylose) 1 105.01 104.96 5.24 (J=7.7) 5.25 (J=7.6) 2 75.09 75.08 3 78.75 78.76 4 70.72 70.71 5 67.67 67.34 Sugar-3 (β-D-xylose) 1 105.83 105.86 5.41 (J=7.6) 5.42 (J=7.7) 2 75.09 75.08 3 79.08 79.11 4 70.88 70.88 5 67.34 67.70 Sugar-5 (aD-rhaminose) 1 102.00 101.98 6.19 6.20 2 72.45 72.46 3 72.72 72.70 4 74.00 73.97 5 69.35 69.36 6 18.46 18.46 1.70 (J=6.1) 1.70 (J=5.8) Sugar-6 (β-D-glucose) 1 105.01 105.03 4.84 (J=7.7) 4.81 (J=7.8) 2 75.20 75.20 3 78.64 78.62 4 71.76 71.65 5 78.53 78.53 6 62.91 62.79 The data in Tables 1-3 are all calibrated by pyridine peak CH'NMR 7.190, ¹³ C MNR 123.55

Tribulus terrestris has been proved by a large number of experiments that the active ingredients of anti-cerebrovascular disorders are saponins with great water solubility, and the main active ingredients are Hecogenin aglycone and Tigogenin aglycone with 5 or 6 sugars after hydrolysis furostanol saponins.

Pharmacological and toxicological tests on the effective components of Tribulus terrestris prove that it has an effect on the prevention and treatment of ischemic cerebral infarction. Oral administration of 2.5-20 mg/kg of active components of Tribulus terrestris has a very obvious protective effect on global cerebral ischemia-reperfusion brain injury caused by bilateral carotid artery ligation combined with hypotension. On the rat model of global cerebral ischemia-reperfusion with ligation of four arteries in the awake state, oral administration of 2.5-10 mg/kg of Tribulus terrestris significantly prolongs the disappearance of EEG after ischemia and reduces the EEG and righting reflex of reperfusion compared with the negative control Reply, compared with nimodipine (5mg/kg orally) and Xinnaoshutong (25, 50mg/kg orally), the effect intensity is significantly higher than that of the control group, suggesting that it has a significant effect on the brain damage caused by global cerebral ischemia in rats. Protective effect, on the middle artery node model in rats, oral administration of 2.5-10mg/kg of Tribulus terrestris extract has obvious therapeutic effect on focal cerebral infarction in rats, and its histological and behavioral evaluations are significantly improved, and There is a certain relationship with the dose, and the effect is significantly stronger than that of nimodipine. The test results show that the extract of Tribulus terrestris has obvious preventive and therapeutic effects on animal experimental cerebral infarction models.

Preventive and therapeutic effects on myocardial infarction

The pharmacodynamic test results of anti-chest pain and heart pain show that administration of 10-40mg/kg duodenum extract of Tribulus terrestris can significantly reduce the degree of myocardial ischemia and narrow the range of myocardial ischemia in dogs with coronary artery ligation , the action intensity has a certain relationship with the dose, the quantitative histological test results are consistent with the results of the epicardial electrogram, compared with the control group, the infarct area is significantly reduced, which proves that Tribulus terrestris has a significant therapeutic effect on the myocardial infarction caused by coronary artery ligation in dogs Effect. The test results showed that Tribulus terrestris caused a dose-dependent decrease in serum LDH ratio, suggesting that Tribulus terrestris had a protective effect on cardiomyocytes. Oral administration of 10-40 mg/kg of Tribulus terrestris extract has obvious preventive effect on rat myocardial ischemia induced by pituitary hormone. The test results support that Tribulus terrestris has obvious preventive and therapeutic effects on chest pain and heart pain.

Experimental studies on the safety of active components of Tribulus terrestris show that the acute toxicity oral LD ₅₀ is greater than 5000mg/kg, intravenous injection is 187.73mg/kg for females and 204.41mg/kg for males, and there is no significant difference between male and female. A 90-day long-term toxicity test was carried out on rats and Beagle dogs. The results of the former showed that the hematology, biochemistry and urine tests of each dose group were basically within the normal range and had nothing to do with the drug dose. It was only considered that the body weight was significantly different from the control at individual times, and no abnormal changes related to drug toxicity were found throughout the experiment. Histopathological examination and visceral coefficient measurement results showed that no pathological changes related to drug toxicity were seen in the animals killed alive at the end of the administration and during the recovery period; , There was no abnormality in general behavioral activities, shiny hair, normal drinking water and food intake. No changes related to drug effects were observed in body weight, body temperature and electrocardiogram of experimental animals. 14 hematological and 15 blood biochemical indexes of the experimental animals had no significant changes. No drug-related pharmacological changes were found in histopathological examination.

Example 1

Extraction of effective components from Tribulus terrestris

For whole grass powder of Tribulus terrestris, add 12 times the amount of water to boil, keep warm at 100°C for 2 hours, release the decoction liquid, add 8 times more water to boil, keep warm for 1-2 hours, release the decoction liquid, combine the second decoction liquid, pack Put it in a cold storage (5-10°C) for 12 hours (add 8 times the amount of water for the third time, cook for 2 hours, and use the decoction as the next ingredient). The next day, use centrifugation to filter, and the filtrate passes through a macroporous resin 1400 type column (medicinal material (g): resin ( ^cm ) = 2: 1). Elute with 60%-70% ethanol until there is no saponin reaction, collect 60%-70% ethanol eluate (the macroporous resin is treated according to the usual method, and finally rinse with water until neutral, and reuse), 60%-70% ethanol The eluate is first recovered from ethanol, and then concentrated under reduced pressure to a concentrated solution with a specific gravity of 1.37-1.40. Add 8 times the amount of 95% ethanol and stir while adding. After the addition, place it in a cold storage (5-10°C) for 10-15 hours , absorb the clarified liquid the next day, the precipitated part becomes viscous, wash and filter twice with an appropriate amount of 60% ethanol, combine the filtrate and the clarified liquid, recover the ethanol, continue to concentrate into a dry extract, and the yield is 2.2-2.5% of the crude drug amount . Dissolve the dry extract with 95% ethanol, mix the solution with a small amount of silica gel, evaporate the ethanol on a water bath, then dry it in an oven at 70°C, take it out after cooling, grind it into a fine powder, sieve it, then pack it into a column, and start washing it with chloroform. Then use chloroform: methanol for gradient elution of 85%-65%: 15%: 35%, and check the control with silica gel thin layer chromatography until the active ingredients are washed out. Combine the washing solution containing the active ingredient, recover the solvent under reduced pressure, continue to dry, dry in a vacuum oven at 70°C, cool down to room temperature, take it out and grind it into a fine powder, pass through an 80-mesh sieve, dry and store for later use, and the yield of the active ingredient is 0.5%.

Example 2

Protective effect of active components of Tribulus terrestris on cerebral ischemia-reperfusion induced by hemorrhagic hypotension combined with bilateral carotid artery ligation in rats

Samples for testing:

Name: Active ingredients of Tribulus terrestris

Source: Shanghai Institute of Materia Medica, Chinese Academy of Sciences

Batch number: 971128

Content: Contains about 50% of medium polar saponins

Appearance: This product is brown-yellow powder

Preparation: After accurately weighing this product, add 1% tragacanth gum solution and grind to prepare suspensions with concentrations of 0.025, 0.5, 1.0 and 2.0 mg/ml respectively.

Control sample:

Name: Nimodipine

Source: Produced by Shandong Xinhua Pharmaceutical Factory

Batch number; 9203033

Content: 101.29%

Appearance: light yellow crystalline powder

Preparation: Grind with 1% tragacanth gum solution to prepare a suspension with a content of 0.5mg/ml

experimental animals

Strain; SD rat

Gender: male

Weight: 250-300 grams

Source: Shanghai Animal Center, Chinese Academy of Sciences

Animal certificate: Shanghai Donghe Zhengzi No. 005

Breeding; constant temperature purification and ventilation animal room, free access to food and water, 25±2°C.

experiment method

The test method refers to the second edition of "Pharmacological Experimental Methodology" P947. Rats were intraperitoneally anesthetized with urethane 1.2g/kg, electrodes were placed on the top of the skull to record EEG, femoral artery cannulation was used to record blood pressure, right external jugular vein was cannulated to the right ventricle, and blood was quickly drawn until the blood pressure dropped to 70mmHg , use an arterial clip to clamp the bilateral carotid arteries, continue to draw blood until the blood pressure is 40-45mmHg, it can be seen that the EEG becomes a flat line, start timing, open the carotid arteries after 10 minutes, and re-inject the drawn blood into the animal body to reduce the blood pressure. Return to normal, observe the EEG recovery time.

The test samples were administered orally, 3 days before the test, and administered 1 hour before the test on the day of the test. The doses were 2.5, 5.0, 10 and 20 mg/kg, and the volume was 10 ml/kg. The negative control group was given normal saline 10 ml/kg, and the positive control group was 5 mg/kg nimodipine.

All data are used X ± S said, statistical analysis using t test.

The results are shown in Table 1.

The EEG recovery time after reperfusion in the normal saline group was 977±134 seconds. The EEG recovery time of Tribulus terrestris at 2.5-20mg/kg oral administration was very significantly shortened, and the recovery time of the high-dose group was less than 1/2 of that of the normal saline group. Compared with the normal saline group, the differences were very significant (P< 0.01). The positive control nimodipine group also significantly reduced the EEG recovery time.

Table 1. The effect of oral administration of Tribulus terrestris on cerebral hemorrhagic hypotension combined with bilateral carotid artery ligation in rats

The effect of EEG recovery time after ischemia reperfusion (X±S). Group Dose (mg/kg) Number of animals (only) EEG recovery time (S) Normal saline 5ml/kg 7 977±134 Nimodipine 5.0 8 415±177 ^*** Tribulus terrestris 2.5 9 604±137 ^{** *}

5.0 8 468±255 ^***

10.0 10 379±112 ^***

20.0 6 375±101 ^***

P＞0.05, ^** P＜0.05, ^*** p＜0.01 compared with normal saline group

Tribulus terrestris has a very obvious protective effect on brain injury caused by global cerebral ischemia-reperfusion in rats.

Example 3

Effects of active components of Tribulus terrestris on cerebral ischemia after middle cerebral artery ligation in rats

experiment material

Test samples and control samples

Tribulus terrestris, nimodipine, source, batch number and preparation method are the same as embodiment 1

Test Instruments

SW-30 radio frequency bipolar coagulator (produced by Shanghai Testing Technology Institute Testing Instrument Factory)

307-6 Desktop Dental Drill Cart (produced by Shanghai Dental Equipment Factory)

SXP-1B Surgical Microscope (produced by Shanghai Medical Optical Instrument Factory)

experimental animals

Male wistar rats, weighing 260-300 grams.

experimental method

Take male Wistar rats with a body weight of 260-300g, 50 in total, and randomly divide them into 5 groups. The animals are anesthetized with 10% chloral hydrate 300mg/kg intraperitoneally, and the cerebral ischemia model is prepared according to the method of Tamura, and compared with postoperative Behavioral scoring was carried out when awake and 24 hours. The single-blind method was used for scoring. Those with postoperative scores lower than 9 points were excluded. The right middle cerebral artery of the rat was blocked for 24 hours. If it is confirmed that the right middle cerebral artery has been burned between the olfactory tract and the inferior cerebral vein and the brain parenchyma has no damage, the sections are stained, the weight of the necrotic area and the right hemisphere of the brain are weighed, and the percentage of the necrotic area and the right hemisphere of the brain is calculated . Statistical analysis of all data was performed by t-test.

The effective components of Tribulus terrestris are divided into three groups: low dose (2.5mg/kg), medium dose (5mg/kg) and high dose (10mg/kg). The samples were orally administered with a volume of 1ml/100g, the positive control group was orally administered nimodipine suspension 5mg/kg, and the negative control group was orally administered the same volume of normal saline, 30 minutes before the operation.

The test results are shown in Table 2.

As can be seen from Table 3, the middle dose group and the high dose group of the effective components of Tribulus terrestris can make the necrosis area brain tissue weight and the percentage of the necrosis area and the right hemisphere significantly reduce (p＜0.01), and the low dose The percentage of cerebral ischemic necrosis of rats in the group was reduced, but the difference was not significant (p=0.098), and the positive control (nimodipine group) could also reduce the range of cerebral ischemic necrosis in rats, but there was no significant difference (p=0.072) . The behavior scores of the rats at 24 hours after operation were also reduced in the middle and high dose groups of Tribulus terrestris, but only the middle dose group had a significant difference (P<0.01).

Table 2 Histological and behavioral effects of active components of Tribulus terrestris on rats with middle artery ligation

Animal behavior score Weight of right hemisphere necrosis area (g) Necrosis percentage group number 24 hours after operation Weight (g) (%) Negative control group 10 9.6±0.5 8.2±1.8 0.742±0.016 0.214±0.079 28.8±10.6 Positive control group 9 9.7±0.5 8.3±2.0 0.739±0.013 0.151±0.051 20.5±6.8 ^* Low-dose group 10 9.7±0.5 8.1±2.0 0.734±0.015 0.152±0.063 20.8±8.8 ^* Middle-dose group 10 9.6±0.5 ^**3 5.6 ±0.012 0.099±0.063 13.4±8.4 ^*** High dose group 9 9.7±0.5 6.7±2.6 0.732±0.020 0.105±0.053 14.2±6.8 ^*** *P＜0.10, **P＜0.05, ***P＜0.01 Compared with the negative control group, the effective components of Tribulus terrestris have obvious therapeutic effects on the focal cerebral infarction caused by ligation of the middle cerebral artery in rats, and the drug effect has a certain relationship with the dosage.

Example 4

Therapeutic Effect of Active Components of Tribulus Terrestris on Coronary Artery Ligation and Myocardial Infarction in Dogs

experiment material

Name: Active ingredients of Tribulus terrestris

Source: Shanghai Institute of Materia Medica, Chinese Academy of Sciences

Batch number; 971128

Content: Contains about 50% saponins

Appearance: This product is brown-yellow powder

Preparation: Accurately weigh this product, add 1% tragacanth gum solution and grind to make suspensions with concentrations of 10, 20 and 40mg/ml respectively

Control sample 1:

Name: Nitropine

Source: Shanghai Seventeenth Pharmaceutical Factory

Batch number; 851118

Content: 99.6%

Appearance: yellow crystalline powder

Preparation: Grind with 1% tragacanth gum solution to prepare a suspension with a content of 5mg/ml

Control sample 2:

Name: Xinnaoshutong

Source: Jilin Aodong Taonan Pharmaceutical Co., Ltd.

Batch number; 990402

Content: The content of each capsule weighs 152.42±3.99mg, containing 15mg of furostanol

Shape: brown yellow powder

Preparation: Grind with 1% tragacanth gum solution to prepare a suspension with a content of 100mg/ml, containing 10mg/ml saponin

Other medicines:

Sodium pentobarbital, batch number 860901, was imported from Foshan Chemical Experimental Factory and sub-packaged, and made into a 3.5% solution with normal saline.

Lidocaine injection, 100mg/5ml, batch number 990402, produced by Shanghai Xudong Haipu Pharmaceutical Co., Ltd.

5% Glucose + 0.9% Sodium Chloride Injection, bottled in 500ml, batch number 9708272, produced by Shanghai Changzheng Pharmaceutical Factory.

Nitrotetrazolium blue chloride, batch number 970421, sub-packaged by the medicinal material supply station of the Academy of Military Medical Sciences, made a 0.1 solution with 0.1M pH7.4 phosphate buffer.

Lactate dehydrogenase (LDH) enzymatic color synthesis, batch number 980906, produced by Shanghai Institute of Chemical Reagents.

Twenty-five hybrid dogs, both male and female, weighing 10.3±1.8kg, were randomly divided into 5 groups. Provided by the animal room of the hospital.

Test Instruments

Mingograf-82 eight-channel physiological recorder, produced by Seimens-Elema.

SC-3 electric artificial respirator, produced by Shanghai No. 4 Medical Device Factory.

GPS-2 high-frequency electric knife, produced by Wujin Radio Factory.

722 type grating spectrophotometer, produced by Shanghai Third Analytical Instrument Factory.

80-2 type centrifuge, produced by Shanghai Surgical Instrument Factory.

Dose settings:

The low dose is 10 mg/kg, the ratio of low, medium and high doses is 1:2:4, and the corresponding medium and high doses are 20 and 40 mg/kg respectively. One positive control is the calcium antagonist nifedipine, with a dose of 5 mg/kg; the other positive control, Xinnaoshutong, has a dose of 100 mg/kg, which is about 10 mg/kg in terms of saponins, which is equivalent to the content of saponins in the dose group in the test samples. The clinical administration route of this product is oral administration, which should be given by gavage consistent with the clinical route. However, considering that the animal’s gastric peristalsis slows down under anesthesia, and the emptying into the intestine is obviously prolonged, which will affect the absorption of the sample, so twelve Intestinal administration.

experiment method:

The dog was anesthetized by intravenous injection of 3.5% pentobarbital sodium 35 mg/kg, and at the same time, an anesthesia tracheal tube (Curity®, inner diameter 9.5 mm, produced in Thailand) was inserted through the mouth, connected to an artificial respirator, and breathed at a frequency of 30 times/min. Artificial respiration was performed at a time ratio of 1.5:1, and the tidal volume was adjusted to a positive respiratory pressure of 15 cmH ₂ O. The animal was fixed on the operating table lying on the left side, the femoral vein was separated at the right groin, and a catheter was inserted for infusion. A midline incision was made on the upper abdomen, the duodenum was carefully found out, a purse string suture was made 3cm from the pylorus and a tube was inserted, and a tee was connected for drug use. Open the thoracotomy at the 4th and 5th intercostal space on the left chest wall, open the chest cavity with a dilator, cut the pericardium 2 cm away from the vagus nerve along the direction of the nerve, and then suture the pericardium to the chest wall to fully expose the heart; Free coronary artery between the third branches, thread two threads under it, and suture the four corners of the 30-point epicardial electrode made according to the literature on the surface of the heart with a non-injured cardiovascular suture needle, the distance between the electrodes is 8 mm, and trace the normal limb leads synchronously I, II, III and pressurized limb leads aVR, aVL, aVF, and then trace the surface ECG at 30 points. When tracing the limb leads, the ECG calibration is 8mm/mv, and the epicardial electrogram calibration is 1mm/mv. Recording paper speed 25mm/s. Harris two-step ligation method: 2 minutes before the first ligation, iv lidocaine 5mg/kg from the femoral vein, a 6-gauge injection needle was added during ligation, and pulled out after ligation; 30 minutes after the first ligation, the second time was performed ligation. 10 minutes after the second ligation, the epicardial electrogram was recorded as the data before administration, and then the test sample was given from the duodenal catheter with a volume of 1ml/kg, and the negative control group was given the same volume of 1% tragacanth gum solution. Record the epicardial electrogram at 0.5, 1.0, 1.5, 2 and 3 hours after the administration, and measure the sum of the ST segment elevation (∑-ST) value of the electrocardiogram at each point and the number of ECG leads with ST segment elevation ≥ 2mv (N-ST). The animal was sacrificed at 3 hours, the heart was quickly taken out, the weight of the whole heart was weighed, the atrium and right ventricle were cut off, the weight of the left ventricle was weighed, put into the refrigerator for quick freezing for 1 hour, and the ventricle was cut parallel to the coronary sulcus under the coronary ligature. Cut into 5 slices of equal thickness, put into 0.1% nitro blue tetrazolium (N-BT) solution, shake and dye in a constant temperature water bath at 37°C for 10 minutes. The normal myocardium was stained dark blue, and the uncolored myocardium in the infarcted area was light red. The infarcted part was carefully cut out, the weight of the infarcted area was weighed, and the percentage of the weight of the infarcted area to the weight of the whole heart and left ventricle was calculated. Before coronary artery ligation and 3 hours after administration, 3ml of right ventricular blood was collected, centrifuged at 3000rpm for 15 minutes, and serum was taken to measure LDH. Add 20 μl of serum to be tested in the sample tube, 20 μl of LDH standard solution in the standard tube, 20 μl of normal saline in the blank tube, then add 2.0 ml of reaction solution in each tube, incubate at 37°C for 15 minutes, add 0.5 ml of stop solution, and adjust to zero with the blank tube. Adjust the reading of the standard tube to 500 in the C position of the spectrophotometer, and the LDH value of each test tube can be read directly on the instrument.

Data are presented as mean ± standard deviation ( X±S) said. The statistical test of the changes before and after the administration was performed by the t-test between the change percentages of each group at different time points and the change rate of the normal saline group at the same time.

test results:

Influence on the degree of myocardial ischemia in epicardial electrogram:

10 minutes after ligation, the sum of ST segments (∑ST) of electrocardiogram in each group increased significantly. The low dose of Tribulus terrestris decreased the degree of myocardial ischemia (∑ST), but there was no significant difference (P=0.088, t=3 hours); the middle dose of ∑ST decreased significantly after 3 hours after administration (P<0.05 ): the degree of myocardial ischemia decreased significantly (P<0.05) in the high-dose group after 1.5 hours of administration, and was very significant in the 3rd hour (P<0.01); the effect of reducing the degree of myocardial ischemia in the high-dose Tribulus terrestris group was greater than that in the low-dose group. In the positive control nifedipine group, after 1.5 hours of administration, there was a significant difference in ΣST segment decline (P<0.05), and after 3 hours, the difference was very significant compared with the negative control group (P<0.01); the positive control Xinnaoshutong There was a significant difference (P<0.05) between the two groups after administration for 3 hours, and the degree of decline was similar to that of the middle-dose group of Tribulus terrestris. In the negative control group, after giving 1% tragacanth gum solution, the ΣST segment rose slightly, and then slightly fluctuated. The test results are shown in Table 3.

The test results show that oral administration of 20 to 40 mg/kg of Tribulus terrestris has a significant therapeutic effect on the degree of myocardial ischemia in the electrocardiogram caused by coronary artery ligation in dogs.

Effects on myocardial ischemic extent (NST) of epicardial electrogram:

After oral administration of Tribulus terrestris 10mg/kg, NST decreased, but there was no significant difference (P=0.060, 3 hours); the NST value of 20mg/kg group decreased significantly compared with the negative control group at 3 hours; The ischemic extent decreased, but there was a significant difference from 2 hours onwards. The range of myocardial ischemia in the positive control nifedipine group was significantly reduced after 1.5 hours of administration (P<0.05); the positive control Xinnaoshutong group had no significant difference (P=0.061), and the degree of decline was slightly lower than that of Tribulus terrestris middle dose group. The range of myocardial ischemia in the negative control group did not change significantly over time. The test results are shown in Table 4.

The test results show that oral administration of 20 to 400 mg/kg of Tribulus terrestris can significantly reduce the range of myocardial ischemia in the electrocardiogram. The statistical method is the same as above.

Table 3 The effect of Tribulus terrestris on the sum of the ST segments (∑ST) of the heart surface electrogram in dogs with coronary artery ligation (mv, X±S, n=5) group Dose mg/kg index Before administration After administration (hours) 0 0.5 1 1.5 2 3 negative control NS1ml/kg X±S change% 164±44 166±621.2±28.0 188±8012.5±36.0 173±752.2±25.4 171±712.3±28.4 164±501.2±22.8 Nifedipine 5 X±S change% 285±122 206±38-18.5±30.5 168±37-34.5±21.5 ^* 142±52-47.0±13.3 ^** 141±4646.1±16.3 ^** 130±35-50.5±13.2 ^** Xinnaoshutong 100 X±S change% 179±59 160±67-11.3±21.8 135±71-26.2±22.6 138±75-24.8±26.7 132±71-27.7±25.8 110±59-39.5±22.3 ^* Tribulus terrestris 10 X±S change% 158±60 160±73-0.3±7.6 167±763.9±11.6 146±48-5.7±14.8 136±57-14.3±11.2 113±44-26.0±21.4 20 X±S change% 202±63 161±89-14.4±23.5 148±62-18.5±16.6 142±48-23.9±10.4 134±46-27.5±9.5 112±38-37.2±9.9 ^* 40 X±S change% 181±83 145±52-30.6±15.4 137±43-33.9±23.5 120±45-39.4±21.2 ^** 111±44-46.0±21.3 ^* 90±34-59.7±17.7 ^{** *} p<0.05, ^** p<0.01, compared with the change value of the normal saline group at the same time point.

Table 4 The effect of Tribulus terrestris on myocardial infarct size (NST) on surface electrogram of dogs with coronary artery ligation (points, X±S, n=5) group Dose mg/kg index Before administration After administration (hours) 0 0.5 1 1.5 2 3 negative control 1ml/kg X±S change% 24.6±2.1 23.6±2.6-3.4±14.6 24.6±1.70.8±13.8 25.4±2.53.7±12.3 24.8±2.80.9±8.7 26.2±3.76.4±10.6 Nifedipine 5 X±S change% 29.0±1.2 27.6±0.9-4.7±4.5 26.4±2.1-8.9±7.0 25.0±3.5-14.0±10.2 ^* 26.2±0.8-9.6±2.6 ^* 26.2±2.0-9.7±4.6 ^* Xinnaoshutong 100 X±S change% 24.6±2.3 24.8±3.11.2±13.1 22.4±4.4-9.0±14.9 23.8±2.7-2.7±13.0 24.0±3.2-2.1±13.1 22.0±4.7-10.9±14.2 Tribulus terrestris 10 X±S change% 25.2±3.3 25.8±2.33.3±12.2 25.0±3.1-0.4±9.1 24.0±3.1-4.0±11.8 23.4±2.1-6.2±11.4 21.8±4.4-12.9±16.6 20 X±S change% 26.5±2.6 25.3±2.8-2.7±3.3 24.3±2.9-8.6±3.9 23.8±3.6-9.7±5.0 24.5±3.9-5.9±4.9 22.5±6.0-7.9±3.8 ^* 40 X±S change% 25.0±5.2 24.0±3.9-9.3±8.6 24.3±4.3-6.0±9.9 22.8±4.6-12.9±21.3 21.8±3.9-16.7±12.8 21.8±4.5-24.2±20.3 ^{* *} p<0.05, ^** p<0.01, compared with the change value of the normal saline group at the same time point.

Example 5

Acute Toxicity Test of Intravenous Injection of Tribulus terrestris

Name: Active ingredients of Tribulus terrestris

Preparation: Accurately weigh 600mg of active components of Tribulus terrestris, add distilled water to 40ml, and then dilute to the required concentration step by step according to 1:0.8.

experiment method

The animals were divided into random groups, each dose group consisted of 10 male and 10 animals, and according to the pre-experimental results, the selected dose range was 122.88-300 mg/kg, and the dose interval was set at a ratio of 1:0.8. The administration group was intravenously injected with a volume of 0.2ml per 10kg body weight, and the control group was intravenously injected with the same volume of normal saline. The observation and recording methods are the same as the oral toxicity test. LD ₅₀ and 95% confidence limits were calculated by Bliss method.

test results

Animals in the control group received intravenous injection of normal saline for 7 days without any death.

After the administration, the animals in the administration group slowly appeared symptoms such as inactivity, loose hair, and poor appetite, without convulsions. Animals in the higher dose group died within 24 to 48 hours, some died within 2-3 days, and some died within 5 days. Autopsies of dead animals showed no obvious abnormalities in various organs. Surviving animals resumed normal activities after 3 days. The anatomical observation of the surviving animals in each group did not reveal any obvious abnormal changes in the main organs until the 8th day. The experimental results are shown in Table 5.

According to the experimental results, the acute LD ₅₀ (95% credible limit) of the effective components of Tribulus terrestris in mice was 187.73 (166.74-211.11) and 204.41 (182.12-230.31) mg/kg for male and female respectively. There were no gender differences in acute toxicity.

Table 5. Death distribution and LD ₅₀ value of intravenous injection of Tribulus terrestris

Dose log Animal Distribution of dead animals (days) Death Mortality Probability LD ₅₀ (95% CI) Sex

mg/kg dose number 4h 1 2 3 3 4 5 6 7 number of animals (%) unit mg/kg

300.00 2.4771 10 0 0 8 2 2 10 100 7.4

240.00 2.3802 10 0 1 5 2 0 0 0 0 0 8 80 5.84

187.73 Female 192.00 2.2833 10 0 0 3 1 0 0 0 5 50 5.00

153.60 2.1864 10 0 0 0 2 1 0 0 0 0 0 0 3 30 4.48 (166.74-211.11)

122.88 2.0895 10 0 0 0 0 0 0 0 0 0 0 10 2.60

300.00 2.4771 10 0 0 0 10 10 100 7.40

240.00 2.3802 10 0 1 6 0 0 0 0 0 0 7 70 5.52

204.41 Xiong 192.00 2.2833 10 0 1 1 0 0 0 0 3 30 4.48

(182.12-230.31)

153.60 2.1864 10 0 0 0 1 1 1 0 0 0 0 0 2 20 4.16

122.88 2.0895 10 0 0 0 0 0 0 0 0 0 0 0 0 2.60

Claims (11)

1. A class of furostanol saponins has the following structure R ₁ is -β-D-Glu _R2 is

_R3 is H; O R' is CH ₃ , H 2. The furostanol saponin according to claim 1, characterized in that the furostanol saponin has the following structure: 1b R ₁ =-β-D-Glu       

R ₃ =O R'＝H 3. The furostanol saponin according to claim 1, characterized in that the furostanol saponin has the following structure: 2a R ₁ =-β-D-Glu      

R ₃ =H R'= _CH3 4. The furostanol saponin according to claim 1, characterized in that the furostanol saponin has the following structure: 2b R ₁ =-β-D-Glu      

R ₃ =H R'＝H 5. The furostanol saponin according to claim 1, characterized in that the furostanol saponin has the following structure: 3a R ₁ =-β-D-Glu      

R ₃ =H R'= _CH3 6. The furostanol saponin according to claim 1, characterized in that the furostanol saponin has the following structure: 3b R ₁ =-β-D-Glu      

R ₃ =H R'＝H 7, a kind of preparation method of furostanol saponin as above, it is characterized in that adopting hydrothermal method to extract macroporous resin, its steps are as follows: a. Boil the whole herb of Tribulus terrestris and extract it twice with water, filter it, extract the residue three times with H ₂ O, filter it, discard the residue, and combine the water extract; b. The above water extract is placed at 5-10°C for 10-15 hours, and then filtered; c. The filtrate is passed through the macroporous resin, the extract is passed through, and then washed with water until the eluent is colorless, the water eluent is discarded, and then eluted with 60%-70% ethanol until no saponin is reacted; d. Remove 60%-70% ethanol eluent by distillation under reduced pressure until the specific gravity of the concentrated solution is 1.37-1.40; e. Add ethanol to the concentrated solution obtained above, and place it at 5-10°C for 10-15 hours; f. The above solution is filtered to remove the filter residue, and the filtrate is concentrated under reduced pressure to obtain dipping and put on the column; g. Rinse with chloroform, then use chloroform:methanol gradient elution to rinse, combine active ingredients, and concentrate under reduced pressure to obtain the product; h. The crude product is subjected to column chromatography with different proportions of silica gel, and 6 furostanols can be separated respectively. 8. the preparation method of furostanol saponin according to claim 7 is characterized in that the macroporous resin that described step C adopts is styrene nonpolar adsorption macroporous resin. 9. The preparation method of furostanol saponin according to claim 7, characterized in that said step g chloroform: methanol gradient elution is 85%-65%: 15%-35%. 10. The use of a class of furostanol saponins as mentioned above in the treatment of anti-ischemic cerebral infarction drugs. 11. The application of a class of furostanol saponins as described above in the treatment of myocardial infarction.

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