CN111067932A - A Prunellae Spica and/or herba Spinaciae effective component and its application in medicine for treating diabetes and diabetic nephropathy - Google Patents
A Prunellae Spica and/or herba Spinaciae effective component and its application in medicine for treating diabetes and diabetic nephropathy Download PDFInfo
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Abstract
The invention relates to a selfheal and/or spinacia oleracea effective component and application thereof in drugs for treating diabetes and diabetic nephropathy, wherein the active component comprises ursolic acid, oleanolic acid and corosolic acid, and is characterized in that the weight ratio of the oleanolic acid, the ursolic acid and the corosolic acid is 0.10-0.38: 0.70-0.95: 0.05-0.25.
Description
Technical Field
The invention relates to an application of natural components, in particular to a selfheal and/or spinacia oleracea effective component and an application thereof in medicines for treating diabetes and diabetic nephropathy.
Background
Diabetes is a serious metabolic disease and a major health problem worldwide. Diabetes affects approximately 4% of the population worldwide, and is expected to increase to 5.4% by 2025. According to statistics, China is one of the most diabetic countries, and Diabetic Nephropathy (DN) is one of common microvascular complications of diabetes, is also one of the complications with the highest fatality rate in diabetes, and is extremely harmful to human bodies, wherein about 30% -45% of patients finally cause end-stage nephropathy (ESRD); its early pathological manifestations are glomerular filtration rate increase, glomerular mesangial area broadening and glomerular capillary basement membrane thickening; the later stage is marked by the decrease of glomerular filtration rate, glomerular and tubulointerstitial fibrosis and the like. According to statistics, by the middle of the 21 st century, the number of diabetic patients will reach 3.66 million worldwide, the number of DN patients will exceed 1 million, and the incidence rate of diabetic nephropathy also rises year by year, so that it is very important to find a medicine capable of effectively treating diabetes and diabetic nephropathy.
The traditional Chinese medicine preparation is used as a complex system, the material basis of the traditional Chinese medicine preparation does not act as a single component, but the medicine effect is exerted by realizing the synergistic effect through multiple components and multiple targets. The traditional Chinese medicine material basis is considered to be divided into three levels from micro level to macro level by the component structure theory, wherein the monomer component is the most basic unit for forming the material basis, the monomer components in the same chemical category form the components, and the whole formed by the interaction of different components forms the material basis of the traditional Chinese medicine preparation. The components in each component have certain composition proportion, so that the quality of the traditional Chinese medicine preparation is controlled, the controllability of the traditional Chinese medicine preparation is realized, and the integrity of the traditional Chinese medicine is reflected. Only within the optimal component/component combination ratio range can the multicomponent drug exert the optimal effect.
The oleanolic acid can directly reduce the blood sugar content by weakening the absorption and the transportation of the body to glucose, reducing the generation of endogenous glucose and increasing the insulin sensitivity of receptor cells, can improve the antioxidation function of diabetic nephropathy rats, can reduce the level of the ursolic acid to reduce STZ-induced diabetic nephropathy rats, KW/BW, BUN and SCr, can enhance the kidney function and reduce the oxidative damage, can treat the inflammatory damage of the kidney by inhibiting the expression of TNF- α, MCP-1 and IL-1 β, can achieve the effect of treating the inflammatory damage of the kidney, and can be used for treating the renal damage of the diabetic nephropathy rats according to a certain proportion of the renal damage and the renal damage inhibition ratio of the renal cell, so the oleanolic acid has a certain effect of inhibiting the proliferation of the renal cell and the renal damage of the spinacia according to the research.
In the literature, selfheal has been used for treating diabetes, but the active ingredients reported are mainly caffeic acid, rosmarinic acid or other triterpenoid ingredients.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a selfheal and/or spinacia oleracea effective component based on component structural characteristics. The invention also aims to disclose the application of the effective component in preparing the medicament for treating diabetes. The invention also aims to disclose the application of the effective component in preparing the medicine for treating diabetic nephropathy.
The technical scheme is as follows: the main active ingredients of the effective component of the selfheal and/or the spinacia oleracea are oleanolic acid, ursolic acid and corosolic acid, wherein the ratio of oleanolic acid: ursolic acid: the weight ratio of the corosolic acid is 0.10-0.38: 0.70-0.95: 0.05-0.25.
The effective components of the selfheal and/or the spinacia oleracea are applied to the preparation of the medicine for treating diabetes.
The effective components of the selfheal and/or the spinacia oleracea are applied to the preparation of the medicine for treating the diabetic nephropathy.
The effective components of the selfheal and/or the spinacia oleracea are mixed with pharmaceutically acceptable auxiliary materials and prepared into capsules, tablets, pills or granules.
The pharmaceutically acceptable auxiliary materials are selected from one or more of fillers, adhesives, disintegrants, solubilizers, solvents or flavoring agents.
The preparation method of the effective component of the selfheal and/or the spinacia oleracea comprises the following steps:
(1) collecting dried cluster of Prunellae Spica and/or herba Spinaciae of Labiatae, and cleaning;
(2) heating and refluxing for extraction;
(3) concentrating the extractive solution, diluting with pure water, loading onto AB-8 type macroporous resin, sequentially eluting with pure water, 35-45% ethanol, and 70-80% ethanol solution, and collecting 70-80% ethanol eluate;
(4) and (4) concentrating the 70-80% ethanol elution part in the step (3), and drying in vacuum to obtain an extract state to obtain the effective component.
The preparation method comprises the steps of heating and reflux extracting twice in the step (2), adding 90-99.8% ethanol in an amount which is 15-25 times of the amount of the ethanol for the first time, reflux extracting for 1-2 hours, adding 90-99.8% ethanol in an amount which is 15-25 times of the amount of the ethanol for the second time, reflux extracting for 0.5-1.5 hours, and combining the extracting solutions of the two times.
The invention has the beneficial effects that the self-heal and/or spinacia oleracea effective components have better effects of reducing blood sugar and increasing weight of rats with diabetic nephropathy, can obviously reduce the contents of blood creatinine, blood urea nitrogen and 24-hour urine protein of the rats with diabetic nephropathy, can obviously inhibit the expression of kidney cell factors TGF- β 1, FN protein and Col IV protein of the rats with diabetic nephropathy, and has good application prospect.
Drawings
FIG. 1 is HPLC chromatogram of effective components of Prunellae Spica and/or herba Spinaciae;
FIG. 2 is a graph showing the weight change of rats in each group, which is the weight of rats in each group given 1 to 8 minus the weight before the administration;
FIG. 3 shows the course of blood glucose changes during the administration period of rats in each group;
figure 4 is a graph of the 24h urine protein content of rats after administration of the beneficial agent (compared to the blank,####p is less than 0.0001; in comparison to the model set,*P＜0.05，**P＜0.01，***P＜0.001)；
figure 5 is a graph of the serum creatinine content of rats after administration of the beneficial agent (compared to the blank,####p is less than 0.0001; in comparison to the model set,**P＜0.01，***P＜0.001,****P＜0.0001)；
figure 6 is a graph of the blood urea nitrogen content of rats after administration of the beneficial agent (compared to the blank,##p is less than 0.01; in comparison to the model set,*P＜0.05)；
figure 7 is a graph of the content of AGEs in rats after administration of the beneficial agent (compared to the blank,###p is less than 0.001; in comparison to the model set,*P＜0.05，**P＜0.01)；
figure 8 is a graph of RAGE content in rats after administration of the beneficial agent (versus blank,###p is less than 0.001; in comparison to the model set,**P＜0.01)；
fig. 9 shows HE staining of rat kidney (a is blank group, b is model group, c is aminoguanidine group, d is selfheal and/or spinacia oleracea alcohol extract high dose group, e is selfheal and/or spinacia oleracea alcohol extract low dose group, and f is selfheal and/or spinacia oleracea effective component group);
FIG. 10 shows PAS staining of rat kidney (a is blank group, b is model group, c is aminoguanidine group, d is Prunella vulgaris and/or spinacia oleracea alcohol extract high dose group, e is Prunella vulgaris and/or spinacia oleracea alcohol extract low dose group, f is Prunella vulgaris and/or spinacia oleracea effective component group);
FIG. 11 shows a TGF- β 1 immunohistochemical chart of rat kidney tissue (a: blank group, b: model group, c: aminoguanidine group, d: Prunella vulgaris and/or spinacia oleracea alcohol extract high dose group, e: Prunella vulgaris and/or spinacia oleracea alcohol extract low dose group, f: Prunella vulgaris and/or spinacia oleracea effective group);
FIG. 12 is a FN immunohistochemical map of rat kidney tissue (a: blank group; b: model group; c: aminoguanidine group; d: Prunella vulgaris and/or spinacia oleracea alcohol extract high dose group; e: Prunella vulgaris and/or spinacia oleracea alcohol extract low dose group; f: Prunella vulgaris and/or spinacia oleracea effective group);
FIG. 13 Col IV immunohistochemistry map of rat kidney tissue; wherein: a is a blank group, b is a model group, c is an aminoguanidine group, d is a selfheal and/or spinacia oleracea alcohol extract high-dose group, e is a selfheal and/or spinacia oleracea alcohol extract low-dose group, and f is a selfheal and/or spinacia oleracea effective component group (a: the blank group, b: the model group, c: the aminoguanidine group, d: the selfheal and/or spinacia oleracea alcohol extract high-dose group, e: the selfheal and/or spinacia oleracea alcohol extract low-dose group, and f: the selfheal and/or spinacia oleracea effective component group);
FIG. 14 shows the change of TGF- β 1, FN, and ColIV proteins in rat kidney tissue, wherein 1 is blank group, 2 is model group, 3 is positive group, 4 is Prunella vulgaris and/or spinacia oleracea alcohol extract high dose group, 5 is Prunella vulgaris and/or spinacia oleracea alcohol extract low dose group, and 6 is Prunella vulgaris and/or spinacia oleracea high effect group (1: blank group; 2: model group; 3: aminoguanidine group; 4: Prunella vulgaris and/or spinacia oleracea alcohol extract high dose group; 5: Prunella vulgaris and/or spinacia oleracea alcohol extract low dose group; and 6: Prunella vulgaris and/or spinacia oleracea high effect group).
Detailed Description
Example 1 preparation of active ingredients of Prunellae Spica and/or spinacia oleracea
Taking 500g of selfheal, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to have no fluidity, and drying in vacuum to be in a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.22: 0.70: 0.25.
taking 500g of spinacia oleracea, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol elution part, concentrating the 75% ethanol elution part to no flowability, and vacuum drying to a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.15: 0.95: 0.21.
taking 500g of mixed medicinal materials of selfheal and spinacia oleracea, heating and refluxing for two times, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the first time, refluxing for 1.5h, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting the extracting solution with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to have no flowability, and drying in vacuum to be in a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.10: 0.95: 0.08.
example 2
Taking 1kg of selfheal, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to have no flowability, and drying in vacuum to be in a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.38: 0.72: 0.18.
taking 1kg of spinacia oleracea, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol elution part, concentrating the 75% ethanol elution part to no flowability, and vacuum drying to a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.37: 0.89: 0.05.
taking 1kg of mixed medicinal materials of selfheal and spinacia oleracea, heating and refluxing for two times, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the first time, refluxing for 1.5h, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting the extracting solution with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in 8 times of column volume in sequence, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to have no flowability, and drying in vacuum to be in a dry extract state, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.30: 0.75: 0.24.
example 3
Taking 1kg of selfheal, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to be free of fluidity, vacuum drying to be in a dry extract state, detecting by HPLC, wherein the content of active components in the extract is 65.2%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.26: 0.78: 0.25.
taking 1kg of spinacia oleracea, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solutions in sequence by 8 times of column volume, collecting 75% ethanol elution parts, concentrating the 75% ethanol elution parts to be free of fluidity, vacuum drying to be in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 65.2%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.16: 0.70: 0.24.
taking 1kg of mixed medicinal materials of selfheal and spinacia oleracea, heating and refluxing for two times, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the first time, refluxing for 1.5h, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting the extracting solution with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts until the eluting parts have no flowability, vacuum drying the eluting parts until the eluting parts are in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 65.2%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.: 0.76: 0.23.
example 4
Taking 1kg of selfheal, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to be free of fluidity, vacuum drying to be in a dry extract state, detecting by HPLC, wherein the content of active components in the extract is 66.4%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.17: 0.94: 0.18.
taking 1kg of spinacia oleracea, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solutions in sequence by 8 times of column volume, collecting 75% ethanol elution parts, concentrating the 75% ethanol elution parts to be free of fluidity, vacuum drying to be in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 66.4%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.38: 0.72: 0.16.
taking 1kg of mixed medicinal materials of selfheal and spinacia oleracea, heating and refluxing for two times, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the first time, refluxing for 1.5h, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting the extracting solution with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts until the eluting parts have no flowability, vacuum drying the eluting parts until the eluting parts are in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 66.4%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.: 0.92: 0.21.
example 5
Taking 1kg of selfheal, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts to be free of fluidity, vacuum drying to be in a dry extract state, detecting by HPLC, wherein the content of active components in the extract is 67.5%, wherein the weight ratio of oleanolic acid to ursolic acid to corosolic acid is 0.25: 0.82: 0.10.
taking 1kg of spinacia oleracea, heating and refluxing for two times, adding 20 times of 95% ethanol for the first time, refluxing for 1.5h, adding 20 times of 95% ethanol for the second time, refluxing for 1h, combining the extracting solutions of the two times, concentrating the extracting solution, diluting with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solutions in sequence by 8 times of column volume, collecting 75% ethanol elution parts, concentrating the 75% ethanol elution parts to be free of fluidity, vacuum drying to be in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 67.5%, wherein the weight ratio of oleanolic acid to ursolic acid to corosolic acid is 0.35: 0.88: 0.13.
taking 1kg of mixed medicinal materials of selfheal and spinacia oleracea, heating and refluxing for two times, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the first time, refluxing for 1.5h, adding 95% ethanol 20 times the amount of the mixed medicinal materials for the second time, refluxing for 1h, combining extracting solutions of the two times, concentrating the extracting solution, diluting the extracting solution with pure water, applying AB-8 type macroporous resin, eluting with pure water, 40% ethanol and 75% ethanol solution in sequence by 8 times of column volume, collecting 75% ethanol eluting parts, concentrating the 75% ethanol eluting parts until the eluting parts have no flowability, vacuum drying the eluting parts until the eluting parts are in a dry extract state, and detecting by HPLC, wherein the content of active components in the extract is 67.5%, wherein the weight ratio of oleanolic acid, ursolic acid and corosolic acid is 0.: 0.85: 0.19.
example 6
Evaluation of Activity of active ingredients with different compatibility ratios
1. Instruments and reagents:
rat mesangial cells (HBZY-1); DMEM (low sugar, high sugar) culture medium, fetal bovine serum, trypsin, PBS buffer solution, CO2Cell culture case, inverted microscope, full-automatic enzyme mark appearance, low temperature ultracentrifuge, superclean bench, micropipette, digital display constant temperature stirring circulation water bath.
2. The experimental method comprises the following steps:
(1) HBZY-1 cell in vitro culture
The high-sugar model control group and administration group are cultured in high-sugar DMEM + 10% PBS culture medium, the blank control group is cultured in low-sugar DMEM + 10% PBS culture medium at 37 deg.C and 5% CO2Cell passage was performed when grown to 80% -90% confluence under conditions. Pouring out the culture medium in a culture vessel to remove the culture medium, washing the cells for 1 time by PBS, discarding the PBS, adding a proper amount of 0.1% pancreatin solution according to the area of the culture medium for digestion, standing at 37 ℃ for 3-5min, observing under an inverted microscope, adding the culture medium to stop digestion after most of the cells become round and suspend, blowing and sucking the cells which are dispersed into clusters for several times, carrying out cell passage according to the proportion of 1:3, and changing the solution for one time after 24 hours of the cells after passage.
(2) MTT (methyl thiazolyl tetrazolium) detection HBZY-1 cell viability
Collecting HBZY-1 cells in logarithmic growth phase, digesting, adjusting to a certain cell concentration, uniformly inoculating on 96-well plate, and culturing at 37 deg.C with 5% CO2The culture box of (1) was cultured for 24 hours, the medium was discarded, the blank group and the high-sugar model control group were not treated, the administration group was administered with the effective component of examples 3-5, and the positive control group was administered with aminoguanidine and 5% CO in the wells2Culturing for 48h under the condition, discarding the liquid medicine, adding 20 μ L (5mg/mL) MTT into each well, culturing for 4h, terminating the culture, sucking out the culture solution in each well, adding 150 μ L DMSO into each well, incubating at 37 ℃ for 10min, and measuring the OD value of each well at the wavelength of 570nm by a full-automatic enzyme labeling instrument. Calculated according to the following formulaAnd (3) calculating the cell survival rate:
(3) results of the experiment
The MTT method is used for detecting the cell survival rates of the effective components of the selfheal and/or the spinacia oleracea in different proportions, the results are shown in the following table, the effective components of the selfheal and/or the spinacia oleracea in different proportions can inhibit the proliferation of mesenteric cells, wherein the inhibition effect of oleanolic acid, ursolic acid and corosolic acid (0.25: 0.82: 0.10) is most obvious, and the inhibition effect is close to the effect of aminoguanidine as a positive drug, so the optimal proportion of the effective components of the selfheal and/or the spinacia oleracea is oleanolic acid: ursolic acid: corosolic acid ═ 0.25: 0.82: 0.10.
example 7 determination of active ingredient of Prunella vulgaris and/or spinacia oleracea
Dissolving the obtained active components of Prunellae Spica and/or herba Spinaciae with methanol, diluting with methanol, and measuring the content of active components by HPLC.
The instrument comprises the following steps: ThermoFisher U3000 HPLC (ThermoFisher Co., USA), SVEA C18 Gold 5 μm250 x 4.6mm column.
Chromatographic conditions are as follows: acetonitrile: isocratic elution with 0.1% phosphoric acid in water (90: 10); the time is 30 min; the column temperature is 25 ℃; the wavelength is 210 nm; the flow rate was 1.0 mL/min.
The active components of Prunellae Spica are shown in figure 1.
EXAMPLE 8 pharmacodynamic experiment
1. Moulding and grouping
36 SPF-grade SD male rats aged 8 weeks are selected, 6 rats serving as a blank group are fed with normal diet, the rest rats are fed with high-sugar high-fat feed for 4 weeks and then injected with STZ (35mg/kg) solution in the abdominal cavity to carry out type II diabetes molding, and the blank rats are injected with sodium citrate buffer solution with the same amount in the abdominal cavity. Injecting STZ solution into abdominal cavity for 72h, 120h and 168h, then cutting tail to collect blood to measure blood sugar of rat, using rat with blood sugar value not less than 16.7mmol/L as diabetes model, collecting 24h urine of rat by metabolism cage method, measuring urine protein content of rat, using 24h urine protein content not less than 20g as kidney model.
2. Administration and treatment
After the model building is finished, rats successfully modeled on diabetic nephropathy are divided into a model group (n ═ 6), an aminoguanidine group (100mg/kg/d, n ═ 6), a high-dose group (6g decoction pieces/kg/d, n ═ 6) of alcohol extracts of selfheal and/or spinacia oleracea, a low-dose group (3g decoction pieces/kg/d, n ═ 6) of alcohol extracts of selfheal and/or spinacia oleracea, and a high-effect group (corosolic acid, ursolic acid and oleanolic acid with the purity of more than 90%) of selfheal and/or spinacia oleracea, which are mixed in the best proportion in example 6, 100mg/kg/d, n ═ 6), a blank group and the model group are administered with pure water, and the rats in each group are subjected to gastric lavage for 8 weeks. Rats body weight and blood glucose were measured weekly during dosing. After 8 weeks of administration, the urine of the rat is taken by a metabolic cage method, and the urine volume of the rat is recorded; anesthetizing with 10% chloral hydrate solution, collecting blood from abdominal aorta, separating serum, and storing in refrigerator at-80 deg.C; two kidneys of a rat are rapidly picked, the left kidney is fixed by 10% formalin solution and used for staining HE pathological sections and analyzing immunohistochemistry, and the right kidney is stored in an ultralow temperature refrigerator at minus 80 ℃ and used for detecting related tissue protein indexes of Western Blot.
3. Index and experimental method
(1) Adopting a tail tip blood sampling method, and measuring the blood sugar value of a rat by using a glucometer; measuring the concentration of the urine protein of the rat by using a urine protein quantitative test box, and calculating the content of the urine protein of the rat for 24 hours; measuring the contents of serum creatinine and blood urea nitrogen in the blood serum of the rat by using a creatinine test box and a blood urea nitrogen test box; the AGEs and RAGE content in rat serum was determined using an ELISA kit.
(2) HE staining was performed according to the following procedure: collecting rat kidney tissue blocks, fixing, embedding with conventional paraffin, and slicing at 4 μm; the slices were dewaxed conventionally with xylene, washed with various grades of ethanol to water: xylene (I)5min → xylene (II) 5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing 2 min; hematoxylin staining for 5 minutes and washing with tap water; ethanol hydrochloride is differentiated for 30 seconds; soaking in tap water for 15 minutes or warm water (about 50 ℃) for 5 minutes; placing in eosin solution for 2 minutes; conventional dehydration, transparency, mounting: 95% ethanol (I) min → 95% ethanol (II) 1min → 100% ethanol (I)1min → 100% ethanol (II) 1min → xylene (I)1min → xylene (II) 1min → neutral resin sealing, and observing the tissue morphology under a microscope.
(3) PAS dyeing was performed according to the following procedure: conventional fixation, usually 10% formalin is adopted, and conventional dehydration embedding is carried out; paraffin section and dewaxing into distilled water; directly adding distilled water into the frozen section; washing with tap water for 2-3 min, and washing with distilled water for 2 times; and then adding periodic acid solution, standing at room temperature for 5-8 min, washing with tap water for 1 time, washing with distilled water for 2 times, adding Schiff reagent, performing dip dyeing in the shade at room temperature for 10-20 min, washing with tap water for 10min, adding hematoxylin dyeing solution, dyeing cell nuclei for 1-2 min, differentiating with acidic ethanol differentiation solution for 2-5 s, washing with tap water for 10-15 min, replacing distilled water for cleaning, returning blue, and performing conventional ethanol dehydration step by step. Xylene was clear, gel-sealed with neutral gum, and the sections were observed under an inverted microscope.
(4) The method comprises the steps of measuring the change conditions of TGF- β 1, FN and ColIV proteins in rat kidney tissues by a Western Blot method, taking 100mg of kidney tissues stored in an ultra-low temperature refrigerator, washing the kidney tissues with precooled physiological saline, grinding the kidney tissues on ice, extracting total proteins by using a protein extraction kit, quantitatively measuring the protein concentration by a Bradford method, denaturing the total proteins of all groups of samples, performing SDS-PAGE gel vertical electrophoresis to perform protein separation, transferring the proteins to a polyvinylidene fluoride membrane, sealing the membrane at room temperature for 2h, incubating the membrane at the temperature of 4 ℃ overnight with a specific primary antibody, incubating the membrane at the temperature of 2h with a secondary antibody, developing the color, taking GAPDH protein as an internal reference standard, imaging by using G: BOXchemi XR5, analyzing protein bands by using ImageJ image analysis software, and expressing the relative expression of the proteins in a ratio of the gray value of the target protein band to the gray value of the internal reference protein band.
(5) The method comprises the steps of taking A tissue block with the kidney tissue of A rat being less than 0.5cm multiplied by 0.1cm, washing with PBS, fixing with 4% paraformaldehyde, washing with 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes twice, 95% ethanol for 30 minutes twice, 100% ethanol for 30 minutes twice, xylene for transparent 15 minutes twice, paraffin for 1 hour and three times at 55 ℃, embedding the tissue block with A stainless steel mold, attaching A tissue slice with the thickness of 5 mu m on A glass slide coated with polylysine, standing overnight at 60 ℃, immersing the tissue slice in xylene for 5 minutes twice, 100% ethanol for 5 minutes twice, 95% ethanol for 5 minutes twice, 90% ethanol for 5 minutes twice, 85% ethanol for 5 minutes twice, 70% ethanol for 5 minutes, washing with tap water for 5 minutes, washing with PBS for twice, washing with citric acid buffer solution, thermal repairing for 20 minutes, washing with PBS for 3 times, washing with 3 times 5 minutes, 3 times with 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, washing with 90% ethanol for 5 minutes, washing with 85% ethanol for 5 minutes, washing with normal horseradish for 5 minutes, washing with PBS, washing with 1 minute, washing with 5 minutes, washing with normal washing with 5 minutes, washing with 1 minute, washing with 5 minutes, washing with HRP 5 minutes, washing with normal staining solution, washing with 5 minutes, washing with normal washing with 5 minutes, 5 minutes of albumin, 5 minutes, washing with 5 minutes.
4. Statistical method
ˉ
The experimental data are expressed in x + -s, statistically processed using Graphpad prism 6.01 software, and the component comparisons were performed using One-Way analysis of variance (One-Way ANOVA).
5. Pharmacodynamic experiment result (1) influence of alcohol extract and effective component of selfheal and/or spinacia oleracea on body weight of diabetic nephropathy rats
After the molding is finished, the weight of the rat successfully molded is obviously reduced, the body is obviously weakened, and the phenomena of hair yellowing, listlessness and the like occur; in the experimental process, the body weight of a model group rat is reduced, the body weight of a positive group and a low-dose group of the selfheal alcohol extract is weakened, and the body weight of a selfheal alcohol/spinacia oleracea extract high-dose group and the body weight of a selfheal and/or spinacia oleracea effective component group are increased, so that the high-dose selfheal and/or spinacia oleracea alcohol extract and the effective component monomer have the effects of effectively relieving the weight loss and the form emaciation of the diabetic nephropathy rat. The body weight changes of the rats during the administration are shown in FIG. 2. (2) Influence of alcohol extract and effective components of Prunellae Spica and/or herba Spinaciae on blood sugar of diabetic nephropathy rat
After the molding is finished, the molded rats are randomly grouped according to blood sugar, and the blood sugar levels of the rats in the model group before administration, the aminoguanidine group, the selfheal and/or spinacia oleracea alcohol extract high-dose and low-dose groups have no obvious difference. The blood sugar of rats in the model group is measured once a week during administration, the blood sugar of rats in the model group is kept stable, the blood sugar change is small during administration, the high-dose and low-dose groups of the alcohol extract of the aminoguanidine group, the selfheal and/or the spinacia oleracea and the effective groups of the selfheal and/or the spinacia oleracea show a descending trend, so that the high-dose and low-dose alcohol extract of the selfheal and/or the spinacia oleracea have the effect of reducing the blood sugar of the rats with diabetic nephropathy, and the blood sugar reducing effect of the high-dose alcohol extract of the selfheal and/or the. (see Table 1, Table 2, and FIG. 3)
TABLE 1 blood glucose Change (mmol/L) for groups of rats during dosing
TABLE 2 blood glucose Change (mmol/L) of rats of each group during administration
Note: in comparison with the blank set, the results,####p is less than 0.0001; in comparison with the set of models,*P＜0.05，**P＜0.01，***P＜0.001,****P＜0.0001
(3) influence of alcohol extract and effective components of Prunellae Spica and/or herba Spinaciae on 24h urine protein of diabetic nephropathy rat
After the molding is finished, the molded rats are randomly grouped according to the content of urine protein for 24h, and the levels of the urine protein of the rats in the model group before administration, the aminoguanidine group, the selfheal and/or spinacia oleracea alcohol extract high-dose and low-dose groups have no obvious difference. After 8 weeks of administration, the urinary protein content of each group of rats is shown in figure 4, compared with the rats in the blank group, the urinary protein content of the rats in the model group is obviously increased, after the administration is finished, the urinary protein content of the rats in the high-dose group of the ethanol extract of the selfheal and/or the spinacia oleracea and the high-effect component group of the selfheal and/or the spinacia oleracea is reduced to a certain degree, and the urinary protein content of the rats in the low-dose group of the ethanol extract of the selfheal and/or the spinacia oleracea is not obviously different.
(4) Influence of alcohol extract and effective components of Prunellae Spica and/or herba Spinaciae on blood creatinine and blood urea nitrogen content of diabetic nephropathy rat
The results are shown in table 3, fig. 5 and fig. 6, and it can be seen from the table that the high dose of alcohol extract of prunella vulgaris and/or spinacia oleracea and the effective component can significantly reduce the blood creatinine content of rats and have certain reducing effect on blood urea nitrogen; low doses of ethanol extracts of prunella vulgaris and/or spinacia oleracea also have a significant effect on the blood creatinine content, but have little effect on the blood urea nitrogen content.
TABLE 3 serum creatinine (SCr) and Blood Urea Nitrogen (BUN) content (mmol/L) in rat groups
Note: in comparison with the blank set, the results,####p is less than 0.0001; in comparison with the set of models,*P＜0.05，**P＜0.01，***P＜0.001,****P＜0.0001
(5) influence of alcohol extract and effective components of Prunellae Spica and/or herba Spinaciae on AGEs and RAGE content of diabetic nephropathy rats
The results are shown in table 4, fig. 7 and fig. 8, and it can be seen from the table that the high dose of alcohol extract of prunella vulgaris and/or spinacia oleracea and the effective component have certain reduction effect on AGEs and RAGE in rat serum, and the low dose of alcohol extract of prunella vulgaris and/or spinacia oleracea has no obvious effect.
TABLE 4 serum AGEs (ng/L) and RAGE (pg/L) levels in rats of each group
Note: in comparison with the blank set, the results,####p is less than 0.0001; in comparison with the set of models,*P＜0.05，**P＜0.01，***P＜0.001,****P＜0.0001
(6) HE (see fig. 9) and PAS (see fig. 10) staining and showed that the rat renal tubular epithelial cells in the blank group (a) were well-aligned, the renal bodies were homogeneous in texture, the renal capsule cavities were clearly visible, no significant mesangial hyperplasia or capillary degeneration was seen, and no inflammation was observed; in the model group (b), part of glomeruli of a rat is lost, mesangial matrix hyperplasia is obvious, renal tubular epithelial cells are vacuolated, part of the renal tubular epithelial cells are exfoliated and necrotized, the tubular cavities of renal tubules are reduced in different degrees, the tubular cavities are narrowed, inflammatory infiltration is obvious, and the morphological influence is the most serious; the renal tubule cavities of the rats in the aminoguanidine group (c) are reduced in different degrees, the tubular cavities are narrowed, the area of the renal corpuscle capsule cavities is increased, epithelial cells are disintegrated, and the model group rat renal tubule is obviously improved; the pathological changes of the rats in the administration group are obviously improved compared with those of the rats in the model group, and are shown in that the phenomena of mesenteric matrix hyperplasia, renal tubular epithelial cell vacuolation and desquamation necrosis are lighter than those of the rats in the model group, particularly the high-dose group (d) is obviously improved compared with the low-dose group (e), the high-dose group is similar to the selfheal and/or spinacia oleracea effective group (f), and the rats in the administration group have no obvious inflammatory infiltration compared with the rats in the model group.
(7) The result of Western Blot detection of the expression of TGF- β 1, FN and ColIV proteins in rat kidney tissues is shown in figure 14 and table 5, compared with rats in a blank group, the expression of TGF- β 1, FN and ColIV proteins in rat kidney tissues of a model group is remarkably up-regulated, selfheal and/or spinacia oleracea alcohol extracts can remarkably down-regulate the expression of the proteins, and the high-dose group has a lower down-regulation trend and is remarkably shown in a low-dose group.
TABLE 5 expression of TGF- β 1, FN, ColIV by the effective components of Prunellae Spica and/or herba Spinaciae extract
Note: in comparison with the blank set, the results,###p is less than 0.001; in comparison with the set of models,*P＜0.05，**P＜0.01。
(8) the results are shown in figures 11-13. compared with the blank group, the TGF- β, FN and Col IV protein expression of the rat kidney of the model group is obviously increased, the high-low dose administration group and the selfheal effective group of the selfheal and/or the spinacia alcohol extract can inhibit the expression of the TGF- β, FN and Col IV protein to different degrees, and the effect of the high dose group is similar to that of the effective component and is superior to that of the low dose group.
Example 9
Pulverizing the dry extract obtained in example 5, adding an appropriate amount of microcrystalline cellulose into the medicinal powder, mixing well, adding an appropriate amount of starch slurry to prepare a soft material, preparing the soft material into wet granules by using a granulator, drying, granulating, adding an appropriate amount of talcum powder and magnesium stearate, mixing well, pressing into tablets, and preparing into tablets, wherein each tablet contains an active ingredient equivalent to 9g of the crude drug of the selfheal, and the usage amount is as follows: orally administered 1 tablet each time, twice a day, and reduced for children.
Example 10
Pulverizing the dry extract obtained in example 5, adding an appropriate amount of starch into the medicinal powder, mixing uniformly, adding an appropriate amount of starch dextrin and polyethylene glycol 6000 to prepare a soft material, preparing the soft material into wet granules by using a granulator, drying, granulating, bagging, and preparing into granules, wherein each bag contains active ingredients equivalent to 9g of crude drug of the selfheal, and the usage amount is as follows: orally administered 1 bag each time, twice a day, and reduced for children.
Example 11
Pulverizing the dry extract obtained in the embodiment 5, adding an appropriate amount of starch into the medicinal powder, fully mixing uniformly, adding an appropriate amount of starch dextrin and polyethylene glycol 6000 to prepare a soft material, preparing wet granules by using a granulator, drying, granulating, filling into a capsule shell of a capsule, and preparing the capsule, wherein each granule contains an active ingredient which is equivalent to 9g of crude drug of the selfheal, and the usage amount is as follows: orally administered 1 granule each time, twice a day, and reduced for children.
Example 12
Crushing the dry extract in the embodiment 5, adding a proper amount of refined honey into the medicinal powder, fully and uniformly mixing to prepare pills with proper hardness and good plasticity, standing the pills for a period of time, kneading the pills into pills, and preparing the pills by using a pill rolling machine, wherein each pill contains 9g of active ingredients, namely the crude drug amount of the selfheal, and the usage amount is as follows: orally administered 1 pill each time, twice a day, and reduced for children.
Claims (7)
1. A selfheal and/or spinacia oleracea effective component comprises main active ingredients of oleanolic acid, ursolic acid and corosolic acid, and is characterized in that the ratio of oleanolic acid: ursolic acid: the weight ratio of the corosolic acid is 0.10-0.38: 0.70-0.95: 0.05-0.25.
2. The use of the effective components of Prunellae Spica and/or herba Spinaciae of claim 1 in preparing medicine for treating diabetes.
3. The use of the effective components of Prunellae Spica and/or herba Spinaciae of claim 1 in the preparation of medicine for treating diabetic nephropathy.
4. The use according to any one of claims 2-3, wherein the effective components of Prunellae Spica and/or herba Spinaciae are mixed with pharmaceutically acceptable adjuvants and made into capsule, tablet, pill or granule.
5. The use according to claim 4, characterized in that the pharmaceutically acceptable excipients are selected from one or more of fillers, binders, disintegrants, solubilizers, solvents or flavoring agents.
6. A method for preparing the effective component of prunella vulgaris and/or spinacia oleracea according to claim 1, characterized by comprising the following steps:
(1) collecting dried cluster of Prunellae Spica and/or herba Spinaciae of Labiatae, and cleaning;
(2) heating and refluxing for extraction;
(3) concentrating the extractive solution, diluting with pure water, loading onto AB-8 type macroporous resin, sequentially eluting with pure water, 35-45% ethanol, and 70-80% ethanol solution, and collecting 70-80% ethanol eluate;
(4) and (4) concentrating the 70-80% ethanol elution part in the step (3), and drying in vacuum to obtain an extract state to obtain the effective component.
7. The process according to claim 6, wherein the step (2) comprises heating and reflux-extracting twice, the first with 15-25 times of 90-99.8% ethanol for 1-2 hours, the second with 15-25 times of 90-99.8% ethanol for 0.5-1.5 hours, and combining the two extracts.
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