CN107551089A

CN107551089A - A kind of medicine for treating hyperlipidemia and preparation method thereof and detection method

Info

Publication number: CN107551089A
Application number: CN201710969758.XA
Authority: CN
Inventors: 梅花·尼合买提; 库丽夏西·马尼; 加尼亚·卡克尔汗; 古妮沙·沙地克; 叶尔扎提·海肉拉; 努尔古丽·卡克木
Original assignee: XINJIANG ALTAY REGION KAZAKH MEDICAL HOSPITAL
Current assignee: XINJIANG ALTAY REGION KAZAKH MEDICAL HOSPITAL
Priority date: 2017-10-18
Filing date: 2017-10-18
Publication date: 2018-01-09
Anticipated expiration: 2037-10-18
Also published as: CN107551089B

Abstract

The invention belongs to the field of Chinese medicines, and in particular to a kind of medicine for treating hyperlipidemia and preparation method thereof and detection method, the medicine are made up of the raw material of following number：9~15 parts of cassia seed, 10~15 parts of the red sage root, 9~12 parts of hawthorn, 6~12 parts of the fleece-flower root, 5~10 parts of feverfew, 3~9 parts of Leaves of Hippophae L, 6~10 parts of rhizoma alismatis, 9~15 parts of giant knotweed, 6~10 parts of wild marjoram, medicine of the present invention has the therapeutic effect of highly significant to hyperlipidemia.Present invention also offers the preparation method of the medicine and the content assaying method of index active component simultaneously.The technology of the present invention be traditional Chinese medical science national medicine management board of Xinjiang Uygur Autonomous Regions emphasis support project, project name：Xinjiang property of Chinese medicine ethnic drug new drug development cultivates project, problem title：Uygur medicine Empirical formula adjusts the clinical observation on the therapeutic effect of the oral side's treatment hyperlipidemia of fat drink, numbering：Q2015‑02‑14.

Description

A kind of medicine for treating hyperlipidemia and preparation method thereof and detection method

Technical field

The invention belongs to the field of Chinese medicines, and in particular to one kind treats hyperlipidemia and preparation method thereof and detection side Method.

Background technology

Hyperlipidemia is a kind of metabolic disease, and strict definition should be blood fat disorder or dyslipidemia, refer in human body The concentration level of serum (slurry) lipid is beyond normal range (NR).Symptom shows as dizziness, spiritlessness and weakness, insomnia forgetfulness, limbs more Numb, uncomfortable in chest, palpitaition etc..Long-term High-fat mass formed by blood stasis can cause fatty liver, liver function damage, occur when heavier have a dizzy spell, head Bitterly, uncomfortable in chest, shortness of breath, nervous, pectoralgia, weak, extremity numbness etc., are the important risk factors for causing atherosclerosis, are hats The main inducing of the cardiovascular and cerebrovascular diseases such as worry, to this, sick treatment is increasingly subject to pay attention to.Active treatment hyperlipidemia is also pre- Anti- cardiovascular and cerebrovascular disease, the important means for reducing the death rate.The medicine dialectical treatment hyperlipidemia that treats in Kazak has curative effect can Lean on, prolonged application safety, the advantage having no toxic side effect, increasingly attract attention.

The invention provides a kind of pharmaceutical composition for treating hyperlipidemia, research has shown that the extract has obvious drop Blood fat.The technology of the present invention be traditional Chinese medical science national medicine management board of Xinjiang Uygur Autonomous Regions emphasis support project, project Title：Xinjiang property of Chinese medicine ethnic drug new drug development cultivates project, problem title：Uygur medicine Empirical formula adjusts fat to drink oral Fang Zhi The clinical observation on the therapeutic effect that (hyperlipidemia) is closed in rare agate Yilan is treated, numbering：Q2015-02-14.

Meanwhile several pharmacy corporations of the innovative technology of this patent with Gansu Province and Jiangsu Province reach Intentionality cooperation Agreement, the innovative technology for this patent develop the preparation that new drug has carried out early stage.According to incompletely statistics, China controls The market capacity of hyperlipemia medicine is treated, it is about 30,000,000,000 yuan every year, preliminary pre- after innovative technology developing new drug of the invention success The annual market sales revenue is surveyed more than 1,000,000,000 yuan, the development to promoting China's Chinese medicine, ethnic drug real economy, will be played very Big impetus.

The content of the invention

There is provided that a kind of quick, effect is good and nothing the invention aims to solve above-mentioned the deficiencies in the prior art A kind of medicine for treating hyperlipidemia of side effect and preparation method thereof and detection method.

The invention provides the flavour of a drug raw material composition of the medicine for the treatment of hyperlipidemia.

Present invention also offers the preparation method of the medicine.

Present invention also offers the detection method of main active in the medicine；

Present invention also offers the pharmaceutical applications of the medicine.

The purpose of the present invention is achieved through the following technical solutions：

A kind of medicine for treating hyperlipidemia, the medicine are made up of the flavour of a drug raw material of following parts by weight：Cassia seed 9~15 Part, 10~15 parts of the red sage root, 9~12 parts of hawthorn, 6~12 parts of the fleece-flower root, 5~10 parts of feverfew, 3~9 parts of Leaves of Hippophae L, rhizoma alismatis 6~ 10 parts, 9~15 parts of giant knotweed, 6~10 parts of wild marjoram.

The medicine of described treatment hyperlipidemia, the medicine are preferentially made up of the flavour of a drug raw material of following parts by weight：Cassia seed 12 parts, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, wild marjoram 8 Part.

The medicine of described treatment hyperlipidemia, the preparation method of the medicine are as follows：

(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, wild marjoram are taken, is added Enter 8~12 times of amount pH for 3~5 aqueous hydrochloric acid solution, Microwave Extraction, 500~700W of microwave power, 15~25min of extraction time, 55~65 DEG C of Extracting temperature, extract solution filtration, filtrate are condensed into medicinal extract, obtain extractum A；The dregs of a decoction after extraction retain, standby；

(2) dregs of a decoction after step (1) extraction are taken, add the bionic extraction solvents of 8~12 times of amounts, 36~38 DEG C of water-baths add Heat, stirs 1.5~2.5h of extraction, centrifuges 25~35min, 4000~4400r/min of centrifugal speed, collects supernatant respectively and sinks Form sediment；Supernatant concentration, obtains medicinal extract B, and standby, precipitate C is standby；Above-mentioned bionic extraction solvent be by 0.05~0.15% sodium chloride, 0.1~0.3% pepsin, 0.1~0.3% trypsase and 0.01molL^-1Hydrochloric acid is configured to solution；

(3) extractum A obtained by step (1) is merged with the medicinal extract B obtained by step (2), precipitate C, stirred, mixed, obtain Mixed extract D；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:13~17, loading volume 10BV~14BV, with ethyl acetate:Third Ketone=90~100:5 eluant, eluent 10BV~14BV elutions, flow velocity is 5BV/h~7BV/h, collects eluent, and eluent concentrates, Dry, produce.

The medicine of described treatment hyperlipidemia, the preparation method of the medicine, is preferably as follows：

(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, wild marjoram are taken, is added Enter the aqueous hydrochloric acid solution that 10 times of amount pH are 4, Microwave Extraction, microwave power 600W, extraction time 20min, 60 DEG C of Extracting temperature, carry Liquid is taken to filter, filtrate is condensed into medicinal extract, obtains extractum A；The dregs of a decoction after extraction retain, standby；

(2) dregs of a decoction after step (1) extraction are taken, add the bionic extraction solvents of 10 times of amounts, 37 DEG C of heating water baths, stirring carries 2h is taken, centrifuges 30min, centrifugal speed 4200r/min, collects supernatant and precipitation respectively；Supernatant concentration, medicinal extract B is obtained, it is standby With precipitate C is standby；Above-mentioned bionic extraction solvent be by 0.1% sodium chloride, 0.2% pepsin, 0.2% trypsase and 0.01mol·L^-1Hydrochloric acid is configured to solution；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 Eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce.

The medicine of described treatment hyperlipidemia, piece agent, pill, hard capsule, soft capsule, oral can be prepared Liquid.

A kind of detection method for treating hyperlipidemia, the medicine are made up of the flavour of a drug raw material of following parts by weight：Cassia Son 8~10 parts, 10~15 parts of the red sage root, 9~12 parts of hawthorn, 6~12 parts of the fleece-flower root, 5~10 parts of feverfew, 3~9 parts of Leaves of Hippophae L, 6~10 parts of rhizoma alismatis, 9~15 parts of giant knotweed, 6~10 parts of wild marjoram；

Its preparation method is：

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:13~17, loading volume 10BV~14BV, with ethyl acetate:Third Ketone=90~100:5 eluant, eluent 10BV~14BV elutions, flow velocity is 5BV/h~7BV/h, collects eluent, and eluent concentrates, Dry, produce；

Determine red sickle rose element, 2,5- dimethoxy-benzoquinones, the Yin Dan in medicine simultaneously using reversed-phased high performace liquid chromatographic Join ketone, cyanidenon, miltionone content, step is as follows：

(1) chromatographic condition and system suitability：Chromatographic column：C₁₈Liquid-phase chromatographic column, mobile phase：Acetonitrile -0.35moL/L Sodium dihydrogen phosphate liquid, gradient elution：From 0min to 15min, acetonitrile is from 5% linear rise to 20%, 0.35moL/L phosphoric acid Dihydro sodium solution liquid is from 95% linear decline to 80%；From 16min to 25min, acetonitrile from 20% linear rise to 40%, 0.35moL/L sodium dihydrogen phosphates liquid is from 80% linear decline to 60%；From 26min to 35min, acetonitrile is linear from 40% 30%, 0.35moL/L sodium dihydrogen phosphates liquid is dropped to from 60% linear rise to 70%；From 35min to 40min, second Nitrile is from 30% linear decline to 20%, 0.35moL/L sodium dihydrogen phosphate liquid from 70% linear rise to 80%；Flow velocity 2.0mL·min^-1, 290~300nm of Detection wavelength, column temperature is 35~40 DEG C, and the μ L of sample size 5~20, theoretical cam curve is with red sickle Rose element meter is not less than 5000；

(2) prepared by reference substance solution：Accurately weighed red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, sweet-scented osmanthus respectively Careless element, the reference substance of miltionone are appropriate, are dissolved with ethyl acetate and quantify dilution, reference substance solution is made, produces；

(3) prepared by need testing solution：This product is taken, finely ground, precision weighs 1~3g, puts in 25~100mL measuring bottles, adds acetic acid Ethyl ester, 35~45min is ultrasonically treated, 250~350W of ultrasonic power, 55~65kHz of supersonic frequency, is let cool, it is dilute with ethyl acetate Release to scale, shake up, miillpore filter filtration, take subsequent filtrate, produce；

(4) determine：Precision draws reference substance solution, each 5~20 μ L of need testing solution, and injection high performance liquid chromatograph is surveyed It is fixed.

The detection method of described treatment hyperlipidemia, the medicine are made up of the flavour of a drug raw material of following parts by weight：Certainly 12 parts of pine torch, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, ox To 8 parts；

Its preparation method is：

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 Eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce；

Determine red sickle rose element, 2,5- dimethoxy-benzoquinones, the Yin Dan in medicine simultaneously using reversed-phased high performace liquid chromatographic Join ketone, cyanidenon, miltionone content, step preferably is as follows：

(1) chromatographic condition and system suitability：Chromatographic column：C₁₈Liquid-phase chromatographic column, model：250mm × 4.6mm, 5 μ m；Mobile phase：Acetonitrile -0.35moL/L sodium dihydrogen phosphate liquid, gradient elution：From 0min to 15min, acetonitrile is linear from 5% 20%, 0.35moL/L sodium dihydrogen phosphates liquid is risen to from 95% linear decline to 80%；From 16min to 25min, second Nitrile is from 20% linear rise to 40%, 0.35moL/L sodium dihydrogen phosphate liquid from 80% linear decline to 60%；From 26min to 35min, acetonitrile is from 40% linear decline to 30%, 0.35moL/L sodium dihydrogen phosphate liquid from 60% linear rise To 70%；From 35min to 40min, acetonitrile from 30% linear decline to 20%, 0.35moL/L sodium dihydrogen phosphate liquid from 70% linear rise is to 80%；Flow velocity 2.0mLmin^-1, Detection wavelength 296nm, column temperature is 38 DEG C, the μ L of sample size 10, theoretical tower Plate number is not less than 5000 in terms of red sickle rose element；

(2) prepared by reference substance solution：Accurately weighed red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, sweet-scented osmanthus respectively Careless element, the reference substance of miltionone are appropriate, dissolved with ethyl acetate and be quantitatively diluted to every 1mL respectively containing 100 μ g of red sickle rose element, μ g of 2,5- dimethoxy-benzoquinone 50, μ g of Cryptotanshinone 50, μ g of cyanidenon 100, the μ g of miltionone 50 mixed solution, are produced；

(3) prepared by need testing solution：This product is taken, finely ground, precision weighs 2g, puts in 50mL measuring bottles, adds ethyl acetate 40mL, 40min, ultrasonic power 300W, supersonic frequency 60kHz are ultrasonically treated, is let cool, is diluted to scale with ethyl acetate, shakes up, 0.22 μ M miillpore filters filter, and take subsequent filtrate, produce；

(4) determine：Precision draws reference substance solution, each 10 μ L of need testing solution, injection high performance liquid chromatograph measure.

A kind of detection method for treating hyperlipidemia, the medicine are made up of the flavour of a drug raw material of following parts by weight：Cassia 12 parts of son, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, wild marjoram 8 Part；Its preparation method is：

It is as follows using the content of gas chromatography measure dihydroactinidiolide, its step：

(1) chromatographic condition：Chromatographic column：HP-5 posts, model：30m × 0.32mm × 0.25 μm, to be crosslinked 5% phenyl methyl Polysiloxanes is stationary phase；Column temperature uses temperature programming：120 DEG C of starting, 20min is kept, rise to 190 DEG C with 10 DEG C/min, protect Hold 5min；Gasification temperature：260℃；Detector temperature：260℃；Carrier gas is nitrogen, flow 22mL/min, flow velocity 0.9mL/ min；The μ L of sample size 10, split ratio 4:1；

(2) measure of correction factor：N-octadecane is taken, it is accurately weighed, add normal hexane that solution of every mL containing 1.0mg is made, As inner mark solution；Dihydroactinidiolide reference substance 2mg is taken, it is accurately weighed, put in 10mL measuring bottles, precision adds inner mark solution 1mL, scale is diluted to normal hexane, is shaken up, taken 10 μ L to inject gas chromatograph, measure, calculate correction factor；

(3) preparation of need testing solution：Take this product precision to weigh 5g, put in conical flask with cover, chlorination imitates 50mL, at ultrasound 20min is managed, filtration, is evaporated with the appropriate chloroform dregs of a decoction and filter, merging filtrate and washing lotion, low temperature, residue adds normal hexane to make Dissolving, puts in 10mL measuring bottles, and precision adds inner mark solution 1.0mL, and scale is diluted to normal hexane, is filtered with 0.45 μm of miillpore filter Cross, take subsequent filtrate as need testing solution；

(4) determine：Take the μ L of need testing solution 10 to inject gas chromatograph, be measured.

A kind of medicine prepare treat hyperlipidemia in application, the medicine by following parts by weight flavour of a drug raw material system Into：12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, giant knotweed 12 Part, 8 parts of wild marjoram；

Its preparation method is：

(4) determine：Precision draws reference substance solution, each 10 μ L of need testing solution, injection high performance liquid chromatograph measure；

A kind of application of medicine in medicament for treating insomnia is prepared, the medicine are made up of the flavour of a drug raw material of following parts by weight： 12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, 8 parts of wild marjoram；

Its preparation method is：

Tested by following for embodying and verifying technical scheme and technique effect, be not intended to limit the present invention's Claimed technical scope.

Experiment one：The medicament screening experiment research of effect for reducing blood fat

1 experiment material

1.1 medicine

1.1.1 test medicine A：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：Take the cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, Wild marjoram, add the aqueous hydrochloric acid solution that 10 times of amount pH are 4, Microwave Extraction, microwave power 600W, extraction time 20min, Extracting temperature 60 DEG C, extract solution filtration, filtrate is condensed into medicinal extract, obtains medicinal extract, dries, produces.

1.1.2 test medicine B：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：Take the cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, Wild marjoram, adding 10 times of bionic extraction solvents measured, (bionic extraction solvent is by 0.1% sodium chloride, 0.2% pepsin, 0.2% Trypsase and 0.01molL^-1Hydrochloric acid is configured to solution), 37 DEG C of heating water baths, stirring extraction 2h, centrifuge 30min, centrifugation speed 4200r/min is spent, collects supernatant and precipitation respectively；Supernatant concentration, medicinal extract is obtained, standby, precipitation is standby；By above-mentioned medicinal extract Merge with precipitation, stir, mix, obtain mixed extract, dry, produce.

1.1.3 test medicine C：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, tiger are taken Cane, wild marjoram, add the aqueous hydrochloric acid solution that 10 times of amount pH are 4, Microwave Extraction, microwave power 600W, extraction time 20min, extraction Temperature 60 C, extract solution filtration, filtrate is condensed into medicinal extract, obtains medicinal extract, standby；

(2) column chromatography for separation chromatography is carried out to the medicinal extract obtained by step (1)；Entered from H-20 type macroporous absorbent resins Row column chromatography for separation, resin column blade diameter length ratio are 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce.

1.1.4 test medicine D：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, tiger are taken Cane, wild marjoram, add 10 times amount bionic extraction solvents (bionic extraction solvent be by 0.1% sodium chloride, 0.2% pepsin, 0.2% trypsase and 0.01molL^-1Hydrochloric acid is configured to solution), 37 DEG C of heating water baths, stirring extraction 2h, 30min is centrifuged, Centrifugal speed 4200r/min, supernatant and precipitation are collected respectively；Supernatant concentration, medicinal extract is obtained, standby, precipitation is standby；Will be upper State medicinal extract with precipitation to merge, stir, mix, obtain mixed extract；

(2) column chromatography for separation chromatography is carried out to the mixed extract obtained by step (1)；From H-20 type macroporous absorptions Resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 wash De- agent 12BV elutions, flow velocity 6BV/h, eluent is collected, eluent concentration, dries, produces.

1.1.5 the tested E of medicine：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, tiger are taken Cane, wild marjoram, add the aqueous hydrochloric acid solution that 10 times of amount pH are 4, Microwave Extraction, microwave power 600W, extraction time 20min, extraction Temperature 60 C, extract solution filtration, filtrate are condensed into medicinal extract, obtain extractum A；The dregs of a decoction after extraction retain, standby；

1.1.6 test medicine F：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Preparation method is：Take the cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, Wild marjoram, 10 times of amount aqueous solution are added, decoct extraction 3 times, 1 hour every time, merge extract solution, extract solution filtration, filtrate is condensed into Extract dry, produce.

Positive drug：Blood fat recovery capsule, produced by Beijing WBL Peking University Biotech Co., Ltd, authentication code：Traditional Chinese medicines Quasi- word Z10950029, specification：0.3g × 12 × 2 plates/box, lot number：20151213-8.

1.2 reagent triglyceride determination kits, T-CHOL measure kit, low-density LP determination reagent box, High-density LP determination reagent box, produced by Beijing Puli's lema gene Technology Co., Ltd..

1.3 animal Kunming mouses, male and female half and half, 18~22g of body weight, carried by Xinjiang Medicine University's Experimental Animal Center For quality certification number：SCXX (new) 2011-0004, raise in temperature (20 ± 2) DEG C, relative humidity 55%~65% environment in.

1.4 instrument B-260 type thermostat water baths, manufactured by Shanghai Jin Qi experimental instruments and equipment limiteds.TDL80-2B types are low Fast centrifuge, by Sichuan, Li Jun JingHua Medicines limited liability company system makes.DG5033A type enzyme-linked immunosorbent assay instruments, by Nanjing China Eastern electronics group's medical equipment limited liability company system makes.

2 experimental methods

The preparation of 2.1 test agent samples

Above-mentioned each 8g of test medicine A to F are weighed, it is 0.5%CMC-Na to add 100mL concentration, and being configured to concentration is 0.08g/mL decoction, it is standby.

The preparation of 2.2 yolk milk solutions

Egg yolk liquid 80mL is drawn, is put in 100mL graduated cylinder, with normal saline dilution to 100mL, is made into 80% egg-nog Homogeneous solution.

2.3 packets and administration

90 Kunming mouses, male and female half and half are taken, body weight is randomly divided into 9 groups, every group 10, divided in the range of 18~22g It is not：Normal group, hyperlipidemia model group, positive controls, test medicine A groups~test medicine F groups, gastric infusion.Normally It is 0.5%CMC-Na solution that control group and hyperlipidemia model group give concentration daily, and it is 0.2mL/10g to give volume；Positive control Group, test medicine A groups~test medicine F groups give relative medicine respectively, and dosage is 1g/kg.Administration 14 days, gives for the last time Two hours after medicine, in addition to Normal group, 80% yolk milk solution 0.02mL/g modelings, modeling 20 is injected intraperitoneally in remaining each group After hour, blood is taken from eyeball, by blood sample in 3000rpmmin^-1, 10min is centrifuged, isolated serum, is consolidated according to total courage Alcohol, triglycerides, low-density lipoprotein and the operation of HDL kit specification, measure T-CHOL (TC), glycerine The concentration of three esters (TG), low-density lipoprotein (LDL-C) and HDL (HDL-C).

2.4 statistical procedures

The data obtained carries out statistical analysis with SPSS11.0 statistical softwares, analysis result withRepresent, with P<0.05 has Statistical significance.

3 experimental results

After various dose drug administration 14 days, T-CHOL (TC), glycerine in each group experiment mice serum are determined respectively The concentration of three esters (TG), low-density lipoprotein (LDL-C) and HDL (HDL-C), experiment are shown in Table 1 with analysis result.

Influence of the various dose drug administration group of table 1 to the corresponding blood lipids index of mouse

Note：Compared with Normal group, ##P<0.01；Compared with hyperlipidemia model group, * * P<0.01, * P<0.05, it is and tested Medicine E groups compare, ★ P<0.05.

Experimental result shows that TC, TG, LDL-C of hyperlipidemia model group water average specific Normal group significantly raise (P< 0.01), the horizontal of HDL-C decreases (P than Normal group<0.01) induced Hyperlipidemia in Mice model modeling success, is shown；With Model group is compared, and test medicine E groups can significantly reduce TC, TG, LDL-C level (P of mouse<0.01) HDL-C water can, be raised Flat (P<0.01), its effect is better than test medicine A groups, test medicine B groups, test medicine C groups, test medicine D groups, test medicine F groups.

4 conclusions

This experimental study shows that reductions of the test medicine E to TC, TG, LDL-C has obvious effect, has to HDL-C significantly Rise acts on, and test medicine E has the definite effect for reducing experimental animal blood fat, and is only specifically made in test medicine E Manufactured medicine under Preparation Method, its effect for reducing blood fat ability highly significant.

Experiment two：Treat the clinical observation on the therapeutic effect of hyperlipidemia

This research method is further to verify that oral Uygur medicine Empirical formula medicine oral liquid of the present invention can effectively alleviate height Various malaise symptoms caused by pionemia, seek, Small side effects higher to hyperlipidemia cure rate and the economy easily side for the treatment of Method.The effectiveness and reliability that in June, 2015 in June, 2018 treats hyperlipidemia to oral medicine oral liquid of the present invention is carried out Clinical observation on the therapeutic effect, is now summarized as follows.

1 data and design

1.1 experimental design：Using random packet, the design of medicine check experiment.

1.2 diagnostic criteria：

(1) Uygur medicine diagnostic criteria：

Reference《Qi Pagereleke Bayans》Card category Si Tedeke, main symptom:Dizzy shortness of breath nose heave, uncomfortable in chest, limb fiber crops are heavy；It is secondary Card:Palpitaition, body are fat, weak, abdominal distension and anorexia, mouth stick, and have nausea be intended to tell, tongue fur is satiny, wiry and rolling pulse.Tool main symptom 2 above , secondary card at least 2 persons, you can diagnosis

(2) Western medicine diagnostic criteria：

Western medicine diagnostic criteria：In the case of normal diet, 2 blood specimens (serum) check in 2 weeks, T-CHOL (TC) >= 5.72mmol/l or triglyceride (TG) >=1.70mmol/l, low-density lipoprotein LDL-C) >=3.64mmol/l or high density fat Albumen (HDL-C)≤1.04mmol/l person can make a definite diagnosis.

In the case of normal diet and habits and customs, the blood lipid level after fasting 12-14 hours is detected, is judging whether to deposit In dyslipidemia, it is necessary to have at least 2 blood specimen detection record results are as foundation in 1-2 weeks.

1.3 case selection

The object of observation is Altay Prefecture's Uygur medicine internal medicine in hospital outpatient service and the patient being in hospital.Primary dyslipidemia is suffered from Person, there is toning fat drug administration history, but had more than more than two weeks and do not taken fat regulation medicine, and blood lipid level still accords with Close the patient of diagnostic criteria.102 high fat that hospital accepts for medical treatment are breathed out by Altay Prefecture during choosing in June, 2015 to 2 months 2017 Mass formed by blood stasis patient, all cases meet inclusive criteria.Using single blind random controls method, choose hyperlipidemia patient and be randomly divided into treatment Group (52), control group (50).180ml2 times/d of the oral Empirical formula for the treatment of group medicine oral liquid of the present invention.Control group is orally pungent Cut down before statin piece 20mg sleeps once, a course for the treatment of is 6 weeks.It is the effect of two groups of observation and bad anti-before medication and after medication 6 weeks Should, the related data of two groups of cases is subjected to statistical analysis.

2 methods：

2.1 patients for including this research must not take other in selected preceding first quarter moon to reduce the medicine of blood fat, and And diet after enrollment and life style all do not make any changes.

2.2 treatment group：Using medicine oral liquid of the present invention, prescription and preparation method are as follows：

Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 Eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, is added appropriate flavouring, add purified water to be diluted to 8000mL, point Dress, every bottle of 10mL, is made 800 bottles of oral liquid.

Usage and dosage：Orally, each 30mL, 3 times a day.

2.3 control group：It is oral using Simvastatin Tablets, 20mg/ times, every night 1 time it is oral.

2.4 the course for the treatment of：Two groups with 6 weeks for a course for the treatment of.

3 criterion：

3.1 breathe out doctor's therapeutic effect of syndrome criterion

Clinic control：Clinical symptoms, sign disappear or basic disappearance, syndrome integral reduce >=95%.

It is effective：Clinical symptoms, sign are obviously improved, and syndrome integral reduces >=70%

Effectively：Clinical symptoms, sign take a favorable turn, and syndrome integral reduces >=30%

It is invalid：Clinical symptoms, sign are not improved, even aggravating, syndrome integral is reduced<30%

Note：Calculation formula (Nimodipine method) is：

(being integrated before (being integrated before treatment after integration-treatment) ÷ treatments) × 100%.

3.2 laboratory examination curative effect determinate standards

Clinic control：Every check in laboratory recovers normal

It is effective：Lipids detection reaches following any one person.TC decline >=20%, TG >=40%, HDL-C of decline risings >= 0.26mmol/L。

Effectively：Lipids detection reaches following any one person.TC decline >=10% but<20%, TG decline >=20% but< 40%, HDL-C rising >=0.104mmol/L but<0.26mmol/L.

It is invalid：Lipids detection is not up to above standard person.

4 statistical procedures：

All data are represented with mean ± standard deviation (x ± s), are compared between group and are examined with t, and multi-group data compares with variance point Analysis, compare carry out q inspections two-by-two；Examined during heterogeneity of variance using t ＇.All data are counted using SPSS11.0 software kits Analysis.P<0.05 is to have significant difference.

Through SPSS statistical analysis, experimental group patient is total effective 34, accounts for 80.91%；Control group patient is total effective 29, Account for 63.04%；Experimental group total effective rate is apparently higher than control group, χ²=6.99, P ＜ 0.05, difference is statistically significant.In detail It the results are shown in Table 2, table 3, table 4.

5 results

5.1 two groups of total effectses compare

5.1.1 the comparison of two groups of clinical efficacies after treating

The comparative example (%) of two groups of clinical efficacies after table 2 is treated

5.1.2 two groups of dizziness and extremity numbness curative effect compare

Two groups of dizzy and extremity numbness curative effect comparative examples (%) after table 3 is treated

6.1.3 two groups of blood fat comprehensive therapeutic effect compares

The blood fat Evaluation of Synthetic Effect of Holistic of 4 two groups of table

Through SPSS statistical analysis, experimental group patient is total effective 44, accounts for 84.61%；Control group patient is total effective 38, Account for 76.04%；Experimental group total effective rate is apparently higher than control group, χ²=6.99, P ＜ 0.05, difference is statistically significant.

Experiment three：RPLC (RP-HPLC) method determines the content of 5 compositions in medicine of the present invention simultaneously

Research shows that the red sickle rose contained in cassia seed is plain, 2,5- dimethoxy-benzoquinones, the hidden red sage root contained in the red sage root Ketone, miltionone, the cyanidenon contained in feverfew, be medicine composite for curing hyperlipidemia of the present invention some activity into Point, so, this detection method is established in 5 kinds of flavour of a drug raw materials in simultaneous quantitative medicine of the present invention using high performance liquid chromatography The method of 5 main active contents.

1 instrument and reagent

1.1 instrument

Waters2695 high performance liquid chromatographs, including it is quaternary pump, PDAD, on-line degassing machine, automatic Injector；KQ-300DV type ultrasonic cleaners, manufactured by Shanghai Qiao Feng Instrument Ltd.；P225D type electronic analytical balances, Manufactured by Shanghai Ping Xuan scientific instrument Co., Ltd；AL2004 type electronic analytical balances, by the limited public affairs of Shanghai Ping Xuan scientific instrument Department's manufacture.

1.2 reagent

Medicine of the present invention：Medicine of the present invention also turns into this product, lot number 160926 in the present invention.

Its preparation method is：

Medicinal material used in the present invention is purchased from this thatched cottage chain pharmacy of Xinjiang, is accredited as through the college of traditional Chinese medicine of Xinjiang Medicine University Certified products.

Reference substance：Red sickle rose element, is produced, lot number 20160315, content by Shenzhen Bo Taier Pharmaceutical Technology Co., Ltd 99.6%；2,5- dimethoxy-benzoquinones, produced by Chengdu standard specimen bio tech ltd, lot number 20160428, content 99.5%；Cryptotanshinone, produced by Jiangsu Yong Jian Pharmaceutical Technology Co., Ltd, lot number 20160223, content 99.3%；Reseda Element, produced by Shanghai Xin Rui bio tech ltd, lot number 20160513, content 99.2%；Miltionone, by Changzhou Bei Yuan Prosperous bio tech ltd's production, lot number 20160318, content 99.4%；Phosphoric acid is pure to analyze, and acetonitrile is chromatographically pure, and water is Ultra-pure water.

2 methods and result

It is prepared by 2.1 solution

2.1.1 prepared by reference substance solution

Accurately weighed red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, pair of miltionone respectively It is appropriate according to product, dissolved with ethyl acetate and be quantitatively diluted to every 1mL respectively containing 100 μ g of red sickle rose element, 2,5- dimethoxy-benzoquinones 50 μ g, μ g of Cryptotanshinone 50, μ g of cyanidenon 100, the μ g of miltionone 50 mixed solution, are produced.

2.1.2 prepared by need testing solution

This product is taken, finely ground, precision weighs 2g, puts in 50mL measuring bottles, adds ethyl acetate 40mL, is ultrasonically treated 40min, ultrasound Power 300W, supersonic frequency 60kHz, lets cool, and is diluted to scale with ethyl acetate, shakes up, and 0.22 μm of miillpore filter filtration, takes continuous Filtrate, produce.

2.1.3 prepared by negative sample solution

Prepared by process and lack cassia seed, the red sage root, the negative sample of feverfew, pressed method under " 2.1.2 " item respectively and grasp Make, the negative sample solution of different quality concentration is made.

2.2 chromatographic conditions and system suitability

Chromatographic column：C₁₈Liquid-phase chromatographic column, model：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.35moL/L di(2-ethylhexyl)phosphates Hydrogen sodium solution liquid, gradient elution：From 0min to 15min, acetonitrile is from 5% linear rise to 20%, 0.35moL/L sodium dihydrogen phosphate Solution liquid is from 95% linear decline to 80%；From 16min to 25min, acetonitrile is from 20% linear rise to 40%, 0.35moL/ L sodium dihydrogen phosphates liquid is from 80% linear decline to 60%；From 26min to 35min, acetonitrile from 40% linear decline to 30%, 0.35moL/L sodium dihydrogen phosphate liquid are from 60% linear rise to 70%；From 35min to 40min, acetonitrile from 30% linear decline to 20%, 0.35moL/L sodium dihydrogen phosphate liquid is from 70% linear rise to 80%；Flow velocity 2.0mL min^-1, Detection wavelength 296nm, column temperature is 38 DEG C, the μ L of sample size 10, and theoretical cam curve is not less than 5000 in terms of red sickle rose element.

Accompanying drawing 1, accompanying drawing 2, accompanying drawing 3, the accompanying drawing that mixing reference substance, sample, negative sample chromatogram are shown in Figure of description 4th, accompanying drawing 5.As a result show, red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, miltionone 5 are to be measured The chromatographic peak of composition and the separating degree of adjacent peak are all higher than 1.8, and the other compositions in sample are to the measure of 5 compositions to be measured without dry Disturb.

2.3 Method validation

2.3.1 linear relationship is investigated

Precision draw mixing reference substance storing solution 0.2mL, 0.4mL, 0.6mL, 1.2mL, 2.4mL, 4.8mL, 9.6mL, 19.2mL, it is respectively placed in 25mL measuring bottles, with methanol dilution to scale, shakes up, produces the mixed reference substance solution of series concentration. It is accurate respectively to draw 10 μ L sample introductions, record chromatogram.With mass concentration X (μ gmL^-1) it is abscissa, peak area Y is ordinate Mapping, obtains regression equation.It the results are shown in Table 5.Mixed reference substance solution is diluted step by step, sample introduction measure, is fixed using 10 times of signal to noise ratio Amount limit, as a result red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, the quantitative limit of miltionone are respectively 0.145、0.065、0.070、0.45、0.085μg·mL^-1。

The red sickle rose element of table 5,2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, miltionone linear relationship result

2.3.2 precision test

It is new that precision draws red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, the red sage root under " 2.3.1 " item Ketone mass concentration is respectively 20 μ gmL^-1、5μg·mL^-1、5μg·mL^-1、20μg·mL^-1、5μg·mL^-1Mixing reference substance The μ L of solution 10, continuous sample introduction 6 times, peak area is determined, it is respectively 1.3%, 1.4%, 1.2%, 1.5%, 2.2% to calculate RSD；Knot Fruit shows that instrument precision is good.

2.3.3 need testing solution stability test

Take with a need testing solution, respectively at 0h, 3h, 6h, 12h, 24h, 48h, peak face is determined by above-mentioned chromatographic condition Product, red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, the RSD of miltionone chromatographic peak area are respectively 1.4%th, 2.2%, 1.5%, 2.3%, 1.2%；As a result show that need testing solution is stable in 48h.

2.3.4 replica test

Same batch of 6 parts of sample (lot number 161012) is taken, according to legal system available test sample solution below " 2.1.2 " item, above-mentioned Sample introduction measure is distinguished under chromatographic condition.Red sickle rose element in results sample, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, reseda Element, miltionone average mass fraction (n=6) are respectively 425.3mgg^-1、85.6mg·g^-1、83.9mg·g^-1、 101.6mg·g^-1、76.5mg·g^-1, RSD is respectively 1.3%, 1.2%, 2.3%, 1.1%, 1.6%；As a result method weight is shown Renaturation is good.

2.3.5 average recovery is tested

The 6 parts of sample for having predicted content (lot number 161012) appropriate particles are taken, finely ground, precision weighs 2g, puts 50mL amounts In bottle, precision adds mixing reference substance storing solution 2.5mL, test solution is prepared according to the method under " 2.1.2 " item, by above-mentioned chromatogram Condition is measured, and calculates the rate of recovery.As a result red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, the red sage root New ketone average recovery rate (n=6) is respectively 98.6%, 97.5%, 101.5%, 10.2.3%, 99.4%, RSD be respectively 0.9%th, 1.2%, 1.9%, 2.1%, 1.3%.

2.3.6 sample size determines

The medicine of the present invention of 10 batches of different lot numbers is taken, each 2 parts, need testing solution is prepared into according to method below " 2.1.2 " item, Sample introduction measure is distinguished under above-mentioned chromatographic condition, external standard method calculates content.It the results are shown in Table 6.

Assay result (the mgg of 5 compositions in 6 medicine of the present invention of table^-1, n=2)

3 conclusions

The method that this method is established can separate red sickle rose element, 2,5- dimethoxy-benzoquinones, Yin Dan in medicine of the present invention simultaneously Join ketone, cyanidenon, the content of miltionone, method is easy, reliable, reproducible, available for medicine of the present invention activity into Divide content detection.

Experiment four：Red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, the red sage root are new in difference group medicine The content of ketone

1 testing sample

1.1 test medicine A：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

1.2 test medicine B：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

1.3 test medicine C：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

1.4 test medicine D：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

The tested E of 1.5 medicines：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

1.6 test medicine F：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, sand Spine leaf 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

2 assay methods

Using the high performance liquid chromatography of the offer of present invention experiment three, red sickle rose element, 2,5- in Simultaneous Determination each group medicine Dimethoxy-benzoquinone, Cryptotanshinone, cyanidenon, the content of miltionone.

3 measurement results

Measurement result, it is shown in Table 7.

The each group sample size measurement result mgg of table 7^-1

4 discuss

As can be seen from the above table, red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, the wood in test medicine E groups Apparently higher than other test medicine groups, this may be also test medicine E in treatment hyperlipemia for rhinoceros grass element, the content of miltionone The effect of aspect is better than the major reason of other test medicines.

Experiment five：Gas chromatography determines the content of dihydroactinidiolide in medicine of the present invention

Premenstruum (premenstrua) research shows that contained dihydroactinidiolide is the important work of medicine reducing blood lipid of the present invention in cassia seed Property composition, so, the present invention content of the dihydroactinidiolide in medicine of the present invention is measured using gas chromatography.

1 instrument and reagent

6890N gas chromatographs (Agilent)；Hydrogen flame detector (FID)；High Purity Nitrogen (purity more than 99.99%)；Two Hydrogen actinidia lactone reference substance, produced by Chengdu standard specimen bio tech ltd, lot number 20160617, through area normalization method Content is determined as 99.95%)；N-octadecane (Tokyo chemical conversion industry Co., Ltd.)；Agents useful for same is that analysis is pure.

Its preparation method is：

2 methods and result

2.1 chromatographic condition

Chromatographic column：HP-5 posts (30m × 0.32mm × 0.25 μm), to be crosslinked 5% methyl-polysiloxane as stationary phase； Column temperature uses temperature programming：120 DEG C of starting, 20min is kept, rise to 190 DEG C with 10 DEG C/min, keep 5min；Gasification temperature： 260℃；Detector temperature：260℃；Carrier gas is nitrogen, flow 22mL/min, flow velocity 0.9mL/min；The μ L of sample size 10, point Stream is than being 4:1；On this condition, the relative position at dihydroactinidiolide peak and n-octadecane peak is suitable, in need testing solution Separating degree between dihydroactinidiolide peak and n-octadecane peak and adjacent peak is all higher than 1.8, and negative control is noiseless；It is theoretical Plate number is calculated by dihydroactinidiolide peak, more than 50000.As a result accompanying drawing 6, accompanying drawing 7, accompanying drawing 8 are seen.

The measure of 2.2 correction factors

N-octadecane is taken, it is accurately weighed, add normal hexane that solution of every mL containing 1.0mg is made, as inner mark solution；Take dihydro Actinidia lactone reference substance 2mg, it is accurately weighed, put in 10mL measuring bottles, precision adds inner mark solution 1mL, is diluted to normal hexane Scale, shake up, take 10 μ L to inject gas chromatograph, measure, calculate correction factor.

The preparation of 2.3 need testing solutions

Take this product precision to weigh 5g, put in conical flask with cover, chlorination imitates 50mL, is ultrasonically treated 20min, filtration, with appropriate The chloroform dregs of a decoction and filter, merging filtrate and washing lotion, low temperature are evaporated, and residue adds normal hexane to make dissolving, are put in 10mL measuring bottles, essence Close addition inner mark solution 1.0mL, scale is diluted to normal hexane, is filtered with 0.45 μm of miillpore filter, takes subsequent filtrate to be used as trying Product solution.

2.4 linear relationships are investigated

Precision weighs dihydroactinidiolide reference substance 20.0mg, puts in 10mL measuring bottles, adds normal hexane to dissolve and be diluted to Scale, it is configured to reference substance solutions of every mL containing 2.0mg.It is accurate respectively to draw dihydroactinidiolide reference substance solution each 0.1, 0.2,0.5,1.0,2.0,4.0mL, put in 5mL measuring bottles, it is accurate respectively to add above-mentioned inner mark solution 0.5mL, add normal hexane to dilute To scale.Determined by above-mentioned chromatographic condition sample introduction, using the peak area ratio of reference substance and internal standard compound as ordinate, with dihydro macaque Concentration (the mgmL of peach lactone^-1) it is abscissa, calculate regression equation.Dihydroactinidiolide regression equation is Y= 0.0086452+3.16824X, r=0.9996, show dihydroactinidiolide concentration in 0.032~1.128mgmL^-1Scope Interior is in good linear relationship.

2.5 precision test

The μ L of mixed solution 1 of same reference substance and internal standard compound are taken, repeat sample introduction 5 times, calculate the peak of reference substance and internal standard compound Area ratio, as a result peak area mean ratio is 1.365, RSD=0.85%, shows that precision is good.

2.6 stability test

Take the mixed solution of same reference substance and internal standard compound, respectively in 0,4,8,12,16h sample introduction, calculate reference substance with it is interior The peak area ratio of thing is marked, as a result peak area mean ratio is 1.354, RSD=0.36%, shows that dihydroactinidiolide exists It is relatively stable in 16h.

2.7 replica test

Take with batch 5 parts of sample, by method parallel test under the preparation of need testing solution and measure item, measure dihydro in sample The average content of actinidia lactone is 0.301mg/g, RSD=1.42%.

2.8 average recoveries are tested

Take this product 1g of known content amount, each accurate a certain amount of dihydroactinidiolide reference substance of addition, by for trying The assay method operation of product solution, calculates the rate of recovery, the results are shown in Table 8.

The average recovery result of the test of table 8

2.9 samples determine

The μ L of need testing solution 10 are drawn, injects in gas chromatograph, determines peak area in accordance with the law, content is calculated by internal standard method, As a result the dihydroactinidiolide content of 3 batches of every gram of samples is respectively 0.301 ± 0.004mg, 0.306 ± 0.007mg, 0.308 ±0.006mg。

3 conclusions

The content of dihydroactinidiolide in medicine of the present invention is determined using GC methods, method is simple, quick, can be used as this hair One of quality control index of bright medicine.

Experiment six：The content of dihydroactinidiolide in difference group medicine

1 testing sample

2 assay methods

Using the gas chromatography of the offer of present invention experiment five, dihydroactinidiolide contains in Simultaneous Determination each group medicine Amount.

3 measurement results

Measurement result, it is shown in Table 9.

The each group sample size measurement result mgg of table 9^-1

4 discuss

As can be seen from the above table, the content of the dihydroactinidiolide in test medicine E groups is tested apparently higher than other Medicine group, this may be also the major reason that effects of the test medicine E in terms of hyperlipemia is treated is better than other test medicines.

Experiment seven：The malicious experimental study of urgency of medicine of the present invention

1 experiment material

1.1 test medicine：Medicine of the present invention：Prescription：It is cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, white Chrysanthemum 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Its preparation method is：(1) take the cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, Giant knotweed, wild marjoram, the aqueous hydrochloric acid solution that 10 times of amount pH are 4 is added, Microwave Extraction, microwave power 600W, extraction time 20min, is carried Temperature 60 C, extract solution filtration are taken, filtrate is condensed into medicinal extract, obtains extractum A；The dregs of a decoction after extraction retain, standby；

(2) dregs of a decoction after step (1) extraction are taken, add the bionic extraction solvents of 10 times of amounts, 37 DEG C of heating water baths, stirring carries 2h is taken, centrifuges 30min, centrifugal speed 4200r/min, collects supernatant and precipitation respectively；Supernatant concentration, medicinal extract B is obtained, it is standby With precipitate C is standby；Above-mentioned bionic extraction solvent be by 0.1% sodium chloride, 0.2% pepsin, 0.2% trypsase and 0.01molL-1 hydrochloric acid is configured to solution；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 Eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce.With 0.8g/mL's during experiment Liquid extract.

1.2 experimental animals and feeding environment：SD mouse 40, body weight are 18~22g, male and female half and half, SPF levels, by Xinjiang Medical university's Experimental Animal Center provides, quality certification number：SCXX (new) 2011-0004, raise in temperature (20 ± 2) DEG C, relatively In the environment of humidity 55%~65%.

Used after mouse cage tool and pallet cleaning and sterilizing.Freely, feed is sterilizing full nutrition particle for drinking water for animals, feed Feed.Animal feeding condition：23 ± 2 DEG C of room temperature, relative humidity 40%~70%, daily illumination 12h.

2 test methods

Take mouse, 40, male and female half and half, a point sex is randomly divided into 2 groups, i.e. blank control group and administration group, every group 20, Each 10 of male and female.Blank control group gavage physiological saline, per 10g body weight gavages 1.0mL；The medicine stream leaching of the present invention of administration group gavage Cream 2.0g crude drugs/mL, per 10g body weight gavage 1.0mL, it is administered 4 times in 1 day, dosing interval 6h.Stood after the first day each administration is administered It is the breathing of quarter, 2h, 4h, 6h Continuous Observation animal activity situation, including mouse, motion, reflection, eyelid indication, salivary secretion, perpendicular Hair, Muscle tensility, stool, urine, dermoreaction, and poisoning or it is dead situations such as, be shown in Table 10.2d~14d after administration, daily The general performance of observation and death condition, Continuous Observation 14d.Weighed during 7d, 14d puts to death mouse after weighing, and will be poisoned to death Animal and execution animal postmortem, the lesion situation of main organs is visually observed, if there is anomalous variation, carries out pathologic examination Record changes of weight.

The animal acute toxicity test of table 10 observes table

3 result of the tests

Oral gavage gives medicine liquid extract daily dose of the present invention and adds up to 8.0g crude drugs/kg, and administration same day mouse activity subtracts Few, stool is in dark brown, just dilute soft, and next day defecation recovers normal.Two groups of mouse breathings are normal, and behavioral activity and autonomic activities are not See exception, cornea, right, stretch reflex is present, absent lacrimation, without exophthalmos.During 14d is observed, animal ordinary circumstance is good, Ingest normal, body weight increase, hair color is smooth, and urine is without significant change.Animal dead is had no during whole experiment.

At the end of experiment, put to death animal, dissect and observe the heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus gland, thyroid gland, gas The internal organs such as pipe, oesophagus, Stomach duodenum, colon, bladder, testis, uterus, ovary, have not seen that obvious pathology sexually revises.It is real Blank control group is consistent with administration group body weight increase during testing, and two groups of animal point sexes are compared, and body weight increase has no difference (p ＞ 0.05), the results are shown in Table 11.It is 8.0g crude drugs/kg to prompt this experiment to measure maximum dosage, clinical equivalent to referrer 20 times of daily dosage.

The acute toxicity mouse weight table (g, Yan x ± s) of table 11

4 conclusions

Result of the test shows, Acute toxicity very little of the present invention.

Brief description of the drawings：

Fig. 1 is to mix reference substance HPLC chromatogram, and No. 1 peak is red sickle rose element in figure；No. 2 peaks are 2,5- dimethoxy benzenes Quinone；No. 3 peaks are Cryptotanshinone；No. 4 peaks are cyanidenon；No. 5 peaks are miltionone.

Fig. 2 is the negative sample HPLC chromatogram for lacking cassia seed, and No. 3 peaks are Cryptotanshinone in figure；No. 4 peaks are reseda Element；No. 5 peaks are miltionone.

Fig. 3 is the negative sample HPLC chromatogram for lacking the red sage root, and No. 1 peak is red sickle rose element in figure；No. 2 peaks are 2,5- dimethoxies Base benzoquinones；No. 4 peaks are cyanidenon.

Fig. 4 is the negative sample HPLC chromatogram for lacking feverfew, and No. 1 peak is red sickle rose element in figure；No. 2 peaks are 2,5- diformazans Epoxide benzoquinones；No. 3 peaks are Cryptotanshinone；No. 5 peaks are miltionone；

Fig. 5 is sample HPLC chromatogram, and No. 1 peak is red sickle rose element in figure；No. 2 peaks are 2,5- dimethoxy-benzoquinones；No. 3 peaks For Cryptotanshinone；No. 4 peaks are cyanidenon；No. 5 peaks are miltionone；

Fig. 6 composes for correcting fluid GC chromatic graphs, and No. 1 peak is dihydroactinidiolide in figure, and No. 2 peaks are n-octadecane；

Fig. 7 composes for test sample GC chromatic graphs, and No. 1 peak is dihydroactinidiolide in figure, and No. 2 peaks are n-octadecane；

Fig. 8 is to lack Pogostemon cablin negative controls GC chromatic graphs spectrum, and No. 2 peaks are n-octadecane in figure

Embodiment

Examples below is used for the further explanation to technical solution of the present invention, but is not restricted to the claims in the present invention Protection domain.

Embodiment 1：

Its preparation method is：

(5) result：Red sickle rose element in this product, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, cyanidenon, miltionone Content is respectively 448.3mgg^-1、85.6mg·g^-1、88.2mg·g^-1、1.10.5mg·g^-1、72.8mg·g^-1。

(4) determine：Take the μ L of need testing solution 10 to inject gas chromatograph, be measured；

(5) measurement result：Dihydroactinidiolide content is 0.313mg in every gram of test sample.

Embodiment 2：

Medicament capsule of the present invention：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Its preparation method is：

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；Inhaled from H-20 types macropore Attached resin carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 Eluant, eluent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, add proper starch, dextrin, mix, Granulation, whole grain, load capsule, hard shell capsules 800 are made.

Embodiment 3：

Medicine oral liquid of the present invention：Prescription：Cassia seed 120g, red sage root 120g, hawthorn 100g, fleece-flower root 90g, feverfew 80g, Leaves of Hippophae L 60g, rhizoma alismatis 80g, giant knotweed 120g, wild marjoram 80g；

Its preparation method is：

Claims

1. a kind of medicine for treating hyperlipidemia, it is characterised in that the medicine is made up of the flavour of a drug raw material of following parts by weight：Cassia Son 9~15 parts, 10~15 parts of the red sage root, 9~12 parts of hawthorn, 6~12 parts of the fleece-flower root, 5~10 parts of feverfew, 3~9 parts of Leaves of Hippophae L, 6~10 parts of rhizoma alismatis, 9~15 parts of giant knotweed, 6~10 parts of wild marjoram.

2. it is according to claim 1 treatment hyperlipidemia medicine, it is characterised in that the medicine by following parts by weight medicine Taste raw material is made：12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, rhizoma alismatis 8 Part, 12 parts of giant knotweed, 8 parts of wild marjoram.

3. the medicine for the treatment of hyperlipidemia according to claim 1 or 2, it is characterised in that the preparation method of the medicine is such as Under：

(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, wild marjoram are taken, add 8~ The aqueous hydrochloric acid solution that 12 times of amount pH are 3~5, Microwave Extraction, 500~700W of microwave power, 15~25min of extraction time, extraction 55~65 DEG C of temperature, extract solution filtration, filtrate are condensed into medicinal extract, obtain extractum A；The dregs of a decoction after extraction retain, standby；

(2) dregs of a decoction after step (1) extraction are taken, the bionic extraction solvents of 8~12 times of amounts is added, 36~38 DEG C of heating water baths, stirs 1.5~2.5h of extraction is mixed, centrifuges 25~35min, 4000~4400r/min of centrifugal speed, collects supernatant and precipitation respectively；On Clear liquid concentrates, and obtains medicinal extract B, standby, precipitate C is standby；Above-mentioned bionic extraction solvent be by 0.05~0.15% sodium chloride, 0.1~ 0.3% pepsin, 0.1~0.3% trypsase and 0.01molL^-1Hydrochloric acid is configured to solution；

(3) extractum A obtained by step (1) is merged with the medicinal extract B obtained by step (2), precipitate C, stirred, mixed, mixed Extract D；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；From H-20 type macroporous absorption trees Fat carries out column chromatography for separation, and resin column blade diameter length ratio is 1:13~17, loading volume 10BV~14BV, with ethyl acetate:Acetone= 90~100:5 eluant, eluent 10BV~14BV elutions, flow velocity is 5BV/h~7BV/h, collects eluent, eluent concentration, does It is dry, produce.

4. the medicine for the treatment of hyperlipidemia according to claim 3, it is characterised in that the preparation method of the medicine is as follows：

(1) cassia seed of recipe quantity, the red sage root, hawthorn, the fleece-flower root, feverfew, Leaves of Hippophae L, rhizoma alismatis, giant knotweed, wild marjoram are taken, adds 10 The aqueous hydrochloric acid solution that amount pH is 4 again, Microwave Extraction, microwave power 600W, extraction time 20min, 60 DEG C of Extracting temperature, extract solution Filtration, filtrate are condensed into medicinal extract, obtain extractum A；The dregs of a decoction after extraction retain, standby；

(2) dregs of a decoction after step (1) extraction are taken, add the bionic extraction solvents of 10 times of amounts, 37 DEG C of heating water baths, stirring extraction 2h, 30min, centrifugal speed 4200r/min are centrifuged, collect supernatant and precipitation respectively；Supernatant concentration, medicinal extract B is obtained, it is standby, Precipitate C, it is standby；Above-mentioned bionic extraction solvent be by 0.1% sodium chloride, 0.2% pepsin, 0.2% trypsase and 0.01mol·L^-1Hydrochloric acid is configured to solution；

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；From H-20 type macroporous absorption trees Fat carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 elution Agent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce.

5. it is according to claim 3 treatment hyperlipidemia medicine, it is characterised in that the medicine can prepare piece agent, Pill, hard capsule, soft capsule, oral liquid.

6. a kind of detection method for treating hyperlipidemia, the medicine is made up of the flavour of a drug raw material of following parts by weight：Cassia seed 8 ~10 parts, 10~15 parts of the red sage root, 9~12 parts of hawthorn, 6~12 parts of the fleece-flower root, 5~10 parts of feverfew, 3~9 parts of Leaves of Hippophae L, rhizoma alismatis 6~10 parts, 9~15 parts of giant knotweed, 6~10 parts of wild marjoram；Its preparation method is：

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；From H-20 type macroporous absorption trees Fat carries out column chromatography for separation, and resin column blade diameter length ratio is 1:13~17, loading volume 10BV~14BV, with ethyl acetate:Acetone= 90~100:5 eluant, eluent 10BV~14BV elutions, flow velocity is 5BV/h~7BV/h, collects eluent, eluent concentration, does It is dry, produce；

Characterized in that, determine red sickle rose element, 2, the 5- dimethoxy benzenes in medicine simultaneously using reversed-phased high performace liquid chromatographic Quinone, Cryptotanshinone, cyanidenon, miltionone content, step are as follows：

(1) chromatographic condition and system suitability：Chromatographic column：C₁₈Liquid-phase chromatographic column, mobile phase：Acetonitrile -0.35moL/L phosphoric acid Dihydro sodium solution liquid, gradient elution：From 0min to 15min, acetonitrile is from 5% linear rise to 20%, 0.35moL/L biphosphate Sodium solution liquid is from 95% linear decline to 80%；From 16min to 25min, acetonitrile from 20% linear rise to 40%, 0.35moL/L sodium dihydrogen phosphates liquid is from 80% linear decline to 60%；From 26min to 35min, acetonitrile is linear from 40% 30%, 0.35moL/L sodium dihydrogen phosphates liquid is dropped to from 60% linear rise to 70%；From 35min to 40min, second Nitrile is from 30% linear decline to 20%, 0.35moL/L sodium dihydrogen phosphate liquid from 70% linear rise to 80%；Flow velocity 2.0mL·min^-1, 290~300nm of Detection wavelength, column temperature is 35~40 DEG C, and the μ L of sample size 5~20, theoretical cam curve is with red sickle Rose element meter is not less than 5000；

(2) prepared by reference substance solution：Accurately weighed red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, reseda respectively Element, the reference substance of miltionone are appropriate, are dissolved with ethyl acetate and quantify dilution, reference substance solution is made, produces；

(3) prepared by need testing solution：This product is taken, finely ground, precision weighs 1~3g, puts in 25~100mL measuring bottles, adds ethyl acetate, 35~45min is ultrasonically treated, 250~350W of ultrasonic power, 55~65kHz of supersonic frequency, lets cool, quarter is diluted to ethyl acetate Degree, shakes up, and miillpore filter filtration, takes subsequent filtrate, produces；

(4) determine：Precision draws reference substance solution, each 5~20 μ L of need testing solution, injection high performance liquid chromatograph measure.

7. the detection method for the treatment of hyperlipidemia as claimed in claim 6, the medicine is former by the flavour of a drug of following parts by weight Material is made：12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, tiger 12 parts of cane, 8 parts of wild marjoram；Its preparation method is：

(4) column chromatography for separation chromatography is carried out to the mixed extract D obtained by step (3)；From H-20 type macroporous absorption trees Fat carries out column chromatography for separation, and resin column blade diameter length ratio is 1:15, loading volume 12BV, with ethyl acetate:Acetone=95:5 elution Agent 12BV is eluted, flow velocity 6BV/h, is collected eluent, eluent concentration, is dried, produce；

(1) chromatographic condition and system suitability：Chromatographic column：C₁₈Liquid-phase chromatographic column, model：250mm × 4.6mm, 5 μm；Stream Dynamic phase：Acetonitrile -0.35moL/L sodium dihydrogen phosphate liquid, gradient elution：From 0min to 15min, acetonitrile is from 5% linear rise To 20%, 0.35moL/L sodium dihydrogen phosphates liquid from 95% linear decline to 80%；From 16min to 25min, acetonitrile from 20% linear rise to 40%, 0.35moL/L sodium dihydrogen phosphate liquid is from 80% linear decline to 60%；From 26min to 35min, acetonitrile is from 40% linear decline to 30%, 0.35moL/L sodium dihydrogen phosphate liquid from 60% linear rise to 70%； From 35min to 40min, acetonitrile is linear from 70% from 30% linear decline to 20%, 0.35moL/L sodium dihydrogen phosphate liquid Rise to 80%；Flow velocity 2.0mLmin^-1, Detection wavelength 296nm, column temperature is 38 DEG C, and the μ L of sample size 10, theoretical cam curve is with red Sickle rose element meter is not less than 5000；

(2) prepared by reference substance solution：Accurately weighed red sickle rose element, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, reseda respectively Element, the reference substance of miltionone it is appropriate, with ethyl acetate dissolve and be quantitatively diluted to every 1mL respectively containing 100 μ g of red sickle rose element, 2, μ g of 5- dimethoxy-benzoquinones 50, μ g of Cryptotanshinone 50, μ g of cyanidenon 100, the μ g of miltionone 50 mixed solution, are produced；

(3) prepared by need testing solution：This product is taken, finely ground, precision weighs 2g, puts in 50mL measuring bottles, adds ethyl acetate 40mL, ultrasound 40min, ultrasonic power 300W, supersonic frequency 60kHz are handled, lets cool, is diluted to scale with ethyl acetate, shakes up, 0.22 μm micro- Hole filter membrane filtration, takes subsequent filtrate, produces；

8. a kind of detection method for treating hyperlipidemia, the medicine is made up of the flavour of a drug raw material of following parts by weight：Cassia seed 12 parts, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, wild marjoram 8 Part；Its preparation method is：

Characterized in that, determining the content of dihydroactinidiolide using gas chromatography, its step is as follows：

(1) chromatographic condition：Chromatographic column：HP-5 posts, model：30m × 0.32mm × 0.25 μm, to be crosslinked the poly- silicon of 5% phenyl methyl Oxygen alkane is stationary phase；Column temperature uses temperature programming：120 DEG C of starting, 20min is kept, rise to 190 DEG C with 10 DEG C/min, keep 5min；Gasification temperature：260℃；Detector temperature：260℃；Carrier gas is nitrogen, flow 22mL/min, flow velocity 0.9mL/min； The μ L of sample size 10, split ratio 4:1；

(2) measure of correction factor：N-octadecane is taken, it is accurately weighed, add normal hexane that solution of every mL containing 1.0mg is made, as Inner mark solution；Dihydroactinidiolide reference substance 2mg is taken, it is accurately weighed, to put in 10mL measuring bottles, precision adds inner mark solution 1mL, Scale is diluted to normal hexane, is shaken up, takes 10 μ L to inject gas chromatograph, measure, calculates correction factor；

(3) preparation of need testing solution：Take this product precision to weigh 5g, put in conical flask with cover, chlorination imitates 50mL, is ultrasonically treated 20min, filtration, is evaporated, it is molten that residue adds normal hexane to make with the appropriate chloroform dregs of a decoction and filter, merging filtrate and washing lotion, low temperature Solution, puts in 10mL measuring bottles, and precision adds inner mark solution 1.0mL, and scale is diluted to normal hexane, is filtered with 0.45 μm of miillpore filter Cross, take subsequent filtrate as need testing solution；

9. a kind of medicine is preparing the application in treating hyperlipidemia, it is characterised in that the medicine is by following parts by weight Flavour of a drug raw material is made：12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, rhizoma alismatis 8 Part, 12 parts of giant knotweed, 8 parts of wild marjoram；

Its preparation method is：

Determined simultaneously using reversed-phased high performace liquid chromatographic red sickle rose element in medicine, 2,5- dimethoxy-benzoquinones, Cryptotanshinone, Cyanidenon, miltionone content, step are as follows：

A kind of 10. application of medicine in medicament for treating insomnia is prepared, it is characterised in that the medicine by following parts by weight flavour of a drug Raw material is made：12 parts of cassia seed, 12 parts of the red sage root, 10 parts of hawthorn, 9 parts of the fleece-flower root, 8 parts of feverfew, 6 parts of Leaves of Hippophae L, 8 parts of rhizoma alismatis, 12 parts of giant knotweed, 8 parts of wild marjoram；

Its preparation method is：