CN101940616B - Preparation method of effective part of Clinopodium chinense (Benth.) O. Kuntze for preventing and treating diabetes and medicine application thereof - Google Patents
Preparation method of effective part of Clinopodium chinense (Benth.) O. Kuntze for preventing and treating diabetes and medicine application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of extract of effective part of Clinopodium chinense (Benth.) O. Kuntze for preventing and treating diabetes and an application thereof. The extract of the effective part is prepared from Clinopodium chinense (Benth.) O. Kuntze as a raw material, a concentrate is obtained by pulverizing, ethanol extraction and reduced pressure recovery, the concentrate is extracted by petroleum ether and ethyl acetate, and the obtained ethyl acetate extract is purified by macroporous resin. The extract of the effective part mainly comprises total flavone and terpenoids, and has obvious effects of reducing blood sugar of rats with streptozotocin diabetes, reducing the content of serum cholesterol, protecting the islet cells, inhibiting the activity of alpha-glucosidase, and protecting the vascular endothelial cells. Thus, the extract of the effective part of Clinopodium chinense (Benth.) O. Kuntze has favorable functions for reducing blood sugar, reducing blood fat, and protecting the islet cells and the vascular endothelial cells, and can be used for preventing and treating diabetes, dyslipidemia induced by diabetes and diabetic cardiovascular and cerebrovascular complications.
Description
One, technical field
The present invention relates to the method for preparing of Chinese medicine Herba Clinopodii Polycephali effective part extract; And the blood sugar lowering of this effective part extract, blood fat reducing, protection islet cells and blood vessel inner skin cell function, and preventing and treating disorders of lipid metabolism that diabetes, diabetes cause and the application aspect the diabetic cardiovascular and cerebrovascular complication.
Two, background technology
Diabetes are the one group of Developmental and Metabolic Disorder syndromes that is caused by the h and E factor interaction.Estimate 1995～2025 years according to WHO, global onset diabetes rate will increase to 3%～5%, estimate to reach 300,000,000 patients, and patient age then tended to 45～54 years old from over-65s.Diabetes can be divided into insulin dependent diabetes mellitus (IDDM) (Insulin Dependent Diabetes Mellitus, IDDM claims 1 type again) and non-insulin-dependent diabetes mellitus (Noinsulin Dependent Diabetes Mellitus, NIDDM claims 2 types again).The main harm of diabetes is its vascular complication, comprises macroangiopathy and microangiopathies.Diabetes trunk complication mainly is meant the pathological changes of cerebrovascular, cardiovascular and lower limb vascular.The probability of the concurrent cardiovascular and cerebrovascular disease of diabetics will be higher than the ND far away.Present research thinks that the macroangiopathy that diabetes cause is the deadly major cause of morbidity of diabetics.
The medicine of treatment diabetes mainly contains sulphanylureas, biguanides, insulin, alpha-glucosidase inhibitor, Thiazolidine ketone etc. at present.These medicines can cause some side effect such as hypoglycemia, liver toxicity, lactic acidosis and diarrhoea.The treatment by Chinese herbs diabetes have remarkable advantages; Its treatment diabetes are comprehensive coordination effects of too many levels, multipath, many target spots; Its medicine source is abundant, cheap, toxic and side effects is little, so the prospect that has an antidiabetic medicine from Chinese medicine screening is very wide.
Herba Clinopodii Polycephali is the herb of Labiatae Clinopodium plant Herba Clinopodii Polycephali (Clinopodium chinense (Benth.) O.Kuntze), the little hardship of Herba Clinopodii Polycephali, puckery, cold.Return Liver Channel.Herba Clinopodii Polycephali is the former plant that the Pharmacopoeia of the People's Republic of China records the Chinese medicine Herba Clinopodii, the effect of have heat-clearing and toxic substances removing, stopping blooding and invigorating blood circulation.Herba Clinopodii Polycephali is among the people in Fujian to be used to treat diabetes.This laboratory early-stage Study finds that Herba Clinopodii Polycephali has hypoglycemic activity; But Shang Weiyou is to the preparation technology of Herba Clinopodii Polycephali effective part extract and blood sugar lowering, blood fat reducing, protection islet cells and the blood vessel inner skin cell function of this effective part extract, and at the report of preventing and treating aspect diabetes and the diabetic cardiovascular and cerebrovascular complication.
Three, summary of the invention
The invention provides a kind of method that from the Chinese medicine Herba Clinopodii Polycephali, prepares effective part extract; And the blood sugar lowering of this effective part extract, blood fat reducing, protection islet cells and blood vessel inner skin cell function, and in the application that prevents and treats aspect diabetes and the diabetic cardiovascular and cerebrovascular complication.
1, the method for preparing of Herba Clinopodii Polycephali effective part extract is got dry Herba Clinopodii Polycephali, pulverizes ethanol or methanol or propanol and their aqueous reflux, extract; Or the dipping extraction, the extracting solution concentrating under reduced pressure is dispersed in spissated extract in the water; Use petroleum ether, ethyl acetate extraction 3～4 times successively; Ethyl acetate extract carries out column chromatography with macroporous resin column, behind the water elution impurity
Ethanol gradient elution is collected ethanol elution, and concentrating under reduced pressure is dry, gets extract.Being divided into step is:
(1) Herba Clinopodii Polycephali herb drying and crushing;
(2) ethanol or methanol or propanol and their aqueous reflux, extract,, or dipping extracts the extracting solution concentrating under reduced pressure;
(3) spissated extract is used petroleum ether, ethyl acetate extraction 3～4 times successively;
(4) ethyl acetate extract carries out column chromatography with macroporous resin column;
(5) behind the water elution impurity, ethanol gradient elution is collected ethanol elution, and concentrating under reduced pressure is dry, gets extract.
The condition and range of above-mentioned method for preparing is: the concentration of ethanol or methanol or propanol is 10～90% in the step (2), and method for distilling is for refluxing, flooding; The ethanol gradient elution scope is 20～80% in the step (5).
2, the Herba Clinopodii Polycephali effective part extract mainly is made up of total flavones and terpenoid, and the main component of flavone is monarda glycoside, naringenin, apigenin, isosakuranetin, luteolin, acacetin etc.; Ursolic acid is the main component of terpenoid.Wherein the mensuration of general flavone content adopts ultraviolet spectrophotometry, with NaNO
2-A1 (NO
3)
3-NaOH chromogenic assay general flavone content, in rutin, the total content of flavone compound is not less than 50%.
Herba Clinopodii Polycephali effective part extract according to the invention can mix with conventional adjuvant and/or excipient, is prepared into various dosage forms, is used for prevention or treatment.Injection, capsule, tablet, granule, dragee, solution etc. all are alternative pharmaceutical dosage form.
Dosage forms such as capsule, tablet, granule, dragee can contain one or more excipient-filler commonly used and bulking agents: like starch, and microcrystalline cellulose etc.; Binding agent: like carboxymethyl cellulose, polyvinylpyrrolidone etc.; Humectant: like glycerol; Disintegrating agent: like calcium carbonate etc.; Absorbent: like Kaolin etc.; Lubricant: like Pulvis Talci etc.
Medicinal excipient can be various forms such as solid, semisolid, liquid, and the prescription adjuvant can be various types of.
Solution can contain general solvent, solubilizing agent, emulsifying agent, antiseptic etc., like water, ethanol, glycerol, Polyethylene Glycol, benzyl benzoate etc.
Dosage forms such as capsule, tablet, granule, dragee, solution can be used known conventional method preparation, can the Herba Clinopodii Polycephali effective part extract be mixed the preparation dosage form with one or more excipient.
3, Herba Clinopodii Polycephali effective part extract blood sugar lowering, blood fat reducing and protection vascular inner skin cell activity
3.1 Herba Clinopodii Polycephali effective part extract (CCE) is to the influence of streptozotocin blood glucose in diabetic mice, cholesterol (TC), serum insulin and islet tissue
Take out 10 of mices at random as normal group, behind all the other mice fasting 16-17h, tail vein injection streptozotocin 160mg/kg; Injection back 72h posterior orbit rear vein beard is got blood, and separation of serum is measured blood glucose; Select 40 of the mices of fasting glucose＞11mmol/L, be divided into 4 groups at random, give CCE 50,100mg/kg by 10ml/kg body weight per os respectively; Metformin (Metformin) 200mg/kg and isopyknic 0.2%CMC-Na, once a day, administration in continuous 12 days.1.5h (fasting 5h) after the last administration in the 6th day, the mouse orbit rear vein beard is got blood, and separation of serum is with determination of glucose oxidase blood glucose.1.5h after the last administration (fasting 5h), mice is plucked eyeball and gets blood, and separation of serum is measured blood glucose, TC, serum insulin with kit method.Separate mice pancreatic, pancreas is immersed in fixing, FFPE in 10% formalin, film-making, histopathological examination is made in HE dyeing.
The blood glucose experimental result is as shown in Figure 1, with normal group relatively, model group streptozotocin blood glucose in diabetic mice extremely significantly raise (P＜0.01).Compare with model group, CCE 100mg/kg and metformin 200mg/kg all can obviously reduce the 6th day blood glucose in diabetic mice (P＜0.05); CCE 50mg/kg dose groups obviously reduces the blood glucose (P＜0.05) of the 12nd day diabetic mice, and CCE100mg/kg and metformin 200mg/kg dose groups all can extremely obviously reduce the blood glucose (P＜0.01) of the 12nd day diabetic mice.
The TC experimental result is as shown in Figure 2, with compared with normal, and the TC content of streptozotocin diabetic mice extremely obviously raise (P＜0.01).Compare with model group, CCE 50mg/kg and metformin 200mg/kg dose groups all reduce the serum TC content (P＜0.05) of diabetic mice significantly, and the CCE 100mg/kg dose groups utmost point reduces the serum TC content (P＜0.01) of diabetic mice significantly.
The experimental result of insulin is as shown in Figure 3, and with compared with normal, the serum insulin content of model group streptozotocin diabetic mice extremely obviously reduces (P＜0.01).Compare CCE100mg/kg dose groups and the metformin 200mg/kg dose groups low insulin content of diabetic mice (P＜0.05) that all gos up significantly with model group.
The tissue pathologies change of streptozotocin diabetic mice pancreas is mainly: endocrine portion islets of langerhans quantity more normally reduces; Volume-diminished, the islet tissue structure that retains is looser, and its inner cell volume increases; Born of the same parents circle owe clearly, and part mice pancreatic exocrine portion acinous cell has cavity to become.CCE 50,100mg/kg and metformin 200mg/kg can alleviate the pathological changes of islets of langerhans; Islets of langerhans quantity increases after the concrete manifestation medicinal application, the islets of langerhans volume increases; Each islets of langerhans inner cell quantity has clear improvement, and the prompting medicine can improve the islets of langerhans pathological changes due to the streptozotocin.
3.2 Herba Clinopodii Polycephali effective part extract (CCE) is to the inhibitory action of alpha-glucosidase
With 4-nitrophenols-α-D-glucofuranose glycosides (PNP-G) is the activity that substrate is measured alpha-glucosidase.With CCE (final concentration is 10,30,60,90,120mg/L) or acarbose (final concentration is 100,300,600,900,1200, and 1500mg/L) 40 μ l and alpha-glucosidase (0.35mg/ml) 30 μ l add 500 μ l KH jointly
2PO
4Buffer (67mmol/L, pH:6.8) in, in 37 ℃ of water-baths, hatch 10min after, add substrate PNP-G (0.01mol/L) 30 μ l to start reaction.Behind 37 ℃ of reaction 10min, add 0.1mol/L Na
2CO
3The 1ml cessation reaction.Under the 405nm wavelength, be determined at the OD value of the p-nitrophenyl (PNP) that from PNP-G, discharges under the enzyme effect.Enzyme activity unit is defined as: at 37 ℃, under the condition of pH 6.8, hydrolysis PNP-G discharges the required enzyme amount of 1 μ mol PNP in the 1min, calculates suppression ratio.The enzyme inhibition test repeats 3 times, each 3 multiple pipes.Suppression ratio to alpha-glucosidase calculates with following formula: suppression ratio=(matched group OD value-drug group OD value)/matched group OD value * 100%.
The result is as shown in table 1: compare with matched group, CCE (30,60,90,120mg/L), acarbose (100,300,600,900,1200,1500mg/L) all have utmost point significant inhibitory effect (P＜0.01) to alpha-glucosidase.CCE, acarbose suppress the IC of alpha-glucosidase
50Be respectively 62.3,666.6mg/L.
Table 1 Herba Clinopodii Polycephali effective part extract (CCE) is to the inhibitory action
of alpha-glucosidase
*P＜0.05,
*P＜0.01vs matched group
3.3, Herba Clinopodii Polycephali effective part extract (CCE) is to the protective effect of the endotheliocyte of high sugar damage
The trophophase HUVEC that takes the logarithm is digested to single cell suspension and is inoculated in 96 porocyte culture plates (1 * 10
4Cell/well), change RPMI 1640 culture medium (containing 10% hyclone) behind the cultivation 24h, cell is divided into 6 groups; Normal group is cultivating naturally of HUVEC, and model group is that CCE and 33mmol/L glucose are cultivated altogether, and all the other groups are cultivated for CCE (1,3,10,30mg/L) and HUVEC and glucose altogether; After cultivating 72h, add the culture medium 20 μ l that contain 0.5%MTT, cultivate 4h again after; Abandon culture fluid, add 150 μ l DMSO stock solutions, vibration 10min; After the dissolving fully to be crystallized, measure the OD value in the 570nm wavelength with the enzyme linked immunological appearance.Experiment repetition 3 times, each 3 multiple holes.
The result is as shown in Figure 4, compares with normal group, and model group HUVEC cell A570 value extremely obviously reduces (P＜0.01).Compare with model group, the A570 value of CCE 3,10,30mg/L group is apparently higher than model group (P＜0.01).It is thus clear that CCE 3,10,30mg/L obviously improve the survival rate of the endotheliocyte of high sugar damage, and the endothelial cell damage due to the high sugar is had protective effect.
According to the invention, effective part extract can be used for preparing disorders of lipid metabolism and the diabetic treating cardiac and cerebral vascular diseases complication that prevention and treatment diabetes, diabetes cause in the Herba Clinopodii Polycephali.
Four, description of drawings
Fig. 1 Herba Clinopodii Polycephali effective part extract (CCE) is to the influence of streptozotocin blood glucose in diabetic mice
Take out 10 of mices at random as normal group, behind all the other mice fasting 16-17h, tail vein injection streptozotocin 160mg/kg; Behind the 72h of injection back; Select the mice of fasting glucose＞11mmol/L, be divided into model group, CCE 50,100mg/kg at random, metformin 200mg/kg; Continuous 12 days oral administrations, once a day.The 6th day and the 12nd day; 1.5h after the last administration (fasting 5h) gets blood respectively; Measure fasting glucose, the result representes with
.
##P＜0.01vs?Normal；
*P＜0.05，
**P＜0.01vs?Control(Student’s-t?test).
Fig. 2 Herba Clinopodii Polycephali effective part extract (CCE) is to the influence of streptozotocin diabetic mice T-CHOL
Take out 10 of mices at random as normal group, behind all the other mice fasting 16-17h, tail vein injection streptozotocin 160mg/kg; Behind the 72h of injection back; Select the mice of fasting glucose＞11mmol/L, be divided into model group, CCE 50,100mg/kg at random, metformin 200mg/kg; Continuous 12 days oral administrations, once a day.The 12nd day; 1.5h after the last administration (fasting 5h) gets blood; Measure serum total cholesterol, the result representes with
.
##P＜0.01vs?Normal；
*P＜0.05，
**P＜0.01vs?Control(Student’s-t?test).
Fig. 3 Herba Clinopodii Polycephali effective part extract (CCE) is to the influence of streptozotocin diabetic mice serum insulin content
Take out 10 of mices at random as normal group, behind all the other mice fasting 16-17h, tail vein injection streptozotocin 160mg/kg; Behind the 72h of injection back; Select the mice of fasting glucose＞11mmol/L, be divided into model group, CCE 50,100mg/kg at random, metformin 200mg/kg; Continuous 12 days oral administrations, once a day.The 12nd day; 1.5h after the last administration (fasting 5h) gets blood; Use the serum measured by radioimmunoassay insulin, the result representes with
.
##P＜0.01vs?Normal；
*P＜0.05vs?Control(Student’s-t?test).
Fig. 4 CCE is to the protective effect of the endotheliocyte of high sugar damage
HUVEC single cell suspension (1 * 10
4Individual/hole) be inoculated in 96 porocyte culture plates, change culture medium behind the cultivation 24h, cell is divided into groups: normal group is cultivating naturally of HUVEC, and model group is that HUVEC and 33mmol/L glucose are cultivated altogether; All the other groups are cultivated with HUVEC and glucose for CCE (1,3,10,30mg/L) altogether, cultivate 72h after, add the culture medium 20 μ l that contain 0.5%MTT; After cultivating 4h again, abandon culture fluid, add 150 μ l DMSO stock solutions; Vibration after the dissolving fully to be crystallized, is measured the OD value.Data are the meansigma methods of 3 experiments among the figure, and each experiment is 3 multiple holes.The result representes with
.
##P＜0.01vs?Normal；
**P＜0.01vs?Control(Student’s-t?test).
Five, the specific embodiment
Describe the present invention below in conjunction with instance.But the present invention is not limited to these given instances.
Embodiment 1: the preparation of Herba Clinopodii Polycephali effective part extract:
Get the dry Herba Clinopodii Polycephali herb of 10kg, be ground into coarse powder, add 80% alcohol reflux 3 times of 6 times of volumes, merge extractive liquid; Concentrating under reduced pressure with concentrated solution, is used petroleum ether, the ethyl acetate extraction 4 times of 3 times of amounts successively, and ethyl acetate extract is carried out column chromatography with macroporous resin column; Behind the water elution impurity, ethanol gradient elution (the eluting scope is 20～80%) is collected ethanol elution; Concentrating under reduced pressure is dry, gets the Herba Clinopodii Polycephali effective part extract, and yield is 1.5%.
Embodiment 2: the preparation of capsule
Herba Clinopodii Polycephali effective part extract 65g among the embodiment 1, microcrystalline Cellulose 135g, ground and mixed 10-30 minute, to even.With the medicated powder of mixing, fill is in No. 1 capsule, and every loading amount of stochastic sampling is controlled at 0.2g, and containing the Herba Clinopodii Polycephali effective part extract is 65mg.
Embodiment 3: the preparation of tablet
Herba Clinopodii Polycephali effective part extract 25g among the embodiment 1, microcrystalline Cellulose 49g, ground and mixed 10-30 minute, to evenly ending; Add magnesium stearate 1g, fully mix; Mixture is pressed into diameter 6mm with single punch tablet machine, the tablet of heavy 300mg, and every contains the Herba Clinopodii Polycephali effective part extract is 100mg.
Embodiment 4: the preparation of granule
Herba Clinopodii Polycephali effective part extract 20g among the embodiment 1 adds corn starch 180 grams, behind the mixing, adds an amount of ethanol (60%) and processes soft material, crosses 12 mesh sieves, after drying, gets granule, and it is 100mg that per 1 gram granule contains the Herba Clinopodii Polycephali effective part extract.
Claims (9)
1. a method for preparing of preventing and treating the Herba Clinopodii Polycephali effective part extract of diabetes is characterized in that: get dry Herba Clinopodii Polycephali, pulverize; With ethanol and aqueous, methanol and aqueous thereof or propanol and aqueous reflux, extract, thereof, or dipping extracts the extracting solution concentrating under reduced pressure; Spissated extract is dispersed in the water, uses petroleum ether, ethyl acetate extraction 3～4 times successively, ethyl acetate extract carries out column chromatography with macroporous resin column; Behind the water elution impurity, ethanol gradient elution is collected ethanol elution; Concentrating under reduced pressure is dry, gets extract.
2. the method for preparing of Herba Clinopodii Polycephali effective part extract according to claim 1 is characterized in that: the ethanol gradient elution scope is 20～80%.
3. the method for preparing of Herba Clinopodii Polycephali effective part extract according to claim 1; It is characterized in that: this extract mainly is made up of total flavones and terpenoid, and the main component of said flavone is monarda glycoside, naringenin, apigenin, isosakuranetin, luteolin and acacetin; The main component of terpenoid is a ursolic acid, adopts ultraviolet spectrophotometry, with NaNO
2-Al (NO
3)
3-NaOH chromogenic assay general flavone content, in rutin, the total content of flavone compound is not less than 50%.
4. a Herba Clinopodii Polycephali effective part extract of preventing and treating diabetes is made by the said method for preparing of arbitrary claim among the claim 1-3.
5. a pharmaceutical composition of treating diabetes is characterized in that by the described extract of claim 4 and one or more adjuvant and/or mixed with excipients.
6. pharmaceutical composition according to claim 5 is characterized in that it is prepared to various dosage forms.
7. pharmaceutical composition according to claim 6, said dosage form are injection, capsule, tablet, granule, dragee or solution.
8. Herba Clinopodii Polycephali effective part extract according to claim 4; It is characterized in that said Herba Clinopodii Polycephali effective part extract can significantly reduce diabetes hyperglycemia, blood fat reducing; The protection islet cells suppresses alpha-glucosidase activity, the protection vascular endothelial cell.
9. the purposes of Herba Clinopodii Polycephali effective part extract according to claim 4 is characterized in that it is used to prepare the disorders of lipid metabolism that prevention and treatment diabetes, diabetes cause or the medicine of diabetic treating cardiac and cerebral vascular diseases complication.
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| US11090313B2 (en) | 2010-05-20 | 2021-08-17 | University Of Iowa Research Foundation | Methods for inhibiting muscle atrophy |
| US9295664B2 (en) * | 2011-06-06 | 2016-03-29 | University Of Iowa Research Foundation | Methods for lowering blood glucose |
| CN103664859A (en) * | 2012-09-05 | 2014-03-26 | 中国医学科学院药用植物研究所 | Novel isopentenyl naphthoquinone obtained in active fraction for promoting blood circulation to remove blood stasis in clinopodium chinense (Benth.) O. Kuntze. and preparation method thereof |
| CN103536615A (en) * | 2013-11-08 | 2014-01-29 | 中国药科大学 | Preparation method of didymin and isosakuranetin, and application thereof in anti-diabetic medicine |
| CN104262155A (en) * | 2014-08-29 | 2015-01-07 | 中国药科大学 | Phenolic acid compound, as well as preparation method and pharmaceutical applications thereof |
| CN105477411A (en) * | 2015-12-14 | 2016-04-13 | 刘玉英 | Nano traditional Chinese medicine composition for treating polycystic kidney and preparation method thereof |
| CN106636245A (en) * | 2016-12-15 | 2017-05-10 | 广东石油化工学院 | Method for preparing naringenin and apigenin by converting exocarpium citrus grandis leaf flavone by using microorganism |
| CN113999272A (en) * | 2021-10-29 | 2022-02-01 | 广西医科大学 | Preparation method and application of melissoside |
| CN115364144A (en) * | 2022-10-07 | 2022-11-22 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Yuqing lobular Kuding tea extract and preparation method and application thereof |
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