CN107789429A - Separation and purification method of bacopa monnieri total saponins extract and its application in the preparation of nervous system drugs - Google Patents

Abstract

The invention provides a method for separation and purification of the total saponins extract of Bacopa monniera: adding 80% ethanol to the medicinal material of Bacopa ^monniera for extraction, combining the extracts to concentrate; Sample liquid: HP-20 macroporous resin, wet packing column loading until complete, collecting the eluate, and evaporating to dryness under reduced pressure to obtain the total saponins extract of Bacopa monnieri. The purity of total saponins was 51.19%-55.67%, and the transfer rate of total saponins was 76.96%-78.29%. The invention provides the application of the total saponins extract of Bacopa monnieri in the preparation of nervous system medicines, including medicines for treating depression and medicines for treating dementia. The medicine of the invention overcomes the toxic and side effects of chemical medicines and has great clinical application prospect. The method for separating and purifying the total saponin extract of bacopa monnieri by adopting macroporous resin in the present invention is easy to operate, suitable for industrial production, and has great application value.

Description

Separation and purification method of bacopa monnieri total saponins extract and its application in the preparation of nervous system drug application

technical field

The invention relates to traditional Chinese medicines, in particular to traditional Chinese medicine extracts, in particular to a separation and purification method of bacopa monnieri total saponins extract and its application in the preparation of nervous system medicines.

Background technique

Bacopa monnieri is the whole herb of Bacopamonnieri (L.) Wettst, a plant of the family Scrophulariaceae, also known as bacopa monnieri. It has the effects of clearing away heat and cooling blood, detoxifying and reducing swelling. Clinically, it can also be used to treat respiratory diseases such as bronchitis and asthma. In the Indian Ayurvedic system of medicine, the plant is also believed to have the effects of enhancing intelligence and improving memory, delaying aging, preventing dementia, calming, hypnotizing, and antidepressant. Preparations made from its standard extracts have been marketed in India as an auxiliary drug for the treatment of memory impairment and senile dementia. Bacopa monnieri mainly contains triterpenoid saponins, and there are five main saponins in Bacopa monnieri saponins (dammarane-type triterpene saponins Bacopaside I (BM28), Bacoside A ₃ (BM37), Bacopaside II (BM29), Bacopasaponin Cisomer (BM35i) and Bacopasaponin C (BM35), the structural formulas of the five saponins are as follows:

Nervous system drugs include anti-Alzheimer's drugs and antidepressant drugs.

Dementia is an acquired and persistent mental retardation syndrome, which is caused by a variety of nervous system degeneration or ischemic processes, and manifests as a variety of high-level brain dysfunction. Dementia is a common disease of the elderly, and its incidence increases gradually with age. Studies have shown that the incidence of dementia is 7.0% at the age of 60, 4.1% at the age of 70, 13.0% at the age of 80, and 32.2% at the age of 90. Clinically, there are three common types of Alzheimer's disease, vascular dementia and mixed dementia. Patients with various dementias and memory impairment, especially those with Alzheimer's disease (AD) and vascular dementia (Vasculardementia, VD) are the most common diseases of cognitive decline. Alzheimer's disease (AD) is currently the leading type of dementia and the fourth leading cause of death in the elderly. With the aging of society, the morbidity and mortality of VD are increasing year by year, and it has become a disease that threatens human beings after heart disease and cancer in the world. on the other hand. With the accelerated pace of life of various social stress factors and the impact of increased pressure, depression presents a rising trend year by year. The lifetime incidence of depression in western developed countries is between 6% and 8%. With the gradual aging of the population, the incidence of depression among people over 60 years old will be as high as 20% to 50%. Under the intensity and fast-paced life and work style, the outbreak of depression patients is becoming more and more concentrated among young people. Depression is becoming a serious global problem. At present, there are many therapeutic drugs for nervous system diseases, including chemical drugs and some compound Chinese medicines. However, due to the adverse reactions of chemical drugs and the therapeutic effects of compound Chinese medicines, people have been working hard to find better therapeutic drugs. Therefore, it is an effective way to discover new drugs by studying natural products with nervous system activity derived from plants to find drugs with high efficiency and low toxicity.

Pharmacological studies in recent years have confirmed that Bacopa monnieri saponin extract has strong neuropharmacological activity and can be used for further development into antidepressant and senile dementia drugs. However, because these ingredients are low in medicinal materials, further purification is required to meet the needs of new drug development. Macroporous adsorption resin is a new type of non-ionic polymer compound, which is widely used in the separation and purification of active ingredients of traditional Chinese medicine. The macroporous resin is used to purify the bacoside extract, which can retain its active ingredients, remove invalid impurities, and improve the purity of total saponins. But Bacopa monnieri has not been developed into any form of medicine so far. The main reason is that the basic research on Bacopa monnieri is insufficient, the material basis of its medicinal effect is not clear, and its pathway of action is not clear, resulting in the lack of scientific theoretical guidance for clinical application and only relying on traditional experience. Therefore, it is necessary to further study the active ingredients of Bacopa monniera extract, clarify the medicinal ingredients of Bacopa monniera, improve its separation and purification method, and investigate its application in the preparation of medicines.

Contents of the invention

The technical problem to be solved by this invention is to overcome the above-mentioned weak points, research and design adopt macroporous resin to separate and purify bacopa monnieri saponin extract, improve the method for total saponin content and purity, develop bacopa monnieri total saponin extract in preparation application in medicine.

The invention provides a method for separating and purifying total saponins of Bacopa monnieri, which comprises the following steps:

(1) Get the Bacopa monnieri medicinal material and place it in an extraction tank, add 10-15 times of 80% ethanol to the weight of the medicinal material each time, extract 2-3 times, each 1-2h, combine the extracts, and concentrate under reduced pressure ( Pressure -0.6Bar, 60℃) to no alcohol smell;

(2) adding 95% ethanol to the extract to adjust the alcohol content to 30%, centrifuging, and suction filtering to prepare a sample solution with a medicinal material content of 0.311 g·mL ⁻¹ ;

(3) Take the pretreated HP-20 macroporous resin, pack it in a wet column (diameter-to-height ratio 1:10), take the sample solution (0.5kg medicinal material/L resin), and adjust the pH value to 2.0 with concentrated hydrochloric acid , load the sample at a flow rate of 0.5BV·h ^-1 until complete, remove impurities with 2BV of 30% ethanol (pH 2.0) and 5BV of 40% ethanol (pH 2.0) in sequence, flow rate 2.5BV/h; then use 2BV of 15% ethanol Rinse to remove acid at a flow rate of 2.5BV/h, then elute with 5BV of 60% ethanol at a flow rate of 2.5BV/h; the pretreatment method of the HP-20 macroporous resin is to wash with 95% ethanol 10 times the weight of the resin;

(4) Collect the eluate and evaporate to dryness under reduced pressure (pressure -0.6 Bar, 60° C.) to obtain the total saponins extract of Bacopa monnieri.

Bacopaside I (BM28), Bacoside A ₃ (BM37), Bacopaside II (BM29), Bacopasaponin C isomer (BM35i) and Bacopasaponin C isomer five triterpenes are mainly contained in the total saponin extract of Bacopa monnieri obtained after separation and purification Saponin compounds. The purity of the total saponins is 51.19%-55.67%, and the transfer rate of the total saponins is 76.96%-78.29%.

Another object of the present invention is to provide the application of the total saponins extract of Bacopa monniera in the preparation of nervous system medicines.

The nervous system medicines in the present invention include medicines for treating depression and medicines for treating dementia.

The invention provides the application of the total saponin extract of Bacopa monniera in the preparation of medicine for treating depression.

The invention provides the application of the total saponin extract of Bacopa monniera in the preparation of medicine for treating dementia.

The preparation of the medicine for treating dementia in the present invention includes the preparation of medicine for treating Alzheimer's disease (AD) and vascular dementia (Vascular dementia, VD).

The present inventor has carried out single-dose antidepressant activity screening of more than 50 kinds of monomers isolated and identified from Bacopa monnieri by using mouse FST and TST experiments, and the results show that the dammarane type triterpene saponin compound Bacopaside I ( BM28), Bacoside A ₃ (BM37), Bacopaside II (BM29), Bacopasaponin C isomer (BM35i) and Bacopasaponin C all have antidepressant activity, wherein bacopaside I (BM28) has the strongest pharmacodynamic effect, therefore, Bacopa monnieri The total saponin extract can be used to prepare medicine for treating depression.

The present inventor isolated and identified more than 40 kinds of monomer components from Bacopa monnieri, and purified them to obtain triterpenoid saponins bacopaside I (BM28), Bacoside A ₃ (BM37), and Bacopaside with strong neuroprotective activity. II (BM29), Bacopasaponin C isomer (BM35i) and Bacopasaponin C isomer. Preliminary pharmacological studies have shown that BM28 has significant protective effects on pheochromocytoma cells (PC12 cells) and rat cortical neurons induced by glutamate (L-Glutamate) and hydrogen peroxide (H ₂ O ₂ ), and its The protective effect is dose-dependent, showing its potential anti-senile dementia activity. Therefore, the total saponins extract of Bacopa monnieri can be used to prepare drugs for treating Alzheimer's disease and vascular dementia.

The medicine of the present invention is a pharmaceutical composition composed of the total saponins extract of Bacopa monnieri as an active ingredient and pharmaceutical adjuvants.

The pharmaceutical composition of the present invention is an oral preparation, an injection or a transdermal administration preparation.

The bacopa monnieri total saponins extract medicine provided by the invention provides a new medicine for treating depression and dementia for clinical use, and the bacopa monnieri total saponin extract is used as an active ingredient and has good drug effect. The medicine of the invention overcomes the toxic effect of chemical medicines and has great clinical application prospect. The method for separating and purifying the total saponin extract of Bacopa monnieri provided by the invention is simple to operate, suitable for industrial production, and has great application value.

Description of drawings

The leakage curve of Fig. 1 embodiment 2

The ordinate represents the volume of sample solution (ml), the abscissa represents the amount of total saponins leaked (mg), and the line represents the leakage trend

Figure 2 Example 2 Gradient elution curves of stock solution with different pH

Wherein the ordinate represents the total saponin elution transfer rate (%), the abscissa represents the elution fraction of different ethanol concentrations, and the line represents the total saponin elution at different pH values (rhombic pH=7.0, square pH=4.5, triangle pH=2.0) trend

Fig. 3 Example 2 Stability investigation of the acid loading stock solution.

Wherein the ordinate represents the total saponin content (mg), the abscissa represents the time (h) after acid addition, and the line represents the trend of total saponin content over time after acid addition

Fig. 4 Examination of the impurity-removing and eluting solvents in Example 2.

The ordinate represents the elution transfer rate (%), the abscissa represents the elution fraction of different ethanol concentrations, and the line represents the influence trend of the elution of different ethanol concentrations on the transfer rate of impurities (triangles) and total saponins (diamonds)

The purity of total saponins in the elution solvent of Fig. 5 embodiment 2.

Wherein the ordinate represents the total saponin purity (%), the abscissa represents the elution fraction of different ethanol concentrations, and the line represents the influence trend of different ethanol concentration elutions on the total saponin purity

Figure 6 Example 3 Bacopa monnieri total saponins extract on the impact of mice swimming immobility time (*P＜0.05)

Wherein the ordinate represents the forced swimming time (second) of the mice, and the abscissa represents the model group, the total saponin administration group, and the fluoxetine (positive drug) administration group

Figure 7 Example 3 Bacopa monnieri total saponins extract on the impact of mouse platform jumping test (*P＜0.05)

Among them, the ordinate represents the number of mistakes made by the mice, and the abscissa represents the normal group, the model group, the total saponins 100mg/kg administration group, and the total saponins 100mg/kg administration group

The protective effect of Fig. 8 embodiment 3 BM28 on glutamate (L-Glutamate)-induced rat cortical neurons and pheochromocytoma cells (PC12 cells) injury

The vertical axis represents the WST-8 conversion rate (%), and the horizontal axis represents the administration of different compounds.

Figure 9 shows that different compounds in Figure 8 are displayed in columns with different fillings, and the specific corresponding relationship is shown in the diagram.

detailed description

Example 1

Example 1-1 Bacopa monniera total saponin content detection method

(1) Instrument

Agilent-1200 high performance liquid chromatography (Agilent, USA); KS5200H ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); BP211D electronic balance (Sartorius, Germany); Milli-Q50SP pure water system (Millipore, USA).

(2) Reagents

Acetonitrile (chromatographically pure, Merck Company); phosphoric acid (chromatographically pure, Tedia Company); ultrapure water was made by Milli-Q pure water machine (Merk Millipore); other reagents were analytically pure.

(3) Test products and standard products

Bacopaside I (purity 95%), Chromadex, purchased from Shenzhen Hongcai Xianggen Biotechnology Co., Ltd.), Bacoside A ₃ , Bacopaside II, Bacopasaponin C isomer and Bacopasaponin C are self-made by the laboratory (purity 95%), after MS, ¹ H -NMR, ¹³ C-NMR determination to determine its chemical structure, the specific spectral data are as follows:

Bacoside A ₃

White needles, mp: 250-252°C (CH ₃ OH/H ₂ O), ESI-MS m/z: 951[M+Na] ⁺ ; ¹ H-NMR(Py-d5)H-3(δH 3.27 , 1H, dd, J=4.0, 12.0Hz), H-13 (δH 2.82, 1H, m), H-18 (δH 1.07, 3H, s), H-19 (δH 0.68, 3H, s), H-21 (δH 1.38, 3H, s), H-23 (δH 5.20, 1H, m), H-24 (δH 5.52, 1H, d, J=8.0Hz), H-26 (δH 1.69, 3H, s), H-27 (δH 1.67, 3H, s), H-28 (δH 1.28, 3H, s), H-29 (δH 1.06, 3H, s). ¹³ C-NMR (Py-d5) δc: 38.4 (C-1), 26.4 (C-2), 88.8 (C-3), 39.4 (C-4), 55.9 (C-5), 18.0 (C- 6), 35.7(C-7), 37.2(C-8), 52.7(C-9), 36.9(C-10), 21.4(C-11), 28.2(C-12), 36.8(C-13 ), 53.4(C-14), 36.5(C-15), 110.2(C-16), 53.7(C-17), 18.5(C-18), 16.0(C-19), 68.2(C-20) , 29.7(C-21), 45.1(C-22), 68.3(C-23), 126.8(C-24), 133.8(C-25), 25.2(C-26), 18.2(C-27), 27.5(C-28), 16.2(C-29), 65.5(C-30); 3-O-Glu: 104.8(C-1), 78.8(C-2), 88.4(C-3), 70.0( C-4), 77.6(C-5), 62.4(C-6); Ara(f): 109.6(C-1), 83.4(C-2), 77.4(C-3), 84.7(C-4 ), 61.8(C-5); G1u: 104.4(C-1), 75.1(C-2), 78.1(C-3), 71.3(C-4), 78.2(C-5), 62.1(C- 6).

Bacopaside II

White needles, mp: 249-251°C (CH ₃ OH/H ₂ O), ESI-MS m/z: 951[M+Na] ⁺ ; ¹ H-NMR(Py-d5)H-3(δH 3.28 , 1H, dd, J=4.0, 11.0Hz), H-13 (δH 2.83, 1H, m), H-18 (δH 1.08, 3H, s), H-22 (δH 2.60, 1H, d, J =8.0Hz), H-23a (δH 3.88, 1H, dd, J=2.0, 10.0Hz), H-23b (δH 4.72, 1H, dd, J=2.0, 10.0Hz), H-24 (δH 5.85, 1H, d, J=11.0Hz), H-26 (δH 1.69, 3H, s), H-27 (δH 1.61, 3H, s), H-28 (δH 1.28, 3H, s), H-29 ( δH 1.04, 3H, s). ¹³ C-NMR (Py-d5) δc: 38.1 (C-1), 26.3 (C-2), 88.8 (C-3), 39.4 (C-4), 55.8 (C-5), 17.9 (C- 6), 35.7(C-7), 37.1(C-8), 52.6(C-9), 36.8(C-10), 21.3(C-11), 28.2(C-12), 36.7(C-13 ), 53.1(C-14), 36.6(C-15), 109.9(C-16), 50.9(C-17), 18.4(C-18), 15.9(C-19), 71.5(C-20) , 26.8(C-21), 45.9(C-22), 65.7(C-23), 123.8(C-24), 132.5(C-25), 25.7(C-26), 18.1(C-27), 27.4 (C-28), 16.2 (C-29), 65.6 (C-30); 3-O-Glu: 104.8 (C-1), 78.7 (C-2), 88.4 (C-3), 70.0 ( C-4), 77.6(C-5), 62.4(C-6); Ara(f): 109.6(C-1), 83.4(C-2), 77.4(C-3), 84.6(C-4 ), 61.8(C-5); Glu: 104.4(C-1), 75.1(C-2), 78.1(C-3), 71.3(C-4), 78.2(C-5), 62.0(C- 6).

Bacopasaponin C isomer

White needles, mp: 250-252°C (CH ₃ OH/H ₂ O), ESI-MS m/z: 921[M+Na] ⁺ ; ¹ H-NMR(Py-d5)H-3(δH 3.23 , 1H, dd, J=4.0, 12.0Hz), H-13 (δH 2.81, 1H, m), H-18 (δH 1.06, 3H, s), H-19 (δH 0.69, 3H, s), H-21 (δH 1.37, 3H, s), H-22a (δH 1.65, 1H, dd, J=2.0, 14.0Hz), H-22b (δH 1.74, 1H, dd, J=2.0, 14.0Hz) , H-23 (δH 5.21, 1H, m), H-24 (δH 5.52, 1H, d, J=8.0Hz), H-26 (δH 1.68, 3H, s), H-27 (δH 1.66, 3H , s), H-28 (δH 1.27, 3H, s), H-29 (δH 1.05, 3H, s). ¹³ C-NMR (Py-d5) δc: 38.4 (C-1), 26.4 (C-2), 88.3 (C-3), 39.5 (C-4), 55.8 (C-5), 17.9 (C- 6), 35.7(C-7), 37.2(C-8), 52.7(C-9), 36.9(C-10), 21.4(C-11), 28.2(C-12), 36.8(C-13 ), 53.4(C-14), 36.5(C-15), 110.2(C-16), 53.6(C-17), 18.5(C-18), 16.0(C-19), 68.1(C-20) , 29.7(C-21), 45.1(C-22), 68.3(C-23), 126.8(C-24), 133.7(C-25), 25.2(C-26), 17.9(C-27), 27.4(C-28), 16.2(C-29), 65.5(C-30); 3-O-Ara: 105.3(C-1), 76.7(C-2), 83.2(C-3), 68.2( C-4), 65.4(C-5); Ara(f): 110.0(C-1), 83.5(C-2), 77.8(C-3), 84.6(C-4), 61.7(C-5 ); Glu: 104.6 (C-1), 74.9 (C-2), 77.6 (C-3), 71.2 (C-4), 78.1 (C-5), 62.2 (C-6).

Bacopasaponin C

White needles, mp: 222-223°C (CH ₃ OH/H ₂ O), ESI-MS m/z: 921[M+Na] ⁺ ; ¹ H-NMR(Py-d5)H-3(δH 3.27 , 1H, dd, J=4.0, 12.0Hz), H-13 (δH 2.83, 1H, m), H-18 (δH 1.09, 3H, s), H-19 (δH 0.75, 3H, s), H-21 (δH 1.38, 3H, s), H-22 (δH 2.62, 1H, d, J=10.0Hz), H-23a (δH 3.87, 1H, dd, J=2.0, 11.0Hz), H- 23b (δH 4.69, 1H, dd, J=2.0, 11.0Hz), H-24 (δH 5.84, 1H, d, J=10.0Hz), H-26 (δH 1.71, 3H, s), H-27( δH 1.63, 3H, s), H-28 (δH 1.29, 3H, s), H-29 (δH 1.07, 3H, s), H-30a (δH 4.29, 1H, d, J=7.0Hz), H-30b (δH 4.16, 1H, d, J = 7.0 Hz). ¹³ C-NMR (Py-d5) δc: 38.5 (C-1), 26.4 (C-2), 88.3 (C-3), 39.5 (C-4), 56.0 (C-5), 18.0 (C- 6), 35.8(C-7), 37.2(C-8), 52.8(C-9), 37.0(C-10), 21.4(C-11), 28.3(C-12), 36.8(C-13 ), 53.2(C-14), 36.6(C-15), 109.9(C-16), 51.0(C-17), 18.5(C-18), 16.0(C-19), 71.5(C-20) , 26.8(C-21), 46.0(C-22), 65.8(C-23), 123.8(C-24), 132.5(C-25), 25.6(C-26), 18.1(C-27), 27.6(C-28), 16.2(C-29), 65.5(C-30); 3-O-Ara: 105.3(C-1), 76.6(C-2), 83.1(C-3), 68.1( C-4), 65.3(C-5); Ara(f): 110.1(C-1), 83.4(C-2), 77.8(C-3), 84.8(C-4), 61.9(C-5 ); Glu: 104.6 (C-1), 74.9 (C-2), 77.8 (C-3), 71.3 (C-4), 78.1 (C-5), 62.4 (C-6).

(4) Preparation of the solution

1. Preparation of reference solution

Accurately weigh Bacopaside I 10.07mg, Bacopaside A ₃ 10.51mg, BacopasideII 10.46mg, Bacopasaponin C isomer 10.47mg, Bacopasaponin C 10.43mg. Dissolve it in methanol, dilute it in a 25ml volumetric flask, and store it in a refrigerator at 4°C for later use.

2. Preparation of the test solution

Sample Bacopa monnieri total saponins extract (self-produced Example 1-2) is directly ground into fine powder, takes 10.0 mg of powder, accurately weighs, adds appropriate amount of methanol to dissolve and settles to a 10ml volumetric flask, shakes up, centrifuges, The supernatant was obtained by passing through a 0.22 μm filter membrane;

(5) HPLC Determination of Bacopa monnieri Total Saponin Extract Content

Adopt AgelaPromsil C ₁₈ (5μm, 4.6mm×250mm) chromatographic column, with 32.5% acetonitrile/67.5% phosphoric acid (NaOH adjust pH=3) as mobile phase, 1ml/min flow rate, 30 ℃ column temperature, 205nm detection wavelength, elution The content determination of five saponin components can be realized simultaneously in 45 minutes.

Embodiment 1-2 prepares Bacopa monnieri total saponins extract

Take 5kg of Bacopa monniera medicinal material and place it in a multifunctional extraction tank, add 50kg of 80% ethanol each time, extract 3 times, each time for 1h, combine the extracts, concentrate (pressure -0.6Bar, 60°C) until there is no alcohol smell , add 95% ethanol to adjust the alcohol content to 30%, centrifuge, and suction filter to prepare a sample solution with a medicinal material content of 0.311 g·mL ⁻¹ . Take the HP-20 macroporous resin that has been pretreated (washed with 95% ethanol 10 times the weight of the resin), and pack it into a column by wet method (diameter-to-height ratio 1:10), with a column volume of 500 mL. Take 800mL of sample solution (0.5kg medicinal material/L resin), adjust the pH value to 2.0 with concentrated hydrochloric acid, load the sample at a flow rate of 0.5BV·h ^-1 until complete, and then use 2BV of 30% ethanol (pH 2.0), 5BV 40% ethanol (pH 2.0) for impurity removal, flow rate 2.5BV/h, use 2BV 15% ethanol, rinse and remove acid, flow rate 2.5BV/h, then use 5BV 60% ethanol for elution, flow rate 2.5BV/h, collect and wash Liquid removal, vacuum evaporation to dryness (pressure-0.6Bar, 60 ℃) to get bacopa monnieri total saponins extract 141g, through HPLC detection, the purity of total saponins is 55.67%, the transfer rate of total saponins is 78.29%.

Through the comparison of the peak retention time of the test product peak and the standard product peak, five triterpenoid saponin compounds Bacopaside I, Bacoside A ₃ , Bacopaside II, Bacopasaponin C isomer and Bacopasaponin C were identified, and each compound was analyzed by HPLC-UV external standard method Quantitative.

Embodiment 1-3 prepares Bacopa monnieri total saponins extract

Take by weighing 5 kg of Bacopa monniera medicinal material and place it in a multi-functional extraction tank, add 60 kg of 80% ethanol at a time, extract 2 times, each 2h, combine the extracts, concentrate (decompression, pressure-0.6Bar60°C) to nothing Alcoholic taste, add 95% ethanol to adjust the alcohol content to 30%, centrifuge, and filter with suction to prepare a sample solution with a medicinal material content of 0.311 g·mL ⁻¹ . Take the HP-20 macroporous resin that has been pretreated (same as above), and pack it into a column by wet method (diameter-to-height ratio 1:10), with a column volume of 500 mL. Take 800mL of sample solution, adjust the pH value to 2.0 with concentrated hydrochloric acid, load the sample at a flow rate of 0.5BV·h ^-1 until complete, and then use 2BV of 30% ethanol (pH 2.0), 5BV of 40% ethanol (pH 2.0 ) to remove impurities, flow rate 2.5BV/h, adopt 2BV 15% ethanol, wash and remove acid, flow rate 2.5BV/h, then use 5BV 60% ethanol to elute, flow rate 2.5BV/h, collect eluate, evaporate to dryness under reduced pressure (Pressure-0.6Bar60 DEG C) obtain bacopa monnieri total saponin extract 146g, through HPLC detection, the purity of total saponin is 51.62%, and total saponin transfer rate is 77.97%.

Embodiment 1-4 prepares Bacopa monnieri total saponins extract

Weigh 5 kg of Bacopa monnieri medicinal material and place it in a multifunctional extraction tank, add 75 kg of 80% ethanol each time, extract 3 times, each 1 hour, combine the extracts, concentrate (decompression, 60 ° C) to no alcohol smell, Add 95% ethanol to adjust the alcohol content to 30%, centrifuge, and filter with suction to prepare a sample solution with a medicinal material content of 0.311 g·mL ⁻¹ . Take the HP-20 macroporous resin that has been pretreated (same as above), and pack it into a column by wet method (diameter-to-height ratio 1:10), with a column volume of 500 mL. Take 800mL of sample solution, adjust the pH value to 2.0 with concentrated hydrochloric acid, load the sample at a flow rate of 0.5BV·h ^-1 until complete, and then use 2BV of 30% ethanol (pH 2.0), 5BV of 40% ethanol (pH 2.0) Impurity removal, flow rate 2.5BV/h, using 2BV 15% ethanol, washing acid removal, flow rate 2.5BV/h, then elution with 5BV 60% ethanol, flow rate 2.5BV/h, collecting eluate, evaporated to dryness under reduced pressure ( 60 DEG C) to obtain 146g of total saponins extract of Bacopa monnieri, detected by HPLC, the purity of total saponins is 51.19%, and the transfer rate of total saponins is 76.96%.

Example 2

Study on separation and purification method of Bacopa monnieri saponin extract

1. Instruments and reagents

Anglient 1200 liquid chromatograph, with quaternary pump, ALS, TCC, VWD and Chemstation workstation (Agilent Technology Co., Ltd., USA), DXTQZ-100 small extraction and concentration unit (Nanjing Renbao Pharmaceutical Equipment Co., Ltd.), R200 rotary evaporator DL-2010 low-temperature coolant circulation pump (Shanghai Shensheng Technology Co., Ltd.), LXJ-IIB high-speed centrifuge (Shanghai Anting Scientific Instrument Factory), PHS-3E pH meter (Shanghai Yidian Scientific Instrument Co., Ltd.) , ZHWY-211B constant temperature culture oscillator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), XP205 analytical balance (0.01mg, Mettler-Toledo Instrument Co., Ltd.);

Bacopaside I reference substance (purity 87%, provided by Shanghai Merrill Chemical Technology Co., Ltd.); Bacopa monniera medicinal material (produced in Zhangzhou, Fujian Province, identified as Bacopa monniera by Professor Zhang Hanming, School of Pharmacy, School of Pharmacy, Second Military Medical University) ; D101 (net type, Tianjin Haiguang Chemical Co., Ltd.), AB-8, NKA-9, H-20, HPD-300 (all purchased from Zhengzhou Qinshi Technology Co., Ltd.), HP-20 (Mitsubishi Chemical Corporation) type macroporous resin; acetonitrile (chromatographically pure, Shanghai Xingke Biochemical Co., Ltd.), concentrated hydrochloric acid, phosphoric acid (both analytically pure, Sinopharm Chemical Reagent Co., Ltd.).

2. Methods and results

2.1 Determination of total saponins content in Bacopa monnieri

2.1.1 Chromatographic conditions

Chromatographic column: Shiseido Capce11-C18 (5μm, 4.6×250mm); mobile phase: acetonitrile-0.2% phosphoric acid (NaOH to adjust pH 5.0)=31.5:68.5; flow rate 1mL·min ^-1 , injection 20μL, column temperature 30℃, The detection wavelength is 205nm.

2.1.2 Preparation of reference solution

Accurately weigh 4.53mg of Bacopaside I reference substance, put it in a 10mL measuring bottle, add an appropriate amount of methanol, after ultrasonic dissolution, let cool, add methanol to constant volume, and shake well to obtain a 0.392mg·mL ^-1 reference substance solution.

2.2 Preparation of sample solution

Weigh 5 kg of Bacopa monniera medicinal material and place it in a multifunctional extraction tank, add 50 kg of 80% ethanol each time, extract 3 times, each time for 1 hour, combine the extracts, concentrate (60 ° C, -0.6 Bar) until no alcohol smell , add 95% ethanol to adjust to alcohol content 30%, centrifugal, suction filtration, make the loading sample solution that medicinal material content is 0.311g mL ^-1 , measure (2.1.1HPLC-UV external standard method) wherein the content of Bacopaside I The content of total saponins is 1.464mg·mL ^-1 , the content of total saponins is 4.684mg·mL ^-1 , and the purity of total saponins is 6.49%.

2.3 Investigation and test of macroporous resin purification process

2.3.1 Macroporous resin pretreatment

The macroporous resin was soaked in 95% ethanol for 24 hours, fully swollen, wet-packed, eluted with ethanol until the effluent was mixed with distilled water in equal amounts and no white turbid precipitate was found, washed with distilled water until no alcohol smell and set aside.

2.3.2 Macroporous resin type screening (static test)

Take pretreated different types of HP-20, H20, AB-8, HPD-300, NKA-9, D101 macroporous adsorption resins (equivalent to 5g of dry resin, converted based on the water content of each resin), put them in a 250mL cone Add 100mL each of the sample solution into a shaped bottle, seal it tightly, shake it in a constant temperature oscillator (100r·min ^-1 ) for 24h, make it reach adsorption equilibrium, let it stand still, draw an appropriate amount of supernatant, and measure the total Saponin content. Suction filter each sample, wash the resin bed with 100mL deionized water respectively, then transfer the resin to a 250mL Erlenmeyer flask, add 100mL95% ethanol, seal it tightly, shake it at the same speed as above for complete desorption, let stand, absorb An appropriate amount of supernatant was used to measure the total saponin content, and calculate the saturated adsorption capacity and desorption rate. See Table 1. The results show that the HP-20 macroporous resin has good adsorption and desorption properties, so HP-20 macroporous resin is selected for the next experiment.

Saturation specific adsorption capacity = (total saponin content in the adsorption stock solution - total saponin content in the supernatant after adsorption)/dry resin amount

Desorption rate = total saponin content in desorption solution/saturated adsorption capacity × 100%

Table 1 Static test results

2.3.3 Inspection of loading capacity

Take the pretreated HP-20 macroporous resin, and wet-pack the column with a column volume of 500mL. Take 1400mL of the original sample solution, load the sample at a flow rate of 0.5BV·h ^-1 , collect once every 200mL, measure the total saponin content in each collected solution, take the total saponin content measured in the collected solution as the ordinate, and the volume of the above sample solution is The leakage curve is drawn on the abscissa, as shown in Figure 1. When the total saponins start to leak, the loading volume of the stock solution is 1100mL, that is, the resin can absorb up to ^2200mL of the stock solution per liter of resin. The saturated loading volume is 1L resin column corresponding to 0.67kg medicinal material. In order to avoid the loss of raw materials caused by overloading of the sample solution, the optimal sample volume is about 0.4-0.5 kg of medicinal materials corresponding to 1 L of resin column.

2.3.4 Inspection of sample mass concentration

Take 3 chromatography columns and load the same amount of macroporous resin with a column volume of 500mL. Take 3 parts of 400mL sample loading stock solution, dilute them with 30% ethanol to volumes of 400, 800, and 1600mL respectively, and then load the samples respectively at a flow rate of 0.5BV·h ^-1 . Rinse the resin bed with 2.5BV each, with a flow rate of 1.5BV·h ^-1 , measure the total saponin content in the effluent and 30% and 80% eluent, calculate the adsorption and desorption rate, and obtain the column adsorption of the original solution diluted to 400, 800, and 1600mL The desorption rates were 94.20%, 98.00%, and 98.00%, respectively. Since the concentration of the loaded drug solution has little to do with its elution law, it is more appropriate to use the undiluted drug solution as the original sample solution in consideration of the sample volume and sample loading time.

2.3.5 Inspection of sample loading flow rate

Take 3 chromatography columns and load the same amount of macroporous resin with a column volume of 500mL. Take 3 parts of 400mL of the original sample solution, and load the sample at different flow rates of 0.5, ^{1, and 2BV·h -1 .} Measure the effluent and 30%, the total saponin content in the 80% eluate, calculate the adsorption and desorption rate, and draw the column adsorption and desorption rate of 0.5, 1, 2BV h ^-1 as the sample loading flow rate is respectively 93.70%, 94.20%, 98.30%. The flow rate of the sample has little relationship with the elution law, but considering the saturated adsorption of the resin, the lower the flow rate of the sample, the more complete the adsorption of the total saponins in the liquid by the resin. With the increase of the sample amount, the possibility of column leakage The smaller the sensitivity, so consider using a flow rate of 0.5BV·h ^-1 for sample loading.

2.3.6 pH investigation of sample solution

Take 3 chromatography columns and load the same amount of macroporous resin with a column volume of 500mL. Take 600mL of 3 parts of the loading stock solution, adjust the pH to 2.0, 4.5, and 7.0 with concentrated hydrochloric acid, and then load the sample respectively at a flow rate of 0.5BV·h ^-1 , and then use 30%, 35%, and 40% ethanol (pH adjusted Same as loading stock solution) each 5BV washes the resin bed, then adopts 70%, 80% ethanol each 5BV washes the resin bed, collects once every 2.5BV, and measures the total saponin content in each eluent. As shown in Figure 2, when the sample pH of the stock solution is 7.0 and 4.5, the transfer rate of total saponins in 30% to 45% ethanol is 33.5%, 13.3%, and the transfer rate in 70% to 80% ethanol is 52.8%, 82.1% %, and when the loading pH of the stock solution was 2.0, the transfer rate of total saponins in 70% ethanol was 100.0%, so adjusting the pH of the stock solution to 2.0 was the best loading.

Adjust the pH of the loading stock solution to 2.0 Stability investigation: Take 40mL of the sample stock solution with an alcohol content of 30%, add concentrated hydrochloric acid to adjust the pH to 2.0, and then take samples at the time points of 1, 2, 4, 6, and 12 hours after adding concentrated hydrochloric acid , Determination of the total saponin content in it, the results are shown in Figure 3, the total saponin content of the liquid medicine does not change much within 12 hours of adding acid, and is relatively stable, so it is determined that it is more appropriate to load the sample within 12 hours after adding acid to the liquid medicine.

2.3.7 Examination of impurity removal and elution solvent and dosage

Take a chromatographic column and load it into a macroporous resin with a column volume of 500mL. According to the optimal loading volume, optimal loading concentration, optimal loading speed and pH loading, use 30% (2BV), 40%, 45%, 50%, 55%, 60%, 65%, Each 5BV of 70% ethanol washes the fat bed (wherein 30%, 40%, 45% ethanol pH is 2.0), collects once every 2.5BV, measures the total saponin content therein respectively, and measures the paste amount to calculate the total saponin purity as shown in Figure 4 And Fig. 5, total saponin begins to elute in 45% ethanol, and impurity is in effluent, and the total transfer rate in 30%～40% ethanol reaches 87.86%, so 40% ethanol (pH 2.0) can be selected as impurity removal solvent , 30% ethanol (pH 2.0) of 2BV, 40% ethanol of 5BV can completely remove this part impurity; The transfer rate of total saponin in 45%-60% ethanol is 96.81%, wherein the total purity of total saponin reaches 47.0%, However, the transfer rate in 40%-70% ethanol was 99.13%, and the purity was not improved. Therefore, 60% ethanol was selected as the elution collection solution, and the volume was set at 5BV.

Investigation on the acid removal steps of the total saponin eluent: adjust the pH of the stock solution and the impurity removal solvent to 2.0, add a certain amount of concentrated hydrochloric acid, and the total saponin collection liquid contains a certain amount of acid, which may affect the properties and storage of the total saponin intermediates in the future. Therefore, it was decided to use low-concentration ethanol to rinse the resin bed after the impurity removal step. After the collection solution was neutral, 60% ethanol was used to elute and collect the total saponins. The experimental results show that the acid in the eluate can be completely removed by washing 2BV with 15% ethanol.

2.3.8 Investigation on the flow rate of impurity removal and elution

Take 3 chromatography columns and load the same amount of macroporous resin with a column volume of 500mL. Load the sample according to the optimal sample volume, optimal sample concentration, optimal sample speed and pH. After completion, use 2BV 30% acidic ethanol (pH 2.0), 5BV 40% acidic ethanol (pH2.0) to remove impurities, 2BV 15% ethanol for acid removal, 5BV 60% neutral ethanol for elution, impurity removal and elution flow rates were 2.5, 4, 6BV·h ^-1 respectively, collected each part, and determined the total saponin content therein, and the total saponin content in each part The transfer rate is shown in Table 2. Compared with different impurity removal and elution flow rates, the transfer rate of total saponins reaches about 80%, which is not much different. However, when the flow rate is 4BV·h ^-1 , the total saponins at 40% ethanol are eluted , so the impurity removal and elution flow rate should not be too large. 2.5BV·h ^-1 is suitable.

Table 2 Investigation of different impurity removal and elution flow rates

2.3.9 Investigation of diameter-to-height ratio

Take 3 chromatographic columns of the same type, and add appropriate amount of treated resin to each, so that the diameter-to-height ratios are about 1:7, 1:11, 1:15 (1BV=44mL, 69mL, 95mL), Take the loading stock solution corresponding to each resin amount and load the sample at the optimal loading speed, then wash the resin bed with 500mL each of 30% and 80% ethanol in sequence at a flow rate of 2.5BV·h ^-1 , measure the effluent and 30%, 80% % total saponin content in the eluent, calculate the adsorption and desorption rate, and obtain the adsorption and desorption rate of the columns whose diameter-to-height ratio is 1: 7, 1: 11, 1: 15, 45.10%, 70.10%, 93.10%, with the column length The increase of , the elution quality of total saponins gradually increases, and the better the elution effect, so choose a column with a smaller diameter-to-height ratio for the experiment, and the better the effect. However, in actual industrial production, the diameter-to-height ratio requirements can be met, so a resin column with a diameter-to-height ratio of 1:10 is selected.

2.3.10 Verification test

Take 3 chromatography columns and load the same amount of macroporous resin with a column volume of 500mL. Test according to the preferred purification process, and measure the amount of paste, calculate the transfer rate of total saponins in 60% ethanol of the three columns to be 77.97%, 76.96%, 78.29%, respectively, and the purity of total saponins to be 51.62%, 51.19%, 55.67% , indicating that the process is stable and the sample quality is controllable.

3. Conclusion

When using the static test to screen macroporous resin models, the adsorption capacity of different types of macroporous resins is quite different, and the total saponins of Bacopa monniera are weakly polar substances, and its content in weakly polar resins (HP-20, HPD-300) The adsorption capacity is significantly higher than that of polar resin (NKA-9) and non-polar resin (D101, H-20). The adsorption and desorption rate of HP-20 in the first two resins is higher, and it is used as another resin The cycle inspection test shows that the adsorption and desorption performance of HP-20 resin remains unchanged after being used 5 times, so HP-20 resin is selected for the test.

Bacopaside in the total saponins of Bacopa monnieri has a sulfonic acid group in its molecular structure, which is more sensitive to the ethanol concentration of the sample solution and impurity removal solvent. Adjusting the pH to acidity will help the Bacopaside solution to exist in the form of free molecules. Enhance the adsorption of the resin, reduce its leakage loss caused by the increase of ethanol concentration in the impurity removal stage, and adjust the pH of the original solution and the impurity removal solvent to 2.0, Bacopaside dew and the other four saponins have the same adsorption and desorption trend, which is more conducive to the purification process Transfer of total saponins. The setting of the low-concentration ethanol acid removal step after impurity removal makes the total saponin intermediates more stable.

Generally, the sample solution used in the macroporous resin column purification process is an alcohol-free solution. During this experiment, it was found that loading the sample with an alcohol-containing drug solution can quickly and effectively remove the alcohol in the drug solution before and during the sample loading process. Large polar impurities, and because the liquid contains alcohol can greatly increase the speed of sample loading, reduce the blockage of large polar impurities on the resin column bed, and shorten the column purification process cycle. According to the experimental investigation, 30% alcohol content can not only ensure the solubility of the target saponin, but also ensure that the target saponin will not be eluted during the sample loading process, which is the best alcohol content. In view of the fact that the alcohol content of the medicinal liquid concentrate cannot be directly and quickly measured during the production process, the process formulated in this step is to concentrate the medicinal material extract to no alcohol and then add alcohol to 30% alcohol by volume.

Example 3

Pharmacodynamic test of total saponins extract from Bacopa monnieri

(1) Research on antidepressant activity

The antidepressant activity of the total saponins extract of Bacopa monnieri prepared in Example 1-2 was studied using the forced swimming test (FST) in mice.

Method and steps: Experimental animals: 20g male ICR mice (commercially available from Shanghai Slack Experimental Animal Co., Ltd.), 12 in each group;

Experimental administration: administration by intragastric administration, continuous administration for six days; a dose group (200 mg/kg) of total saponins, positive drug fluoxetine (12 mg/kg), and an equivalent amount of normal saline were given to the model group.

Experimental process: 40 minutes after the administration of the mice on the sixth day, a forced swimming test was performed, and the mice were adapted for 2 minutes to record the immobility time within 4 minutes.

From the experimental results (see Table 1-1), it can be seen that the total saponins extract of Bacopa monnieri significantly reduces the swimming immobility time of mice, indicating that the total saponins extract of Bacopa monnieri has a significant antidepressant effect.

(2) Anti-senile dementia activity research,

The total saponins extract of Bacopa monnieri prepared in Example 1-2 was subjected to a mouse platform test.

Experimental animals: 20g male ICR mice, 12 in each group;

Experimental administration: intragastric administration was adopted for six consecutive days; two dosage groups (100 and 200 mg/kg) were set up for total saponins, and the model group was given an equal amount of normal saline. Experimental process: The animal was shocked by a 36V alternating current, and the normal reaction was to jump to the platform to avoid the noxious stimulus. After training for 5 minutes, record the number of times the mice received electric shocks (the number of errors) as a learning result. The experiment was repeated after 24 hours, which was a memory retention experiment. Record the number of animals subjected to electric shock, the latency period for jumping off the platform for the first time, and the total number of errors within 3 min.

From the experimental results (see Fig. 6, Fig. 7), it can be seen that the total saponin extract of Bacopa monniera is significant

Reduced the number of mistakes made by mice jumping off the platform.

The present inventors isolated and identified more than 40 monomer components from Bacopa monnieri, and purified them to obtain a triterpenoid saponin compound bacopaside I (BM28, whose structure is shown in formula I) with strong neuroprotective activity. The results of previous pharmacological studies (Figure 8) showed that BM28 had significant effects on the injury of pheochromocytoma cells (PC12 cells) and rat cortical neurons induced by glutamate (L-Glutamate) and hydrogen peroxide (H ₂ O ₂ ). Protective effect, and its protective effect is dose-dependent, showing its potential anti-senile dementia activity.

At the same time, the inventors used mouse FST and TST experiments to screen more than 50 monomers isolated and identified from Bacopa monniera for antidepressant activity in a single dose, and the results showed that BM28 has the strongest pharmacodynamic effect (Table 1-1, compound 50 mg/kg; positive drug fluoxetine (commercially available, Lilly Suzhou Pharmaceutical Co., Ltd.) 10 mg/kg).

Table 1-1 Screening results of BM28 for antidepressant animal models

In summary, the antidepressant and anti-senile dementia activity of Bacopa monniera is mainly due to its saponin components, and one of the main saponin components, Bacopaside I, has the best activity after screening.

(2) Study on the antidepressant and anti-senile dementia effects of Bacopaside I

Based on the above pharmacological activity research, the research group conducted a systematic study on the pharmacodynamics and mechanism of action of Bacopaside I on antidepressant and anti-senile dementia.

1. Bacopaside I antidepressant pharmacodynamics and mechanism of action

Bacopaside I has a significant antidepressant effect in animal models of depression induced by behavioral despair (TST, FST) and in animal models of depression induced by drugs (reserpine, 5-HTP). Further research found that its antidepressant effect may be related to the following mechanisms: 1) It may involve the norepinephrine system and increase the levels of NE and DA in the brain; 2) It may improve the activity of anti-oxidative stress enzymes in the brain; 3) It may not involve 5- 4) It may not work by inhibiting the activity of monoamine oxidase (MAO) in the brain.

2. Study on the pharmacodynamics and mechanism of action of Bacopaside I in anti-senile dementia

First, the dosage of Bacopaside I with significant anti-senile dementia activity was screened through the scopolamine-induced short-term memory loss model. Then according to this dose, Bacopaside I was used to treat APP/PS1 double transgenic Alzheimer's mice. Through Morris water maze, thioflavin staining and Nissl staining, it was clear that Bacopaside I treated APP/PS1 double transgenic Alzheimer's mice. The efficacy of the drug in mice; and the conclusion of the research on its mechanism of action is as follows: 1) Bacopaside I improves the activity of anti-oxidative stress enzymes in the brain of APP/PS1 double transgenic Alzheimer mice; 2) its mechanism of action may not be through the inhibition of acetylcholine in the brain Esterase activity.

Secondly, the anti-ischemia effect of Bacopaside I was evaluated by middle cerebral artery ischemia-reperfusion injury (MCAO) model. The experiment found that Bacopaside I has a protective effect on MCAO rats through neurological function score, degree of cerebral edema, cerebral ischemic area, pathological tissue section and brain enzyme activity detection, and its effect may be related to the following mechanisms: 1) Increase the antioxidant capacity in the brain 2) clear the lipid reaction product MDA in the brain and increase the content of the cerebral vasodilator NO; 3) improve the energy metabolism and energy metabolism-related enzymes in the brain of injured rats.

(3) Summary

Although Bacopaside I has a good antidepressant and anti-senile dementia effect, the preparation of Bacopaside I is very difficult, time-consuming and labor-intensive. Bacopaside I is one of the main saponins of Bacopa monnieri, so Bacopa monnieri saponin extract is used as The research and development of new drugs for the prevention and treatment of Alzheimer's disease and anti-depression has great advantages and good prospects.

Claims (10)

1. the isolation and purification method of bacopa monnieri total saponin extracts, it is characterised in that this method comprises the following steps：

(1) take bacopa monnieri medicinal material to be placed in extractor, add 80% ethanol W/V of 10-15 times of medicinal material amount every time, extract 2- 3 times, each 1-2h, merge extract solution, be concentrated under reduced pressure, -0.6Bar, 60 DEG C to without alcohol taste；

(2) it is 30% that extract solution, which adds 95% ethanol and is adjusted to alcohol content, centrifuges, filters, it is 0.311gmL that medicinal material content, which is made,^-1 Sample solution；

(3) the HP-20 macroreticular resins that pretreatment finishes are taken, wet method dress post, blade diameter length ratio 1: 10, take sample solution 0.5kg medicinal materials/L trees Fat, it is 2.0 to be adjusted to pH value with concentrated hydrochloric acid, with 0.5BVh^-1Flow velocity loading to complete, successively with 2BV 30% ethanol pH 2.0, 5BV 40% ethanol pH 2.0 cleans, flow velocity 2.5BV/h；Then deacidified with the alcohol flushings of 2 BV 15%, flow velocity 2.5BV/h, The ethanol elutions of 5BV 60%, flow velocity 2.5BV/ are used again；The preprocess method of the HP-20 macroreticular resins is with 10 times of resin weights 95% alcohol flushing of amount；

(4) eluent is collected, is concentrated under reduced pressure, -0.6Bar, 60 DEG C obtains bacopa monnieri total saponin extracts.

2. the isolation and purification method of bacopa monnieri total saponin extracts according to claim 1, it is characterised in that described point From containing bacopaside IBM28, Bacoside A in obtained bacopa monnieri total saponin extracts₃BM37, Bacopaside II BM29, Bacopasaponin C isomerBM35i and Bacopasaponin C five triterpene saponin chemical combination of isomer Thing；Structural formula is as follows：

。

3. the isolation and purification method of bacopa monnieri total saponin extracts according to claim 1, it is characterised in that obtain The purity of bacopa monnieri total saponin extracts total saposins is 51.19%-55.67%, and the total saposins rate of transform is 76.96%- 78.29%.

4. bacopa monnieri total saponin extracts are preparing the application in treating drugs for nervous.

5. application according to claim 4, it is characterised in that described to prepare drugs for nervous to prepare treatment depression Or the medicine for the treatment of dementia.

6. bacopa monnieri total saponin extracts are preparing the application in treating strongly fragrant disease drug.

7. bacopa monnieri total saponin extracts are preparing the application in treating dull-witted disease drug.

8. application according to claim 7, it is characterised in that described prepare treats dull-witted disease drug to prepare treatment A Er Zi Haimo disease drugs or vascular dementia medicine.

9. according to the application described in claim 4-8, it is characterised in that the medicine is to be by bacopa monnieri total saponin extracts The pharmaceutical composition of active component and pharmaceutic adjuvant composition.

10. application according to claim 9, it is characterised in that described pharmaceutical composition is oral formulations, injection or warp Percutaneous drug delivery preparation.