CN106176834A - A kind of lichens extract for treating diabetes - Google Patents
A kind of lichens extract for treating diabetes Download PDFInfo
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- CN106176834A CN106176834A CN201610741667.6A CN201610741667A CN106176834A CN 106176834 A CN106176834 A CN 106176834A CN 201610741667 A CN201610741667 A CN 201610741667A CN 106176834 A CN106176834 A CN 106176834A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/09—Lichens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses a kind of lichens extract for treating diabetes, method for preparing extractive includes: (a), by dry lichens 75% ethanol solution circumfluence distillation, united extraction liquid, is concentrated into and obtains ethanol extraction concentrated solution without alcohol taste;B (), by step (a) gained ethanol extraction concentrated solution dilute with water, successively with petroleum ether, ethyl acetate and water saturated n-butanol extraction, concentrating under reduced pressure, respectively obtains petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;C () n-butyl alcohol extract water dissolution, filters, uses macroporous resin enrichment active component, first remove big polar component with 10 column volumes of 10% alcohol flushing, then with 12 column volumes of 65% ethanol elution, collect 65% eluent, concentrating under reduced pressure, obtains lichens extract.This extract can reduce blood glucose and the blood fat of diabetic mice, improves diabetic mice oxidation resistance, can develop into the medicine for the treatment of diabetes.
Description
Technical field
The present invention relates to Chinese medicine extract field, be specifically related to a kind of lichens extract and the medicine system containing this extract
Agent, this lichens extract can apply to the treatment of diabetes.
Background technology
Lichens is the important monoid of rudimentary plant, for helotism body.Fungal component therein is based on ascomycetes, and minority is
Basidiomycetes, phycobiont is cyanophyceae or chlorella.Owing to the biological characteristics of lichens is mainly the embodiment of fungus in helotism body, therefore
It is also called Lichenized fungi.Lichens is considered as typical case the most perfect in biological mutualism.The known lichens in the whole world 500
Multiple genus, plant for more than 26000, and there is 232 genus 1766 kinds in China.Lichens is used as medicine and has a long history in China, in " Book of Songs " just
It is related to the record that Usnea is medicinal.Record according to various books on Chinese herbal medicine and relevant information, there are about 200 kinds of lichens and be used as medicine.
The symbiosis complex that lichens is fungus and algae highly combines, due to the particularity of lichenism, at the beginning of it contains
Raw metabolite (polysaccharide) and secondary metabolite (phenolic acids) constitute the monoid that natural organic-compound is unique.In recent years
The many active noval chemical compounds of isolated from lichen, including: lichenin, lichenic acid (depside, contracting
Phenolic acid cyclic ethers class, depsidone class and dibenzofurans class) and other compositions such as quinones.
Lichens is used as medicine in the existing long history of China, in " Book of Songs " of 600 BC Western Zhou period, just has pine
The record of trailing plants;Litmus (Herba Cladoniae rangiferinae) described in Northern and Southern Dynasties' beam TAO Hong-Jing written " Mingyi Bielu " " materials for improving vision, benefit vital essence ";Li Shi
Precious Compendium of Material Medica describes the form of multiple lichens, habit and drug effect, as " it is cruel that female trailing plants (Usnea) can treat expectorant heat temperature, can
For telling soup, dredging water passages ", " litmus has secretion replenishing and throat moistening, antipyretic reduces phlegm ".In recent years pharmacological research finds, lichens has antitumor, disease-resistant
Many biological activitys such as poison, radioprotective, antibacterial, antioxidation.
Summary of the invention
It is an object of the invention to provide a kind of lichens extract, its preparation method and liquid phase analysis method, carry containing this
The pharmaceutical preparation taking thing and the purposes of the medicine utilizing this extract preparation treatment diabetes.
The above-mentioned purpose of the present invention is achieved by techniques below scheme:
A kind of lichens extract, this extract prepared by following methods:
A (), by dry lichens 75% ethanol solution circumfluence distillation, united extraction liquid, is concentrated into and obtains second without alcohol taste
Alcohol extraction concentrated solution;B (), by step (a) gained ethanol extraction concentrated solution dilute with water, uses petroleum ether, ethyl acetate and water successively
Saturated n-butanol extraction, concentrating under reduced pressure, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;
C () n-butyl alcohol extract water dissolution, filters, uses macroporous resin enrichment active component, first with 10 cylinders of 10% alcohol flushing
The big polar component of long-pending removing, then with 12 column volumes of 65% ethanol elution, collect 65% eluent, concentrating under reduced pressure, obtain lichens
Extract.
Further, macroporous resin described in step (c) is AB-8 type macroporous resin.
In order to by the method setting up HPLC finger printing control the lichens extract prepared of different batches batch between
Difference, the liquid phase analysis method of described lichens extract is:
Chromatographic column: Diamonsil C18 post (4.6mm × 250mm, 5 μm);
Flowing phase: A is acetonitrile, and B is 0.1% phosphoric acid solution (triethylamine regulation pH value to 6.2);
Gradient elution program: 0.01~70min, A 20% → 80%;
Flow rate of mobile phase: 1.0mL min-1;
Detection wavelength: 240nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Pharmaceutical preparation, the described lichens extract containing therapeutically effective amount and pharmaceutically acceptable carrier.
The application in the medicine of preparation treatment diabetes of the described lichens extract.
Described pharmaceutical preparation application in the medicine of preparation treatment diabetes.
When extract of the present invention is used as medicine, can directly use, or use in the form of a pharmaceutical preparation.
Described pharmaceutical preparation contains the lichens extract of the present invention of therapeutically effective amount, remaining be the most acceptable,
Pharmaceutically suitable carrier nontoxic and inert to humans and animals 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.The pharmaceutical preparation of the present invention is used with the form of per weight dose.Extract of the present invention can
It is applied to need the patient for the treatment of by oral or injection form.During for being administered orally, tablet, slow releasing tablet, control can be made into
Release sheet, capsule, drop pill, micropill, suspensoid, Emulsion, powder or granule, oral liquid etc.;During for injecting, can be made into sterilizing
Aqueous or oily solution, aseptic powder injection, liposome or Emulsion etc..
Advantages of the present invention: the present invention by lichens is extracted, remove impurity, enrichment process, can obtain that there are diabetes
The extract of therapeutical effect;The liquid phase analysis method that the present invention provides may be used for setting up the finger printing of this extract, is used for
Control the differences between batches of extract prepared by different batches.
Accompanying drawing explanation
Fig. 1 is lichens extractive HPLC chromatograms.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit the present invention with this and protect model
Enclose.Although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, can be right
Technical scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
Embodiment 1: prepared by lichens extract
Crude drug source: lichens is purchased from Hui nationality's Chinese Medicinal Materials Markets, Shaanxi, the place of production.
Main agents: food-grade ethanol is purchased from Shanghai Ling Feng chemical reagent company limited;Pharmaceutical grade AB-8 macroporous resin is purchased
From sky tunami letter resin company limited;Acetonitrile is HPLC level, is purchased from TEDIA;85% phosphoric acid is HPLC level, is purchased from TEDIA;Chromatograph
It is heartily pure water with pure water.
Preparation method: the lichens that 10kg is dried 75% ethanol solution circumfluence distillation (25L × 3 time), united extraction
Liquid, is concentrated into and obtains ethanol extraction concentrated solution (3L) without alcohol taste;Gained ethanol extraction concentrated solution is diluted with water to 4L, uses successively
Petroleum ether (4L × 3 time), ethyl acetate (4L × 3 time) and water saturated n-butyl alcohol (4L × 3 time) extraction, concentrating under reduced pressure, respectively
Obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract 275g;(c) n-butyl alcohol extract 1L water dissolution,
Medical absorbent cotton filters, with AB-8 macroporous resin (2kg, column volume 1.5L) deposition activity composition, first with 10% alcohol flushing 10
Individual column volume (15L) removes big polar component, then with 12 column volumes (18L) of 65% ethanol elution, collects 65% eluent, subtract
Pressure concentrates, and obtains lichens extract 115g.
Embodiment 2: liquid-phase chromatographic analysis
Need testing solution is prepared: in lichens extract 5mg to the 50mL brown volumetric flask that Example 1 method prepares, add
30mL 20% acetonitrile solution ultrasonic dissolution, after being cooled to room temperature, continues to add 20% acetonitrile solution constant volume.
Analysis method:
High performance liquid chromatograph: Agilent 1260, binary pump;
Chromatographic column: Diamonsil C18 post (4.6mm × 250mm, 5 μm);
Flowing phase: A is acetonitrile, and B is 0.1% phosphoric acid solution (triethylamine regulation pH value to 6.2);
Gradient elution program: 0.01~70min, A 20% → 80%;
Flow rate of mobile phase: 1.0mL min-1;
Detection wavelength: 240nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
Being analyzed with the lichens extract of 10 batches of preparation, carry out chromatographic peak coupling, 1~No. 9 peak is at 10 batches as a result
Sample chromatogram figure all occurs.Therefore demarcating 9 peaks is total chromatographic peak, sets up the HPLC quality control chart of this extract accordingly
Spectrum, result is shown in Fig. 1.The extract pharmacologically active of this 10 batch is similar.
Embodiment 3: lichens extract pharmacological testing
One, material and method
1, material
(1) laboratory sample: lichens extract (MGC) is made by oneself.
(2) laboratory animal: male Kun Ming mice, body weight (22 ± 2) g, Nanjing University's Experimental Animal Center provide.
(3) main agents: alloxan is purchased from Sigma company;Diabetes pill is purchased from Guangzhou No.1 Chinese Pharmacy Factory;
Blood sugar kit is purchased from Beijing Zhong Sheng biotechnology company;
SOD, GSH-Px, MDA test kit builds up Bioengineering Research Institute purchased from Nanjing;
TC, TG, HDL-C testing cassete is purchased from glad biotechnology research institute of Shanghai section.
(4) key instrument: Shimadzu UV-265FW ultraviolet-visible spectrophotometer, Japan.
2, method
(1) duplication of experimental alloxan diabetes mouse model
Alloxan is made into the normal saline solution that volume fraction is 2%.200mg kg is pressed after mice fasting 24h-1Dosage
Carry out alloxan lumbar injection, after 72h, detect glucose in urine with Tes-Tape, detect fasting glucose, choosing with blood sugar detection test kit
Take blood glucose value more than 11.1mmol L-1, glucose in urine be that the mice of strong positive (+++) is as hyperglycemia model mice.
(2) the lichens extract impact on alloxan diabetes mouse blood sugar
Test-type diabetic mice is randomly divided into 7 groups, i.e. Normal group (Control), hyperglycemia model group
(Alloxan), positive controls and 4 test group.During experiment, Normal group and hyperglycemia group gavage normal saline;4
Test group gavage lichens extract (MGC), dosage is respectively 50,100,300,500mg kg-1BW;Positive controls gavage disappears
Thirsty ball (XKW), dosage is 840mg kg-1BW, freely drinks water during test and ingests, experimental period 30d.After last is administered, fasting
12h, eye Dou Jing clump takes blood, separates serum and makees the detection of blood glucose and blood lipids index.After putting to death animal, take out the liver group of animal immediately
Knit and be made liver homogenate to measure superoxide dismutase (SOD), glutathion peroxidase (GSHPx), malonaldehyde (MDA).
(3) observation index and method
The mensuration of blood glucose (BG): take a blood sample after each experimental mouse fasting 10h, uses glucose oxidase method to carry out blood sugar detection.
Serum triglycerides (TG), serum total cholesterol (TC) and serum High Density Lipoprotein Cholesterol (HDL-C) measure:
Separate serum, carry out by kit method, with the ultraviolet-visible spectrophotometer colorimetric determination of Shimadzu UV-265FW.
Liver superoxide dismutase (SOD), glutathion peroxidase (GSH-Px) and malonaldehyde (MDA) measure: point
It is made liver tissue homogenate's liquid from hepatic tissue, carries out by kit method.
(4) statistical procedures: result withRepresent.SAS statistical software is used to carry out variance analysis.
Two, result and analysis
1, the lichens extract impact on tissue of experimental diabetic mice blood glucose
As shown in table 1, compared with the blood glucose value of hyperglycemia model group, 100,300mg kg-1BW dosage group and XKW group are bright
Aobvious reduction (P < 0.01), 500mg kg-1BW group difference has significance (P < 0.05), 50mg kg-1Bw group no significant difference,
Show that lichens extract (MGC) has the hypoglycemic effect of fall to hyperglycemia model mice, and this blood sugar reducing function is with middle dose
Amount 100mg kg-1BW best results, its hypoglycemic effect is suitable (P > 0.01) with the hypoglycemic effect of diabetes pill group.In table 1,*P<
0.05,*P < 0.01vs Alloxan,△△P<0.01vs MGC100;▲▲P<0.01vs XKW。
The table 1 lichens extract (MGC) impact on hyperglycemia model blood glucose in diabetic mice
2, the lichens extract impact on tissue of experimental diabetic mice blood fat
Diabetes are often along with hyperlipemia.After this experiment utilizes alloxan (Alloxan) to replicate diabetic mice, by
Shown in table 2, hyperglycemia model group mice TC and TG be higher than blank group (P < 0.01), HDL-C less than blank group (P <
0.01).After giving MGC and XKW, each dosage group mice TC, TG and HDL-C have substantially recovery, wherein 100mg kg-1Bw group
TC, TG and HDL-C compare with hyperglycemia model group, and difference has significance (P < 0.01);300mg·kg-1Bw group and the TC of XKW group
Being below hyperglycemia model group (P < 0.05), TG is less than hyperglycemia model group (P < 0.01), and HDL-C is higher than hyperglycemia model group (P
<0.01);100mg·kg-1Bw group is suitable with XKW group to the regulation effect of diabetic mice blood fat, TC, TG, HDL-C of two groups
There are no significant for content difference (P > 0.01).Result shows that lichens extract can effectively facilitate the extensive of diabetic mice blood lipid level
Multiple.In table 2,*P < 0.05,*P<0.01vs Alloxan;△P < 0.05,△△P<0.01vs MGC100。
The impact on tissue of experimental diabetic mice blood fat of the table 2 lichens extract
3, the lichens extract impact on tissue of experimental diabetic mice oxidation resistance
SOD, GSH-Px and MDA content of body is the important indicator evaluating its oxidation resistance.As shown in Table 3, with just
Often group is compared, and hyperglycemia model group SOD, GSH-Px activity reduce (P < 0.01), and MDA content rises, and shows that hyperglycemia model is little
The oxidation resistance of Mus is significantly lower than normal mouse.But after giving MGC and XKW, each dosage group SOD in Mice, GSH-Px and MDA have
Substantially recover, wherein 100,300mg kg-1Bw group and XKW group SOD activity have significance (P with hyperglycemia model group comparing difference
< 0.05), GSH-Px activity has significance (P < 0.01) with hyperglycemia model group comparing difference, and MDA content reduces (P < 0.01).
100、300mg·kg-1The SOD content of bw group and XKW group no significant difference (P > 0.01), 100mg kg-1The GSH-of bw group
Px, MDA content and XKW group no significant difference (P > 0.01).Result shows that lichens extract can effectively facilitate diabetic mice
The recovery of liver oxidation resistance, wherein 100mg kg-1Bw to the restitution of diabetic mice liver oxidation resistance with
XKW is suitable.In table 3,*P < 0.05,*P<0.01vs Alloxan;△P < 0.05,△△P<0.01vs MGC100。
The impact on tissue of experimental diabetic mice oxidation resistance of the table 3 lichens extract
Embodiment 4: the preparation of tablet
First prepare extract by embodiment 1 method, add excipient with excipient weight than the ratio for 1:10 in it, system
Grain tabletting.
Embodiment 5: the preparation of oral liquid
First preparing extract by embodiment 1 method, oral liquid preparation method makes oral liquid routinely.
Embodiment 6: capsule or the preparation of granule
First prepare extract by embodiment 1 method, add excipient with excipient weight than the ratio for 1:9 in it, system
Become capsule or granule.
Embodiment 7: the preparation of injection
Preparing extract by embodiment 1 method, inject and use water, fine straining, injection is made in embedding sterilizing.
Embodiment 8: the preparation of aseptic powder injection
First preparing extract by embodiment 1 method, be dissolved in sterile water for injection, stirring makes molten, uses aseptic suction funnel
Filter, more aseptic fine straining, it being sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains injectable powder.
Claims (6)
1. a lichens extract, it is characterised in that described extract is prepared by following methods:
A (), by dry lichens 75% ethanol solution circumfluence distillation, united extraction liquid, is concentrated into and obtains ethanol without alcohol taste and carry
Take concentrated solution;B (), by step (a) gained ethanol extraction concentrated solution dilute with water, uses petroleum ether, ethyl acetate and water saturation successively
N-butanol extraction, concentrating under reduced pressure, respectively obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract;C () just
Butanol extract water dissolution, filters, and uses macroporous resin enrichment active component, first removes with 10 column volumes of 10% alcohol flushing
Big polar component, then with 12 column volumes of 65% ethanol elution, collect 65% eluent, concentrating under reduced pressure, obtain lichens extract.
Lichens extract the most according to claim 1, it is characterised in that: macroporous resin described in step (c) is AB-8 type
Macroporous resin.
Lichens extract the most according to claim 1, it is characterised in that: the liquid phase analysis method of described lichens extract
For:
Chromatographic column: Diamonsil C18 post (4.6mm × 250mm, 5 μm);
Flowing phase: A is acetonitrile, and B is 0.1% phosphoric acid solution (triethylamine regulation pH value to 6.2);
Gradient elution program: 0.01~70min, A 20% → 80%;
Flow rate of mobile phase: 1.0mL min-1;
Detection wavelength: 240nm;
Column temperature: 30 DEG C;
Sample size: 10 μ L.
4. pharmaceutical preparation, it is characterised in that: lichens extract described in the claim 1 containing therapeutically effective amount and pharmaceutically may be used
The carrier accepted.
5. the application in the medicine of preparation treatment diabetes of the lichens extract described in claim 1.
6. the application in the medicine of preparation treatment diabetes of the pharmaceutical preparation described in claim 4.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103249410A (en) * | 2010-10-07 | 2013-08-14 | 韩国海洋研究院 | Pharmaceutical and food composition for preventing or treating diabetes or obesity |
CN105125645A (en) * | 2015-08-28 | 2015-12-09 | 林天样 | Lichen extract for treating viral influenza |
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- 2016-08-28 CN CN201610741667.6A patent/CN106176834A/en active Pending
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Publication number | Priority date | Publication date | Assignee | Title |
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CN103249410A (en) * | 2010-10-07 | 2013-08-14 | 韩国海洋研究院 | Pharmaceutical and food composition for preventing or treating diabetes or obesity |
CN105125645A (en) * | 2015-08-28 | 2015-12-09 | 林天样 | Lichen extract for treating viral influenza |
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Application publication date: 20161207 |