CN103340916B - Lindley eupatorium extract as well as preparation method and application thereof - Google Patents
Lindley eupatorium extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a lindley eupatorium extract and a preparation method thereof. Compared with the prior art, the lindley eupatorium extract is higher in transfer rate of total flavone, and meanwhile a total flavone offer has good activity for hyperlipidemia and liver cirrhosis diseases.
Description
Technical field
The present invention relates to a kind of Chinese medicine extract, relate to a kind of Herba Eupatorii Lindleyani extract, preparation method and application thereof in particular.
Technical background
Herba Eupatorii Lindleyani is the ground drying nest of feverfew Eupatorium lindleyanum (Eupat/rium Lindleyanum DC.), bitter in the mouth, and property is put down, and has effect of clearing away lung-heat to relieve cough, resolving phlegm and relieving asthma, blood pressure lowering, blood fat.Contained by Herba Eupatorii Lindleyani herb, chemical composition is based on flavone compound, also has alkaloids, volatile oil and coumarin, sesquiterpenoids ester etc.The biological activity that flavone compound has obvious antiulcer, spasmolytic, antiinflammatory and blood fat reducing etc. serial, its preparation prepared, has treatment valuable to the disease such as cardiovascular and cerebrovascular vessel, arteriosclerosis, " three-hypers " and long-term taking has no side effect.Current research finds, in Herba Eupatorii Lindleyani, flavone compound and alkaloid have analgesic activity; Flavone compound can increase the pharmacological actions such as leukocyte count.Medicine and food are widely used, for fully developing this resource comprehensively, have very important significance so extract total flavones from Herba Eupatorii Lindleyani.
The method of existing extraction purification total Flavonoids from Eupatorium Lindleyanum DC, as " research of lindley eupatorium herb general flavone purifying process " literary composition of " R&D of modern TCM with put into practice " the 25th volume the 5th phase in 2011, disclosed method is: the total flavones in ethanol extraction Herba Eupatorii Lindleyani, after D101 macroporous resin adsorption eluting, obtain total flavones.The major defect of the method is: 1. purification procedures is single, effectively can not remove impurity; 2. the purity of product is not high, and impurity is more, and color and luster is bad; 3. use organic reagent, do not recycle, cause serious problem of environmental pollution; 4. use high concentration ethanol in a large number, improve production cost, waste energy not environmentally.
Summary of the invention
For solving the problem, the invention provides a kind of preparation method of Herba Eupatorii Lindleyani extract, comprising the following steps:
1) Herba Eupatorii Lindleyani crude drug is pulverized, use 70-90% alcohol reflux, obtain extracting solution;
2) merge extractive liquid, reclaims ethanol to without alcohol taste, adds water and is diluted to every gram of crude drug extract and is dissolved in the water of 5-10 times of weight, obtain dilute aqueous solution;
3) filter dilute aqueous solution, removing solid residue, obtains clear liquid, by D101 or AB-8 macroporous resin column on clear liquid, and with being greater than the water elution macroporous resin column of 5 times of column volumes;
4) by the ethanol elution macroporous resin column of 20-60%, merge ethanol elution, reclaim ethanol to dry, extract low-temperature freeze drying is prepared Herba Eupatorii Lindleyani extract lyophilized powder.
After Herba Eupatorii Lindleyani crude drug being pulverized in step 1), before alcohol reflux, also carry out infiltrating Herba Eupatorii Lindleyani crude drug powder with alkaline water, make the pH value of Herba Eupatorii Lindleyani crude drug powder be 9-11.
Before Herba Eupatorii Lindleyani crude drug being pulverized in step 1), also with petroleum ether or gasoline reflux, extract, Herba Eupatorii Lindleyani crude drug.
Described alkaline water is Ca/H or NaC/
3aqueous solution.
A kind of Herba Eupatorii Lindleyani extract, adopt method described above to prepare Herba Eupatorii Lindleyani extract, wherein the general flavone content of Herba Eupatorii Lindleyani extract is higher than 50%.
The application of described Herba Eupatorii Lindleyani extract in preparation treatment atherosclerosis medicine.
The application of the Herba Eupatorii Lindleyani extract that described method prepares in preparation treatment liver cirrhosis medicine.
Advantageous Effects of the present invention is: the invention provides the preparation method that a kind of Herba Eupatorii Lindleyani extracts, the method hinge structure, the rate of transform of total flavones is higher.Total flavones provides thing to hyperlipidemia regulating liver-QI sclerotic conditions simultaneously, has good activity.
Detailed description of the invention
Embodiment 1 different pre-treatments mode is on the impact of the total flavones rate of transform
1, total flavones detection method
Take Herba Eupatorii Lindleyani ethanol extract respectively, control substance of Rutin add ethanol in proper amount dissolve, add 5%NaN/
21mL, places 6min, then adds 10%AL (N/
3)
31mL, places 6min, adds 1m/lL
-1na/H10mL, adding distil water is settled to scale, with blank after 15min, scans at 400-600nm wavelength place with spectrophotography.Result shows: Herba Eupatorii Lindleyani 70% ethanol extract is maximum in wavelength 490nm place trap, and control substance of Rutin is maximum in 487nm place trap, therefore selects 487nm place to survey absorption of sample degree.
Respectively precision measure control substance of Rutin stock solution 0.5,1,2,3,4,5,6mL in 25mL volumetric flask, adding distil water, to 10mL, adds 5%NaN/
21mL, places 6min, then adds 10%AL (N/
3)
31mL, places 6min, adds lm/lL
-1na/H10mL, adding distil water is settled to scale.With corresponding solution for blank, after 15min, measure trap at 487nm wavelength place.Be vertical coordinate with rutin content, take trap as abscissa, calculating regression equation is:
(, γ=0.9998), the rutin range of linearity is 0.10365-1.2438mg.Accurate absorption concentration is 0.2073mgmL
-1control substance of Rutin 2mL, operation repetitive 6 times, the same standard curve of assay method, RSD is 0.435%.
Take wild horse medical material, clay into power, cross No. six sieves (100 order), take powder 5.012g, as censorship group, add 7/% alcohol solvent 150mL by solid-to-liquid ratio (1:30) and infiltrate, after microwave treatment 15min, reflux in the water-bath of 7/ DEG C lixiviate 1h, lixiviating solution filter, centrifugal, pipette sample liquid 2.0/mL; By rutin standard curve algoscopy, with reagent for blank reference, under the wavelength of 487nm, survey its absorbance A, and draw rutin concentration C from the relation of rutin concentration and absorbance, through the total flavones rate of transform obtained in sample that converts.
The total flavones rate of transform (mg/g dry weight)=(milliliter number is drawn in C × 25/) × V/W
Wherein, V represents extracting liquid volume (mL), and W represents sample dry weight (g).
The rate of transform of the total flavones adopted in the former medicine of ultrasonic extracting method Herba Eupatorii Lindleyani is after testing 19.33mg/g.
Take Herba Eupatorii Lindleyani crude drug 3 groups respectively, each 500.0g, respectively called after 70% alcohol reflux group, alkaline pretreated group and defat pretreated group;
The process of 70% alcohol reflux group is: by pulverizing medicinal materials, be the ratio of 1:10 according to the ratio of the volume (mL) of Herba Eupatorii Lindleyani powder (g) ︰ 70% alcoholic solution, alcoholic solution is added in Herba Eupatorii Lindleyani powder, stir, pump in reflux, extract, device, carry out reflux, extract, 3 times, each 60-100 minute, merge reflux extracting liquid, collect for subsequent use.
The process of alkalescence pretreated group is: by pulverizing medicinal materials, infiltrates medical material 5h with 25 DEG C of saturated limewater clear liquid 250mL of dilution 3 times; Other operations are identical with the process of 70% alcohol reflux group.
The process of defat pretreated group is: by the petroleum ether 1000mL reflux, extract, medical material 1h of medical material 60-90%, volatilize the organic solvent on medical material, and by pulverizing medicinal materials, other operations are identical with the process of alkaline pretreated group.
Detect the general flavone content of each group respectively, specifically as shown in table 1.
Table 1 different medical material preliminary treatment mode is on the impact of the medical material total flavones rate of transform
As shown in Table 1, after adopting alkaline pretreatment, the rate of transform of total flavones significantly improves, and meanwhile, carries out the rate of transform that ungrease treatment also effectively can improve total flavones before extraction to medical material.
The separation and purification of embodiment 2 lindley eupatorium herb general flavone
1, the pretreatment before macroporous resin column chromatography
70% alcohol reflux group embodiment 1 prepared, alkaline pretreated group and defat pretreated group pump in Rotary Evaporators, carry out being concentrated into without till ethanol taste, collect respectively and reclaim ethanol and concentrated clear paste.The extraction in embodiment 1 in method can be continued on for for the recovery ethanol collected; For the concentrated clear paste collected, according to the ratio that the ratio of the volume (mL) of clear paste (g) ︰ pure water is 1 ︰ 10 ~ 15, first in clear paste, add pure water, stir, fully dissolve clear paste, then lysate is pumped in sucking filtration machine, carry out sucking filtration, collect filtering residue and filtrate respectively, for the filtrate of collecting, be upper prop liquid; For the filtering residue collected, be chlorophyll, for the additive of health food after drying.
2, macroporous resin column chromatography process
Activate suitable macroporous resin (i.e. D101, AB-8 macroporous resin or polyamide) loaded in chromatographic column by commercially available, first recoil with the isopyknic pure water of macroporous resin column, after discharging the bubble in macroporous resin column, pump into the 70% alcohol reflux group of having filtered, alkaline pretreated group and defat pretreated group filtrate again, upper prop liquid adsorbs, until cross in post effluent there is flavone time till, collected post effluent.After having adsorbed, again pump into and wash with the pure water of macroporous resin column volume 2 ~ 6 times, washing is entrained in the impurity of interlaminar resin, collects scrub stream fluid respectively and has completed the macroporous resin column of absorption.After the scrub stream fluid of collecting and mistake post effluent are merged, carry out Aerobic Process for Treatment, rear discharge up to standard.
Again toward completing in the macroporous resin column of absorption, the ethanol contend concentration pumping into 2 ~ 5 times of column volumes be 5 ~ 10% alcoholic solution carry out eluting, the flow velocity of ethanol elution is 3 ~ 5 times/hour of the macroporous resin column volume adsorbed, and collects the macroporous resin column after eluting remove impurity.For the macroporous resin column after eluting remove impurity, the ethanol contend concentration pumping into 2 ~ 6 times of column volumes be again 20 ~ 60% alcoholic solution carry out eluting, eluting is adsorbed in the total flavones in macroporous resin column, without till during flavone in eluting effluent, the flow velocity of ethanol elution is 3 ~ 5 times/hour of the macroporous resin column volume adsorbed.Collect the macroporous resin column after total flavones effluent and eluting total flavones respectively.For the total flavones effluent collected, for the preparation of total flavones lyophilized powder; For the macroporous resin column of eluting total flavones, can reuse after activation.
3, lyophilized powder is prepared
After macroporous resin column chromatography process completes, the each group of total flavones effluent that macroporous resin column chromatography process is collected pumps in vacuum concentrating apparatus, vacuum be-0.06 ~-0.09MPa, at temperature is 75 ~ 90 DEG C, carry out vacuum concentration respectively, without till during ethanol abnormal smells from the patient in concentrated solution, collect total flavones concentrated solution and evaporated liquor (ethanol namely reclaimed).For the ethanol reclaimed, can reuse after regulating its concentration; For the total flavones concentrated solution collected, first pre-freeze 5 ~ 6 hours in the refrigerator of-18 DEG C respectively, and then be placed in freezer dryer respectively, temperature be-50 ~-60 DEG C, under vacuum is the condition of 25 ~ 50Pa, dry 24 ~ 30 hours respectively, just prepare purity for being greater than 50% total flavones lyophilized powder respectively; Detect the content of each group of total flavones lyophilized powder total flavones respectively, detection method adopts the total flavones detection method described in embodiment, detect and find that the total flavones yield of defat pretreated group and alkaline pretreated group is higher than 70% alcohol reflux group, but in lyophilized powder, the content of total flavones is lower than 70% alcohol reflux group, and particular content is as shown in table 2.
Table 2 different pretreatments method is on the impact of total flavones purification
Embodiment 3 total flavones lyophilized powder is investigated in the activity for the treatment of hyperlipidemia
Choose qualified mice 60, be divided into 6 groups at random, often organize 10.The Rhizoma Coptidis lyophilized powder that administration embodiment 2 prepares.70% alcohol reflux group, alkaline pretreated group and defat pretreated group press respectively crude drug 25.12,25.56,25.28gkg
-1dosage gavage total flavones lyophilized powder; Atorvastatin group (positive controls) is by 2.20mgkg
-1gavage; Blank group and model group such as to gavage respectively at 1% hydroxy methocel (CMC) of capacity, 0.5mL10g
-1, 1 d
-1, continuous 30d.2h after last gavages medicine, each experimental group (except blank group), mice is lumbar injection 75% egg-nog respectively, 0.5mL only, take a blood sample through eye socket after 20h, the corresponding index of mice is respectively organized, the results detailed in Table 3 with serum cholesterol test kit, serum triglycerides test kit and HDL-C, LDL-C kit measurement.
The level of table 3 each group mice serum cholesterol, triglyceride, HDL-C and LDL-C compares
Compared with model group:
*p ﹤ 0.05
*p ﹤ 0.01
As shown in Table 1, total flavones lyophilized powder is respectively organized and all obviously can be reduced egg-nog induced mice serum cholesterol, triglyceride, LDL-C rising effect, can also obviously raise HDL-C level simultaneously, compare with model group and there is significant difference (P ﹤ 0.05 or P ﹤ 0.01).
Choose qualified rat 60, be divided into 6 groups at random, often organize 10.In the normal feedstuff of mice, add cholesterol 2%, yolk powder 5%, Adeps Sus domestica 10%, methylthiouracil 0.2%, be made into high lipid food.Choose qualified rat 6/, be divided into 6 groups at random, only often organize l/.Blank group feeds normal diet, feeds high lipid food for all the other 5 groups, while nursing high lipid food, give test medicine.70% alcohol reflux group, alkaline pretreated group and defat pretreated group press respectively crude drug 25.43,25.12,25.19gkg
-1dosage ig administration, atorvastatin group presses 1.10mgkg
-1gavage; Blank group and model group such as to gavage at the 1%CMC of capacity.2 groups of administration capacity are 0.5mL100g
-1, 1 d
-1, continuous 30d.Fasting 24h after last gavages medicine, through eye socket blood sampling, measures the serum cholesterol of each group of rat, triglyceride and HDL-C, LDL-C, the results detailed in Table 4.
The level of table 4 each group rat blood serum cholesterol, triglyceride, HDL-C and LDL-C compares
Compared with model group:
*p ﹤ 0.01
As shown in Table 2, total flavones lyophilized powder obviously can reduce Experimental Hyperlipemia rat blood serum cholesterol, triglyceride, LDL-C level, can obviously raise HDL-C level simultaneously, compare have significant difference (P ﹤ 0.01) with model group.
Embodiment 3 total flavones lyophilized powder is investigated in the activity for the treatment of liver cirrhosis
The SD rat 60 in 6 months ages of Mus, male and female half and half, weight (280-25) g, raise indoor temperature and remain on 24 ~ 26 DEG C, humidity maintains 50%-60%, and special room is raised, and special messenger is responsible for.60 rats are divided into 6 groups at random, i.e. Normal group, model control group, positive controls, total flavones lyophilized powder 70% alcohol reflux group, alkaline pretreated group and defat pretreated group press respectively crude drug 25.12,25.56,25.28gkg
-1dosage ig administration, often organize 10, male and female half and half.
Normal group gives normal saline by 3mLkg
-15mL/kg is pressed with normal saline after subcutaneous injection
-1gavage, 1 d
-1.Model control group gives 600gL
-1carbon tetrachloride Oleum Arachidis hypogaeae semen, by 3mLkg
-1subcutaneous injection, first dose doubles, and 3d injects 1 time; After initial injection, press 5mLkg with normal saline
-1gavage, 1 d
-1.Positive controls, by front method injection carbon tetrachloride, gives 28mgL simultaneously
-1colchicine presses 5mLkg
-1(140gkg
-1) gastric infusion, 1 d
-1.0.5kgL
-1group is to 2kgL
-1group is respectively total flavones lyophilized powder 70% alcohol reflux group, alkaline pretreated group and defat pretreated group, and 3 groups, all by front method injection carbon tetrachloride, are given corresponding dosage total flavones lyophilized powder 5mL/kg simultaneously
-1gastric infusion.Each group of equal successive administration of rat 7 weeks, at the end of experiment, rat all opens abdomen under the flat sleeping state of anesthesia, enters pin MS302 systematic survey portal pressure, then get serum and make every biochemical analysis from portal vein; Get the organs such as liver, kidney, spleen, thymus, adrenal gland and claim quality, calculate following organ index: liver index=liver quality/weight × 100; Renal index=kidney quality/weight × 1/0; Index and spleen index=spleen weight/weight × 1/0; Thymus index=thymic factor D injection/weight × 1000; Weight-index=adrenals mass/weight × 1000.Get leftlobe of liver Bo Shi liquid-solid fixed, paraffin embedding, after section, hematoxylin=eosin stains and Van-Gies/nShi picric acid and the dyeing of acid fuchsin method, make pathologic finding.
Quantum evaluation: the observation to rat ordinary circumstance: the general status observing rat every day, changes in diet, 7d claims weight 1 time.Measure the activity of alanine aminotransferase, aspartat aminotransferase with reitman-frankel method, measure total serum protein with biuret method, measure serum albumin levels with Bromocresol green, measure the content of Serum hyaluronic acid with radioimmunoassay.
Total flavones lyophilized powder on the impact of rat weight, liver quality and other organs in table 5.Total flavones lyophilized powder on the impact of rat blood serum biochemical indicator in table 6.
Table 5 carbon tetrachloride and total flavones lyophilized powder are on the impact of gland quality on rat liver,kidney,spleen, thymus and hail
After testing 7 weeks, the weight of each group rat all has increase, but total flavones lyophilized powder various dose group weight obviously increases than other groups, and difference has significance (P ﹤ 0.05-0.01) compared with Normal group, model control group.The liver quality increase (P ﹤ 0.01) more remarkable in Normal group of model control group, positive controls, 70% alcohol reflux group group, but alkaline pretreated group and defat pretreated group no significant difference difference compared with Normal group, and alkaline pretreated group and defat pretreated group obviously alleviate (P ﹤ 0.05) than model control group.Model control group liver index obviously increases than Normal group, alkalescence pretreated group and defat pretreated group and Normal group difference nonsignificance, its liver performance figure decline (P ﹤ 0.05) more obvious than positive controls, model control group renal index, index and spleen index increase (P ﹤ 0.05) than Normal group, thymus index and Weight-index change does not have a significance, and other four groups and Normal group, model control group two groups of comparing differences are not significantly (P ﹥ 0.05).
Table 6 carbon tetrachloride and total flavones lyophilized powder are on the impact of rat blood serum biochemical indicator
Compared with model group:
*p ﹤ 0.05
*p ﹤ 0.01
Alkalescence pretreated group and defat pretreated group alanine aminotransferase are than model control group, positive controls two groups decline, and aspartat aminotransferase declines than model control group, wherein 2kg/L
-1group aspartat aminotransferase obviously lower than positive controls (P ﹤ 0.05); Positive controls, alkaline pretreated group and defat pretreated group group hyaluronic acid all low than model control group (P ﹤ 0.05); Model control group, positive controls two groups of albumin are obviously low than Normal group, but alkaline pretreated group and defat pretreated group low unlike Normal group (P ﹥ 0.05) and higher than model control group; 70% alcohol reflux group is significantly not active for indices.
Claims (3)
1. a preparation method for Herba Eupatorii Lindleyani extract, is characterized in that: comprise the following steps:
1) Herba Eupatorii Lindleyani crude drug is pulverized, use 70-90% alcohol reflux, obtain extracting solution;
2) merge extractive liquid, reclaims ethanol to without alcohol taste, adds water and is diluted to every gram of crude drug extract and is dissolved in the water of 5-10 times of weight, obtain dilute aqueous solution;
3) sucking filtration dilute aqueous solution, removing solid residue, obtains clear liquid, by D101 or AB-8 macroporous resin column on clear liquid, and with being greater than the water elution macroporous resin column of 5 times of column volumes;
4) by the ethanol elution macroporous resin column of 20-60%, merge ethanol elution, reclaim ethanol to dry, extract low-temperature freeze drying is prepared Herba Eupatorii Lindleyani extract lyophilized powder;
Before Herba Eupatorii Lindleyani crude drug being pulverized in step 1), also with petroleum ether or gasoline reflux, extract, Herba Eupatorii Lindleyani crude drug; After Herba Eupatorii Lindleyani crude drug is pulverized, infiltrate Herba Eupatorii Lindleyani crude drug powder with alkaline water, make the pH value of Herba Eupatorii Lindleyani crude drug powder be 9-11, then carry out using 70-90% alcohol reflux.
2. the preparation method of Herba Eupatorii Lindleyani extract according to claim 1, is characterized in that: described alkaline water is Ca (OH)
2or Na
2cO
3aqueous solution.
3. a Herba Eupatorii Lindleyani extract, is characterized in that: adopt the method described in any one of claim 1,2 to prepare Herba Eupatorii Lindleyani extract, wherein the general flavone content of Herba Eupatorii Lindleyani extract is higher than 50%.
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| CN108159099B (en) * | 2014-06-04 | 2021-03-26 | 苏州大学 | Application of eupatorium sesquiterpene part in preparation of anti-hepatitis B virus medicine |
| CN106309529B (en) * | 2016-10-25 | 2019-11-12 | 南京中医药大学 | A kind of preparation method of eupatorium lindleynun var. trifoliolatum active component |
| CN106943442B (en) * | 2017-04-13 | 2020-02-14 | 重庆市中药研究院 | Antiviral traditional Chinese medicine extract and preparation method thereof |
| CN113588855A (en) * | 2021-07-16 | 2021-11-02 | 陕西中医药大学 | Quality detection method of lindley eupatorium herb |
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