CN105079093A - Wild chrysanthemum flower extract and medical application thereof for treating blood stasis disease - Google Patents
Wild chrysanthemum flower extract and medical application thereof for treating blood stasis disease Download PDFInfo
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- CN105079093A CN105079093A CN201510540886.3A CN201510540886A CN105079093A CN 105079093 A CN105079093 A CN 105079093A CN 201510540886 A CN201510540886 A CN 201510540886A CN 105079093 A CN105079093 A CN 105079093A
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- chrysanthemi indici
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Abstract
The invention discloses a wild chrysanthemum flower extract and medical application thereof for treating a blood stasis disease, belongs to the field of traditional Chinese medicines and in particular relates to a wild chrysanthemum flower extract and a medicinal preparation containing the extract, wherein the wild chrysanthemum flower extract can be used for preparing medicines for treating the blood stasis disease. An active component enriched extract is obtained by adopting a method of performing liquid-liquid extraction preliminary enrichment, macro-porous resin impurity removal and then further enrichment to the ethanol extract of wild chrysanthemum flowers, and has remarkable anticoagulation and antithrombus activity. A liquid phase analysis provided by the invention can be used for establishing fingerprints of the extract to control bath-to-batch consistency of different batches of extracts.
Description
Technical field
The present invention relates to the field of Chinese medicines, be specifically related to a kind of Flos Chrysanthemi Indici extract and the pharmaceutical preparation containing this extract, this Flos Chrysanthemi Indici extract can be used for preparing the medicine for the treatment of blood stasis disease.
Background technology
Flos Chrysanthemi Indici is the head inflorescence of feverfew Herba Dendranthematis indici ChrysanthemumindicumL. HERBA CHRYSANTHEMI LAVANDULAEFOLII C.borealeMak. or lavandulaleaf dendranthema herb C.lavandulaefolium (Fisch.) Mak..In " Chinese Pharmacopoeia " 2010 editions and " Chinese medicine voluminous dictionary " second edition, the source of Flos Chrysanthemi Indici is all only the dry capitulum of feverfew Herba Dendranthematis indici ChrysanthemumindicumL..Flos Chrysanthemi Indici is cool in nature, bitter in the mouth, pungent, returns lung Liver Channel, has effect of wind and heat dispersing, subduing swelling and detoxicating, cure mainly anemopyretic cold, hypertension, pneumonia, aphtha, carbuncle furuncle etc.
Flos Chrysanthemi Indici contains number of chemical composition.Flos Chrysanthemi Indici is more containing ter penoids, and mainly contain monoterpene, sesquiterpene, dimerization sesquiterpene, triterpene and containing oxygen derivative thereof etc., wherein major part is present in volatile oil.Flavone compound is active ingredient important in Flos Chrysanthemi Indici.From Flos Chrysanthemi Indici, flavone compound sweet-scented osmanthus flavonoid glycoside is just isolated as far back as nineteen forty-two people.
The medicinal efficacy of Flos Chrysanthemi Indici is all described in numerous books such as Flos Chrysanthemi Indici is with a long history in China, China's Chinese medicine book Shennong's Herbal.The traditional Chinese medical science thinks that Flos Chrysanthemi Indici fragrant taste is slightly pained, the property of medicine is more plain, having clears away the dampness and heat removes effect of toxin, reducing swelling and alleviating pain, for the damp and hot hotness brought of summer weather emit with hot-tempered and because of its occur aphthosis have therapeutical effect well, liver-fire can be gone, control headache rhinorrhea, avoid heat disappear heat effect.Flos Chrysanthemi Indici also has to the red and swollen pustule after summer mosquito bite the effect that sterilization goes to swell in addition.Summer the more can eat Flos Chrysanthemi Indici, calmly, especially to the working clan of every day in the face of computer, can alleviate the fatigue of eye, nourish the effect of eyes and vision protection.Flos Chrysanthemi Indici is not only edible also can the effect of external application, Flos Chrysanthemi Indici is pulverized the effect having sterilization and eliminate pustule.In addition modern medicine also active research Flos Chrysanthemi Indici to the therapeutical effect of modern disease; research display Flos Chrysanthemi Indici has very strong resistivity to antibacterial; can resist in extraneous cell entry body, there is prophylactic effect, all there is protective effect to the heart of human body, brain and blood vessel.Often drink the effect that wild chrysanthemum tea also has reduction and steady blood pressure in addition, for hypertension, there is certain therapeutical effect.
Summary of the invention
The object of the present invention is to provide a kind of Flos Chrysanthemi Indici extract, the preparation method of this extract and liquid phase analysis method, the pharmaceutical preparation containing this extract and utilize this extract to prepare the purposes for the treatment of blood stasis disease medicament.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
A kind of Flos Chrysanthemi Indici extract, this extract is prepared by following methods:
Step one: by weight, every 100 parts of medical materials comprise Flos Chrysanthemi Indici 80 ~ 90 parts and Radix Sophorae Flavescentis 10 ~ 20 parts, co-grinding;
Step 2: extract with 95% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste;
Step 3: petroleum ether, ethyl acetate and water saturated n-butanol extraction are used successively to step 2 concentrated solution, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
Step 4: n-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry.
In order to can by setting up HPLC fingerprint
figurethe Flos Chrysanthemi Indici extract quality that method controls Different sources, prepared by batch medical material of spectrum, the liquid phase analysis method of described extract is:
Chromatographic column: AgilentTCC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.1% formic acid solution (triethylamine adjust ph to 6.0);
Gradient elution program: 0 ~ 5min, A35%; 5 ~ 40min, A35% → 70%; 40 ~ 57min, A70% → 95%; 57 ~ 65min, A95%;
Flow rate of mobile phase: 1.0mLmin
-1; Determined wavelength: 240nm; Column temperature: 30 DEG C; Sample size 10 μ L.
Pharmaceutical preparation, the described Flos Chrysanthemi Indici extract containing treatment effective dose and pharmaceutically acceptable carrier.
The application of described Flos Chrysanthemi Indici extract in the medicine of preparation treatment blood stasis disease.
The application of described pharmaceutical preparation in the medicine of preparation treatment blood stasis disease.
When extract of the present invention is used as medicine, directly can uses, or use in the form of a pharmaceutical preparation.
Described pharmaceutical preparation contains the Flos Chrysanthemi Indici extract of the present invention for the treatment of effective dose, and all the other are acceptable on materia medica, nontoxic to humans and animals and pharmaceutically suitable carrier of inertia and/or excipient.
Described pharmaceutically suitable carrier or excipient are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation adjuvant.Pharmaceutical preparation of the present invention is used with the form of per weight dose.Extract of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into aqueous or oily solution, aseptic powder injection, liposome or the Emulsion etc. of sterilizing.
Advantage of the present invention: Flos Chrysanthemi Indici extract provided by the invention, has significant anticoagulation and antithrombotic acitivity, can be used for being developed to the medicine for the treatment of blood stasis disease.Liquid phase analysis method provided by the invention may be used for the fingerprint setting up this extract
figurespectrum, with control different batches extract batch between concordance.
Accompanying drawing explanation
fig. 1for the HPLC chromatograph of Flos Chrysanthemi Indici extract
figure.
Detailed description of the invention
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Main agents and material: the Flos Chrysanthemi Indici prepared slices of Chinese crude drugs and the Radix Sophorae Flavescentis prepared slices of Chinese crude drugs are purchased from Hui nationality's Chinese Medicinal Materials Markets; Food-grade ethanol is purchased from Shanghai Ling Feng chemical reagent company limited; Pharmaceutical grade D101 macroporous resin is purchased from sky tunami letter resin company limited; Acetonitrile is HPLC level, is purchased from TEDIA; Formic acid is HPLC level, is purchased from TEDIA; Chromatographic grade pure water is heartily pure water.
Embodiment 1: Flos Chrysanthemi Indici extract preparation (Flos Chrysanthemi Indici 85 parts, Radix Sophorae Flavescentis 15 parts)
Get Flos Chrysanthemi Indici 8.5kg and Radix Sophorae Flavescentis 1.5kg and shine dry grinding, extract 3 times with 95% alcohol heat reflux, each extraction 2 hours, at every turn with 95% ethanol 25L, merge extractive liquid, is concentrated into without alcohol taste (4L); Concentrated solution uses petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) to extract successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; N-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry (about 750g).
Embodiment 2: Flos Chrysanthemi Indici extract preparation (Flos Chrysanthemi Indici 80 parts, Radix Sophorae Flavescentis 20 parts)
Get Flos Chrysanthemi Indici 8.0kg and Radix Sophorae Flavescentis 2.0kg and shine dry grinding, extract 3 times with 95% alcohol heat reflux, each extraction 2 hours, at every turn with 95% ethanol 25L, merge extractive liquid, is concentrated into without alcohol taste (4L); Concentrated solution uses petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) to extract successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; N-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry (about 750g).
Embodiment 3: Flos Chrysanthemi Indici extract preparation (Flos Chrysanthemi Indici 90 parts, Radix Sophorae Flavescentis 10 parts)
Get Flos Chrysanthemi Indici 9.0kg and Radix Sophorae Flavescentis 1.0kg and shine dry grinding, extract 3 times with 95% alcohol heat reflux, each extraction 2 hours, at every turn with 95% ethanol 25L, merge extractive liquid, is concentrated into without alcohol taste (4L); Concentrated solution uses petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) to extract successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; N-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry (about 750g).
Embodiment 4: Flos Chrysanthemi Indici extract preparation (Flos Chrysanthemi Indici 75 parts, Radix Sophorae Flavescentis 25 parts)
Get Flos Chrysanthemi Indici 7.5kg and Radix Sophorae Flavescentis 2.5kg and shine dry grinding, extract 3 times with 95% alcohol heat reflux, each extraction 2 hours, at every turn with 95% ethanol 25L, merge extractive liquid, is concentrated into without alcohol taste (4L); Concentrated solution uses petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) to extract successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; N-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry (about 750g).
Embodiment 5: Flos Chrysanthemi Indici extract preparation (Flos Chrysanthemi Indici 95 parts, Radix Sophorae Flavescentis 5 parts)
Get Flos Chrysanthemi Indici 9.5kg and Radix Sophorae Flavescentis 0.5kg and shine dry grinding, extract 3 times with 95% alcohol heat reflux, each extraction 2 hours, at every turn with 95% ethanol 25L, merge extractive liquid, is concentrated into without alcohol taste (4L); Concentrated solution uses petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) to extract successively, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; N-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry (about 750g).
Embodiment 6: liquid-phase chromatographic analysis
Need testing solution is prepared: in the brown volumetric flask of extract 5mg to 50ml that Example 1 method is obtained, add 30ml35% acetonitrile solution ultrasonic dissolution, after being cooled to room temperature, continue to add 35% acetonitrile solution standardize solution.
Analytical method:
High performance liquid chromatograph: Agilent1260, binary pump;
Chromatographic column: AgilentTCC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.1% formic acid solution (triethylamine adjust ph to 6.0);
Gradient elution program: 0 ~ 5min, A35%; 5 ~ 40min, A35% → 70%; 40 ~ 57min, A70% → 95%; 57 ~ 65min, A95%; Flow rate of mobile phase: 1.0mLmin
-1; Determined wavelength: 240nm; Column temperature: 30 DEG C; Sample size 10 μ L.
The extract prepared with 10 batches of Different sources, batch Flos Chrysanthemi Indici and Radix Sophorae Flavescentis is analyzed, sets up fingerprint
figurespectrum is mated, and 1 ~ No. 10 peak is in 10 batch sample chromatographs as a result
in figureall occur, and all reach baseline separation with surrounding chromatographic peak.Therefore demarcate 10 peaks for total fingerprint peaks, set up the HPLC fingerprint of this extract accordingly
figurespectrum, for controlling the quality of this extract.HPLC
figuresee
fig. 1.
Embodiment 7: Flos Chrysanthemi Indici extract pharmacological testing
1, material and reagent:
new westblue large ear rabbit, male, body weight 1.6 ~ 2.5kg.Thrombin and Fibrinogen (prepared before use) are Shanghai Vaccine and Serum Institute's product.
2, the preparation of sample: Flos Chrysanthemi Indici extract 10.80mg prepared by Example 1 adds 10% alcoholic solution and is diluted to different quality concentration, and called after Flos Chrysanthemi Indici extract 1..Same method, the Flos Chrysanthemi Indici extract 10.80mg that respectively prepared by Example 2 ~ 5 adds 10% alcoholic solution and is diluted to different quality concentration, called after Flos Chrysanthemi Indici extract 2. ~ 5..
3, In Vitro Anti Blood clotting:
(1) on the impact of plasma in rabbit recalcification time.Rabbit auricular vein gets blood 4.5mL, adds in the 10mL centrifuge tube containing 38g/L sodium citrate 0.5mL, and mixing, the centrifugal 10min of 1000r/min, gets supernatant for subsequent use.Separately get 96 orifice plate 1, the every hole of experimental group adds pooled plasma 0.1mL and different quality concentration Flos Chrysanthemi Indici extract 1. (0.216,0.108,0.054mg/mL) each 0.1mL, matched group adds pooled plasma 0.1mL and 10% alcoholic solution 0.1mL, after 37 DEG C of incubation 1min, add 2.775g/LCaCl respectively to every hole
2solution 0.1mL, timing, until stop timing when fiber protein yarn being detected, repeats 10 times, averages, be blood plasma recalcification time.Be measured in the same method Flos Chrysanthemi Indici extract 2. ~ 5..
(2) on the impact of thrombin time.The samely prepare pooled plasma, add pooled plasma and different quality concentration Flos Chrysanthemi Indici extract 1. (0.216,0.108,0.054mg/mL) each 0.1mL, 37 DEG C of incubation 3min at 96 orifice plates, then add 1 × 10 of temperature in advance
4u/L thrombin 0.1mL, mixing, record clotting of plasma required time.With 10% alcoholic solution for contrast, often organize repetition 10 times, average.Be measured in the same method Flos Chrysanthemi Indici extract 2. ~ 5..
4, external anti thrombotic action:
(1) thrombolytic effect.Rabbit ear edge vein exploitating blood is put in test tube, and 1:9 adds mass concentration is by volume 38g/L liquor sodii citratis, then adds fibrinogen solution (mass fraction is 0.5%), CaCl successively
2solution (55.5g/L) and thrombin solution (1.0 × 10
5u/L), addition is add fibrinogen solution 20 μ L, CaC1 in every milliliter of blood
2solution 10 μ L and thrombin solution 20 μ L.Mixing, being injected into internal diameter is in the teat glass of 8mm, removal of thromboses after 37 DEG C of water-bath 20min, be cut into the segment of 5mm, put into the test tube that different quality concentration Flos Chrysanthemi Indici extract 1. (0.216,0.108,0.054mg/mL) and each 2mL of 10% alcoholic solution are housed, 37 DEG C of incubations, in 0,4,12,24,48h measures the quality of residue thrombosis section respectively, calculate thromboembolism rate, replication 10 times.Be measured in the same method Flos Chrysanthemi Indici extract 2. ~ 5..
(2) to the dissolution of whole blood grumeleuse.Rabbit Heart blood sampling 20mL is placed in surface plate, is cut into 0.3cm after 2h after natural coagulation
3fritter, stand-by with normal saline drip washing 3 times.By different quality concentration Flos Chrysanthemi Indici extract, 1. (0.216,0.108,0.054mg/mL) and each 2mL of 10% alcoholic solution add in different cillin bottle, often organize 5 bottles, 2 clots are added at random again in each bottle, 37 DEG C of water bath heat preservations, observe clot dissolution situation, respectively at 0,12,24,48h surveys the quality of clot respectively, calculates whole blood thrombolysis rate.Be measured in the same method Flos Chrysanthemi Indici extract 2. ~ 5..
5, result: the Flos Chrysanthemi Indici extract of different quality concentration 1. ~ 3. all can significant prolongation plasma in rabbit recalcification time and thrombin time (see
table 1, remarks: compare with 10% ethanol group, each administration group P < 0.01); Compare with matched group, Flos Chrysanthemi Indici extract 1. ~ 3. all demonstrate the effect of stronger dissolving whole blood grumeleuse in different time points, and along with the prolongation of administration time, whole blood thrombolysis rate increases (
table 2~ 4, remarks: compare with 10% ethanol group, each administration group P < 0.01).And 4. and 5. with 10% ethanol group the Flos Chrysanthemi Indici extract of different quality concentration does not more all have significant difference, can not extend plasma in rabbit recalcification time and thrombin time (
table 5), can not dissolve whole blood grumeleuse (
table 6~ 7).
Result shows, Flos Chrysanthemi Indici extract prepared by the present invention can extend plasma in rabbit recalcification time and thrombin time, can promote to dissolve whole blood grumeleuse, this effect is the coefficient result of Flos Chrysanthemi Indici and Radix Sophorae Flavescentis, the two has synergism, and this synergism is subject to the scale effect of Flos Chrysanthemi Indici and Radix Sophorae Flavescentis.
table 1flos Chrysanthemi Indici extract 1. ~ 3. on the impact of plasma in rabbit recalcification time and thrombin time
table 2flos Chrysanthemi Indici extract is 1. to the dissolution of external thrombus and whole blood grumeleuse
table 3flos Chrysanthemi Indici extract is 2. to the dissolution of external thrombus and whole blood grumeleuse
table 4flos Chrysanthemi Indici extract is 3. to the dissolution of external thrombus and whole blood grumeleuse
table 5flos Chrysanthemi Indici extract 4. ~ 5. on the impact of plasma in rabbit recalcification time and thrombin time
table 6flos Chrysanthemi Indici extract is 4. to the dissolution of external thrombus and whole blood grumeleuse
table 7flos Chrysanthemi Indici extract is 5. to the dissolution of external thrombus and whole blood grumeleuse
Embodiment 8: the preparation of tablet
By embodiment 1 method first obtained extract, add excipient, pelletizing press sheet in itself and excipient weight than the ratio for 1:8.
Embodiment 9: the preparation of oral liquid
By embodiment 1 method first obtained extract, oral liquid method for making makes oral liquid routinely.
Embodiment 10: the preparation of capsule or granule
By embodiment 1 method first obtained extract, add excipient in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 11: the preparation of injection
Obtain extract by embodiment 1 method, inject with water, fine straining, injection is made in embedding sterilizing.
Embodiment 12: the preparation of aseptic powder injection
By embodiment 1 method first obtained extract, be dissolved in sterile water for injection, stirring makes molten, filters with aseptic suction funnel, more aseptic fine straining, and be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains injectable powder.
Claims (5)
1. a Flos Chrysanthemi Indici extract, is characterized in that described extract is prepared by following methods:
Step one: by weight, every 100 parts of medical materials comprise Flos Chrysanthemi Indici 80 ~ 90 parts and Radix Sophorae Flavescentis 10 ~ 20 parts, co-grinding;
Step 2: extract with 95% alcohol heat reflux, merge extractive liquid, is concentrated into without alcohol taste;
Step 3: petroleum ether, ethyl acetate and water saturated n-butanol extraction are used successively to step 2 concentrated solution, obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively;
Step 4: n-butyl alcohol extract D101 macroporous resin enrichment, first with 10% alcohol flushing, 10 column volume removing polysaccharide, then uses 70% ethanol elution, 8 column volumes, collects 70% eluent, concentrating under reduced pressure, spraying dry.
2. Flos Chrysanthemi Indici extract according to claim 1, is characterized in that: the liquid phase analysis method of described extract is:
Chromatographic column: AgilentTCC18 post (4.6mm × 250mm, 5 μm);
Mobile phase: A is acetonitrile, and B is 0.1% formic acid solution (triethylamine adjust ph to 6.0);
Gradient elution program:
0～5min，A35％；5～40min，A35％→70％；
40～57min，A70％→95％；57～65min，A95％；
Flow rate of mobile phase: 1.0mLmin
-1;
Determined wavelength: 240nm; Column temperature: 30 DEG C;
Sample size 10 μ L.
3. pharmaceutical preparation, is characterized in that: described preparation contains the Flos Chrysanthemi Indici extract according to claim 1 for the treatment of effective dose and pharmaceutically acceptable carrier.
4. the application of Flos Chrysanthemi Indici extract as claimed in claim 1 in preparation treatment blood stasis disease medicament.
5. the application of pharmaceutical preparation as claimed in claim 3 in preparation treatment blood stasis disease medicament.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106389501A (en) * | 2016-08-28 | 2017-02-15 | 成都宝科生物科技有限公司 | Chrysanthemum indicum extract for treatment of high blood pressure |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106389501A (en) * | 2016-08-28 | 2017-02-15 | 成都宝科生物科技有限公司 | Chrysanthemum indicum extract for treatment of high blood pressure |
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Application publication date: 20151125 |