CN108245538A - A kind of ginkgo biloba p.e and its medical usage - Google Patents
A kind of ginkgo biloba p.e and its medical usage Download PDFInfo
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- CN108245538A CN108245538A CN201611242639.6A CN201611242639A CN108245538A CN 108245538 A CN108245538 A CN 108245538A CN 201611242639 A CN201611242639 A CN 201611242639A CN 108245538 A CN108245538 A CN 108245538A
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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
- A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
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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
- A61K2236/30—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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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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
Abstract
The present invention relates to a kind of ginkgo biloba p.e and its medical usages; itself and existing ginkgo biloba p.e are compared by specific embodiment; it was found that the ginkgo biloba p.e in the present invention not only has apparent therapeutic effect to myocardial ischemia caused by pituitrin; and can effectively protect rat reperfusion injury; infarct size is obviously reduced, there is better therapeutic effect to angiocardiopathy.In addition, the high income of ginkgo biloba p.e preparation process products obtained therefrom provided by the invention, stable quality, and extraction process is simple, is suitble to industrialized production, facilitates the popularization and application of ginkgo biloba p.e of the present invention.
Description
Technical field
The present invention relates to a kind of ginkgo biloba p.e and its medical usages, belong to field of medicaments.
Background technology
Data is shown according to statistics, on illness rate, incidence, the death rate of China's cardiovascular and cerebrovascular disease continue over 30 years
Rise, by the 7th position that leaps to the first in the sixties, China die of every year cardiovascular and cerebrovascular disease there are about 2,500,000 people, account for population
The 50% of total case fatality rate, slightly below industrializes developed country.Though separately having some patientss to survive through rescue, majority leaves residual
Disease be can't take care of oneself, and cause seriously to bear to relatives and society.Therefore, the original new drug of cardiovascular and cerebrovascular disease is found always
It is a hot spot of the world of medicine.In numerous treating cardiovascular disease drugs, searching is a kind of curative for effect, and side effect is low to be had
The direction for person's effort that effect drug is always drug research.Since Chinese medicine has, treating both manifestation and root cause of disease, toxic side effect is small, can take for a long time
Advantage is increasingly taken seriously in the treatment of cardiovascular and cerebrovascular disease.
Ginkgo (Ginkgo biloba L.) also known as Gong Sunshu, gingko, are gymnosperm Ginkgoaceae plants, are the current earth
On one of most ancient seeds.Ginkgo whole body is all precious, and collection fruit use, Ye Yong, timber-used, pollen are used, protection is used and watched for one
Body.The medical value of ginkgo, Ancient Times in China with regard on the books,《Compendium of Materia Medica》It records, gingko " can enter lung channel, beneficial temper, determine cough
Asthma, reduce just, flat property and sweet taste it is bitter, it is slightly poisonous ", asthma, productive cough, leukorrhea, seminal emission, gonorrhoea, frequent urination etc. can be controlled.Ginkgo is
A kind of high plant of medical value, and the research of the active constituent in ginkgo leaf receives pro-gaze.
The active ingredient of ginkgo biloba p.e (GBE, EGB, Extractor ofGinkgoBiloba) is mainly flavonoid glycoside
Class and terpene substances.Flavonoid glycoside is based on Quercetin, Kaempferide, Isorhamnetin；Terpene is with ginkgolides (GA, GB, GC), white
Based on fruit lactone (BB).The terpenoid of ginkgo biloba extract has the function of anti-platelet aggregation, and the silver in terpene lactone
Apricot lactone B is the most strong platelet-activating factor antagonist found so far, clinically treats thrombus, acute pancreatitis and the heart
Vascular diseases, curative effect are better than other similar medicines.Generally acknowledged GBE quality standards are in the world at present：Total flavonoids content >=
24%, terpene lactone contents >=6%, present standard has become the quality mark of world many countries inlet and outlet gingko leaf preparation
It is accurate.
Invention content
First purpose of the present invention is to provide one kind compared with existing ginkgo biloba p.e, has better healing to disease
The ginkgo biloba p.e of effect.
The present invention, which adopts the following technical scheme that, completes above-mentioned purpose：
The ginkgo biloba p.e of a kind of bilobalide-containing and ginkgo free Flavonoids is obtained by following extraction process：
A. it extracts
A certain amount of ginkgo leaf is weighed, it is 1 to press solid-liquid ratio with the ethanol solution of 20%-70%:5-1:20 ratio carries out
Alcohol extracting, refluxing extraction twice, merge extracting solution and are concentrated into density as 1.1-1.2；
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate, column volume is 1L/kg raw medicinal herbs, is first eluted with water 6 times of volumes, then with 60% ethyl alcohol
8 times of volumes of elution simultaneously collect alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:0.2-1:2 ratio is extracted
It takes, same method extraction three times, merges ester phase, respectively obtains water phase and ester phase；
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with the ethyl alcohol of 20%-70%, volume is 0.01-2L/kg raw medicinal herbs, and adds in work
Property charcoal, addition be raw medicinal herbs 0.1%-0.6%, 60-90 DEG C stirring 1-6h carry out depickling.Same condition carries out secondary de-
Acid dries after filtrate concentration and obtains terpene lactone；
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, adds in hydrochloric acid and carries out sour water solution, 60-90 DEG C of reaction temperature, reaction time 2-6h.
After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, column volume are 0.5L/kg raw medicinal herbs.First use water
4 times of volumes are washed, then 8 times of volumes are washed with 60% alcohol, it is dry after collecting alcohol eluen and concentrating, obtain total free Flavonoids；
E. content calculation
Terpene lactone merges total free Flavonoids, detects through HPLC, is calculated according to mass fraction, terpene lactone >=25%, always
Free Flavonoids >=25%, total ginkgoic acid≤5ppm.
It is respectively preferably a step above：
A. it extracts
A certain amount of ginkgo leaf is weighed, it is 1 to press solid-liquid ratio with the ethanol solution of 20%-70%:10-1:15 ratio carries out
Alcohol extracting, refluxing extraction twice, merge extracting solution and are concentrated into density as 1.1-1.2；
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate, column volume is 1L/kg raw medicinal herbs, is first eluted with water 6 times of volumes, then with 60% ethyl alcohol
8 times of volumes of elution simultaneously collect alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1 ratio is extracted, equally
Method extraction three times, merge ester phase, respectively obtain water phase and ester phase；
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, volume is 0.05-0.2L/kg raw medicinal herbs, adds in and adds in work
Property charcoal, measure the 0.1%-0.6% for raw medicinal herbs, 80 DEG C of stirring 1-2h carry out depicklings.Same condition carries out secondary depickling, filtrate
Drying obtains terpene lactone after concentration；
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, adds in hydrochloric acid and carries out sour water solution, 80 DEG C of reaction temperature, reaction time 2-6h.Instead
After answering, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, column volume are 0.5L/kg raw medicinal herbs.First it is washed with water 4
Times volume, then 8 times of volumes are washed with 60% alcohol, it is dry after collecting alcohol eluen and concentrating, obtain total free Flavonoids；
E. content calculation
Terpene lactone merges total free Flavonoids, detects through HPLC, is calculated according to mass fraction, terpene lactone >=25%, always
Free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Macroreticular resin in the step of wherein described B and D is preferably D101, AB-8 or DA201；Add in the step D
A concentration of 3-7mol/L of the hydrochloric acid entered, volume are the 1/20-1/2 of water phase.
The high income of this method products obtained therefrom, stable quality, and extraction process is simple, is suitble to industrialized production.
It is a further object to provide use of the ginkgo biloba p.e of the present invention in terms of disease therapeuticing medicine is prepared
On the way, wherein the disease can be a few days ago disclosed all diseases of the present patent application.
Preferably, the disease is cardiovascular and cerebrovascular relevant disease.
It is furthermore preferred that the cardiovascular and cerebrovascular relevant disease is angina pectoris or myocardial infarction.
Be it is more of the invention in ginkgo biloba p.e and bilobanone ester dropping pills and existing ginkgo biloba p.e it is anti-acute
Myocardial ischemia effect carries out the test of pesticide effectiveness that pituitrin modeling causes SD myocardial ischemia in rats.9 result of the test table of embodiment
It is bright：(1) compared with model group, ginkgo biloba p.e group of the present invention can significantly reduce LDH and CK it is horizontal (##P ＜ 0.01), and reduce
Effect be significantly stronger than bilobanone ester dropping pills group and existing ginkgo biloba p.e group (△P ＜ 0.05；§P ＜ 0.05,§§P ＜ 0.01)；
(2) compared with model group, ginkgo leaf of the present invention carry group can significantly increased SOD and GSH-Px level (##P ＜ 0.01), and increase
Effect be significantly stronger than bilobanone ester dropping pills group and existing ginkgo biloba p.e group (△P ＜ 0.05；§P ＜ 0.05,§§P ＜ 0.01)；
(3) the J points caused by ginkgo biloba p.e group of the present invention can significantly reduce myocardial ischemia compared with model group move up (##P ＜ 0.01),
And reduction effect be significantly stronger than bilobanone ester dropping pills group and existing ginkgo biloba p.e group (△P ＜ 0.05；§P ＜ 0.05,§§P ＜
0.01)。
After acute myocardial infarction AMI, the change of every parameters of left ventricular function and its recovery extent and speed are in myocardial infarction area
Negative correlation, thus reduction myocardial infarction area can be as the curative effect index of medicaments for resisting myocardial ischemia.
To investigate ginkgo biloba p.e of the present invention with bilobanone ester dropping pills and existing ginkgo biloba p.e to myocardial infarction face
Long-pending influence, make ischemia-reperfusion injury model, embodiment 10 the result shows that：(1) ginkgo biloba p.e group and model group of the present invention
Compared to can be reduced significantly myocardial infarction area (##P ＜ 0.01), and effect is significantly stronger than bilobanone ester dropping pills group and existing ginkgo
Leaf extract group (△P ＜ 0.05；§§P ＜ 0.01)；(2) ginkgo biloba p.e group of the present invention can significantly reduce compared with model group
LDH and CK levels (#P ＜ 0.05,##P ＜ 0.01), and reduction effect is significantly stronger than bilobanone ester dropping pills group and existing ginkgo leaf carries
Take object group (△P ＜ 0.05；§P ＜ 0.05,§§P ＜ 0.01).
The present invention has carried out such as 9 He of embodiment the ginkgo biloba p.e for removing other compositions described in embodiment 9 and 10
The experiment of embodiment 10, the results showed that ginkgo biloba p.e of the present invention causes myocardial ischemia and rat again to pituitrin
Perfusion injury is respectively provided with significant protective effect, and effect is significantly stronger than existing ginkgo biloba p.e and bilobanone ester dropping pills.
The ginkgo biloba p.e that the present invention obtains is compared with existing ginkgo leaf Related product, in the treatment of cardiovascular and cerebrovascular disease
Aspect has following advantage：
1. flavonol glycosides are converted into flavone aglycone by the present invention by acid hydrolysis process, compared with flavonol glycosides, flavonoid glycoside
Member has many advantages, such as to be more conducive to absorb, and bioavilability is high, therefore, compared with existing ginkgo biloba p.e, the silver in the present invention
Apricot leaf extract can preferably play a series of function of resisting myocardial ischemia such as anti-oxidant and removing free radical.
2. the ginkgo biloba p.e in the present invention is demonstrated by embodiment 9 and embodiment 10, not only to pituitrin
Caused myocardial ischemia has apparent therapeutic effect, and can effectively protect rat reperfusion injury, hence it is evident that infarct size is reduced,
Compared with existing ginkgo biloba p.e, there is better therapeutic effect to angiocardiopathy.
Specific embodiment
The present invention is further described, but the present invention is not restricted to following embodiment below by way of specific embodiment, appointed
The equivalent replacement of what form is all apparent for those of ordinary skills and among the present invention.
Embodiment 1
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 20% ethanol solution:15 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (D101), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, volume is 0.05L/kg raw medicinal herbs, and adds in activated carbon, is added
Enter 0.1% that amount is raw medicinal herbs, 80 DEG C of stirring 1h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
Obtain terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 7mol/L carries out sour water solution, adds in 1/20 of volume for water phase,
80 DEG C of reaction temperature, reaction time 2h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 2
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 50% ethanol solution:15 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (D101), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:0.2 ratio into
Row extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, and volume is 0.2L/kg raw medicinal herbs, and adds in activated carbon, is added in
0.6% for raw medicinal herbs is measured, 60 DEG C of stirring 6h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
To terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 3mol/L carries out sour water solution, adds in 1/2 of volume for water phase,
80 DEG C of reaction temperature, reaction time 6h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 3
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 70% ethanol solution:10 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (AB-8), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:2 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, and volume is 0.1L/kg raw medicinal herbs, and adds in activated carbon, is added in
0.3% for raw medicinal herbs is measured, 90 DEG C of stirring 1h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
To terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 5mol/L carries out sour water solution, adds in 1/5 of volume for water phase,
80 DEG C of reaction temperature, reaction time 4h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total yellow free ketone aglycon up to ginkgo biloba p.e.It is detected through HPLC, according to mass fraction meter
It calculates, terpene lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 4
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 70% ethanol solution:5 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (DA201), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:2 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 20% ethyl alcohol, volume is 2L/kg raw medicinal herbs, and adds in activated carbon, addition
It is the 0.1% of raw medicinal herbs, 80 DEG C of stirring 2h carry out depickling.Same condition carries out secondary depickling, dries and obtains after filtrate concentration
Terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 5mol/L carries out sour water solution, adds in 1/10 of volume for water phase,
90 DEG C of reaction temperature, reaction time 2h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 5
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 20% ethanol solution:10 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (AB-8), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1.5 ratio into
Row extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 70% ethyl alcohol, volume is 0.01L/kg raw medicinal herbs, and adds in activated carbon, is added
Enter 0.6% that amount is raw medicinal herbs, 80 DEG C of stirring 1.5h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
It is dry to obtain terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 7mol/L carries out sour water solution, adds in 1/20 of volume for water phase,
90 DEG C of reaction temperature, reaction time 4h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 6
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 20% ethanol solution:20 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (DA201), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 20% ethyl alcohol, and volume is 0.2L/kg raw medicinal herbs, and adds in activated carbon, is added in
0.1% for raw medicinal herbs is measured, 80 DEG C of stirring 2h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
To terpene lactone.
D. the hydrolysis of total flavonoids member and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 7mol/L carries out sour water solution, adds in 1/2 of volume for water phase,
60 DEG C of reaction temperature, reaction time 6h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 7
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 50% ethanol solution:15 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (D101), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:2 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase.
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, volume is 0.05L/kg raw medicinal herbs, and adds in activated carbon, is added
Enter 0.6% that amount is raw medicinal herbs, 70 DEG C of stirring 4h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
Obtain terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 7mol/L carries out sour water solution, adds in 1/20 of volume for water phase,
60 DEG C of reaction temperature, reaction time 6h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
Embodiment 8
A. it extracts
8kg ginkgo leaves are weighed, it is 1 to press solid-liquid ratio with 50% ethanol solution:10 ratio carries out alcohol extracting, refluxing extraction two
It is secondary, merge extracting solution and be concentrated into density as 1.1-1.2.
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate (AB-8), column volume is 1L/kg raw medicinal herbs, 6 times of volumes is first eluted with water, then use
60% 8 times of ethanol elution volume simultaneously collects alcohol eluen.Eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1 ratio carries out
Extraction, same method extraction three times, merge ester phase, respectively obtain water phase and ester phase
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ethyl ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, volume is 0.05L/kg raw medicinal herbs, and adds in activated carbon, is added
Enter 0.1% that amount is raw medicinal herbs, 90 DEG C of stirring 2h carry out depickling.Same condition carries out secondary depickling, is dried after filtrate concentration
Obtain terpene lactone.
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, and the hydrochloric acid for adding in 7mol/L carries out sour water solution, adds in 1/2 of volume for water phase,
80 DEG C of reaction temperature, reaction time 2h.After reaction, it is 5-6 to be neutralized to PH with NaOH.Macroreticular resin on neutralizer, cylinder
Product is 0.5L/kg raw medicinal herbs.4 times of volumes are first washed with water, then 8 times of volumes are washed with 60% alcohol, collect alcohol eluen and are done after concentrating
It is dry, obtain total free Flavonoids.
E. content calculation
Terpene lactone merges total free Flavonoids up to ginkgo biloba p.e.It detects through HPLC, is calculated according to mass fraction, terpene
Class lactone >=25%, total free Flavonoids >=25%, total ginkgoic acid≤5ppm.
9 ginkgo biloba p.e of embodiment causes pituitrin the effect of rats with myocardial ischemia
1. experimental animal and reagent
Experimental animal：SPF grades of Jinan friends of SD rats please experimental animal breeding Co., Ltd
Reagent：Ginkgo biloba p.e X of the present invention1Containing terpene lactone >=25% (by ginkalide A, ginkolide B, ginkgo
Lactone C and Bilobalide composition), total free Flavonoids >=25% (being made of Quercetin, Kaempferide, Isorhamnetin)
Ginkgo biloba p.e X of the present invention2Containing terpene lactone >=25% (by ginkalide A, ginkolide B, ginkalide C
Composition), total free Flavonoids >=25% (being made of Quercetin, Kaempferide, Isorhamnetin)
Existing ginkgo biloba p.e X0Containing terpene lactone >=6%, total flavonoids >=24%
Bilobanone ester dropping pills (more than 24% flavonol glycosides, more than 20% free Flavonoids, more than 6% terpene lactone, ginkgoic acid
Below 5ppm), lot number：150606；Source：Chinese laws and institutions medicine
Pituitrin lot number：151147；Source：Anhui Hongye Medicine Co., Ltd
2. animal packet and administration
Animal is randomly divided into Normal group, ischemia model control group, extract X1Low dosage (40mg/kg), extraction
Object X1High dose group (80mg/kg), extract X2Group (40mg/kg), extract X0Group (80mg/kg) and bilobanone ester dropping pills group
(80mg/kg) (above each administration group dosage is calculated by Bilobanoate amount), every group 10, be half male and half female, and administration group is every
Its gastric infusion is primary, and Normal group and ischemia model control group give same volume with same administering mode and the frequency
0.5% sodium carboxymethylcellulose, equal successive administration 7 days.
3. the foundation of Model of Acute Myocardial Ischemia
After the 7th time is administered 30min, rat body is anaesthetized by 45mg/kg (1.5ml/kg) with 3% yellow Jackets
Afterwards, it lies on the back and is fixed on rat operation platform, II lead electrocardiogram, model comparison are detected using SP2006 electrocardiograph systems
Group, ginkgo biloba p.e X1Group, ginkgo biloba p.e X2Group, ginkgo biloba p.e X0Group and the intravenous injection of bilobanone ester dropping pills group
Pit1U/kg, fast injection (injection finishes in 5s), Normal group intravenous injection same volume physiological saline, 30min after injection
II lead electrocardiogram is recorded again, and (J points are the terminal portions minute of QRS complex to electrocardiogram J point voltage change before and after counting rat ischemia
With the terminal of the interface point of ST sections of startings, i.e. S waves, with PR sections for baseline, the changing value of J point displacements).
3. Serum LDH, the measure of CK, SOD and GSH-Px
30min after rat modeling, femoral artery take the centrifugation of 5000r/min4 DEG C of blood, take supernatant, LDH, CK, SOD to be measured and
GSH-Px。
5. result of the test
Influence of 1 ginkgo biloba p.e of table to treating myocardial ischemia damage Rat Ecg J points
Compared with Normal group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01,；With Bilobanoate
Dripping pill is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05,§§P ＜ 0.01.
From table 1 it follows that model group, compared with Normal group, J points significantly move up, and show that myocardial infarction and ischemia model is answered
Work(is made, and compared with model group, the J points caused by bilobanone ester dropping pills group can significantly reduce myocardial ischemia move up, further table
Bright modeling success；Ginkgo biloba p.e X1Group and ginkgo biloba p.e X2Group can significantly reduce myocardial ischemia institute compared with model group
The J points of cause move up, and reduction effect is significantly stronger than bilobanone ester dropping pills group and ginkgo biloba p.e X0Group.
Influence of 2 ginkgo biloba p.e of table to treating myocardial ischemia damage rat LDH and CK activity
| Group | Size of animal | LDH/(U·L-1) | CK/(U·L-1) |
| Normal group | 10 | 218.23±105.23 | 387.44±103.78 |
| Ischemia model control group | 10 | 608.45±123.09※※ | 702.23±127.76※※ |
| Bilobanone ester dropping pills group | 10 | 399.46±143.07## | 500.56±109.98## |
| Ginkgo biloba p.e X0Group | 10 | 408.67±156.21## | 478.78±125.06## |
| Ginkgo biloba p.e X1Low dose group | 10 | 275.36±113.66##△§ | 405.89±139.81##△§ |
| Ginkgo biloba p.e X1High dose group | 10 | 222.45±144.26##△§§ | 389.34±123.82##△§ |
| Ginkgo biloba p.e X2Group | 10 | 268.44±107.99##△§ | 410.33±121.00##△§ |
Compared with Normal group,※※P ＜ 0.01；Compared with model group,##P ＜ 0.01；Compared with bilobanone ester dropping pills,△
P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05,§§P ＜ 0.01.
From Table 2, it can be seen that model group, compared with Normal group, LDH and CK are significantly raised, show myocardial ischemia mould
Type replicates successfully, and compared with model group, and bilobanone ester dropping pills group can significantly reduce LDH and CK levels, further demonstrate that modeling
Success；Ginkgo biloba p.e X1Group and ginkgo biloba p.e X2Group can significantly reduce LDH and CK levels compared with model group, and drop
Low effect is significantly stronger than bilobanone ester dropping pills group and ginkgo biloba p.e X0Group.Ginkgo leaf in the result above prompting present invention carries
It takes object that there is stronger protective effect to myocardial cell membrane, the leakage of LDH and CK can be reduced.
Influence of 3 ginkgo biloba p.e of table to treating myocardial ischemia damage rat SOD and GSH-Px activity
Compared with Normal group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01；With Bilobanoate
Dripping pill is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05,§§P ＜ 0.01.
From table 3 it is observed that model group, compared with Normal group, SOD and GSH-Px are substantially reduced, show that cardiac muscle lacks
The success of blood model copy, and compared with model group, the horizontal significantly raising of bilobanone ester dropping pills group SOD and GSH-Px, further table
Bright modeling success；Ginkgo biloba p.e X1Group and ginkgo biloba p.e X2Group can significantly increased SOD and GSH-Px compared with model group
Level, and raising effect is significantly stronger than bilobanone ester dropping pills group and ginkgo biloba p.e X0Group.In the result above prompting present invention
Ginkgo biloba p.e can preferably improve cardiac muscular tissue's oxidation resistance, protect myocardial cell membrane, and then ischemic is protected to be damaged
Cardiac muscle.
Influence of 10 ginkgo biloba p.e of embodiment to rat reperfusion injury
1. rat is in the foundation of body coronary artery ligation and Reperfu- sion myocardial infarction and ischemia model
Cleaning grade SD rats 60, are randomly divided into 7 groups：Normal group, ischemia model control group, extract X1Low dosage
Group 40mg/kg, extract X1High dose group 80mg/kg, extract X2Group (40mg/kg), extract X0Group 80mg/kg and ginkgo
Ketone ester dropping pill group 80mg/kg (above each administration group dosage is calculated by Bilobanoate amount), every group 10, half male and half female.Rat
It is anaesthetized with 10% chloraldurate (0.3mL/100g) ip, dorsal position is fixed, neck median incision, detaches right carotid
(CCA), promoting the circulation of qi cannula, connection animal respirator (during breathing ratio for 1.5: 1,60 times/min, tidal volume 70mL/kg), in chest
3~5 rib of bone left border opens thoracic cavity, and exposure heart opens pericardium, at pulmonary conus left border and left auricle of heart lower edge 2mm into
Needle is pierced by with atraumatic suture through superficial layer of myocardium, makes ramus descendens anterior arteriae coronariae sinistrae (LAD) disposed thereon.Stablize 10min, put on
Double-layer plastic casing, tightening binding complete arteria coroaria sinistra occlusion and cause myocardial ischemia.The criterion of myocardial ischemia is electrocardio
(ECG) there is myocardial infarction performance (ST section raise or T wave height sharp) and heart part cyanosis.After myocardial ischemia 30min in extraction
Layer casing unclamps knot and implements Reperfu- sion 60min.Wherein sham-operation group is only threaded and is not ligatured.Medication is each by reagent
Group rat 20min before coronary ligation is injected intravenously corresponding test medicine, and sham-operation group and model group give the NS of equivalent.
2. the calculating of related biochemical indicator and infarct size
After the test, abdominal aortic blood, and heart is taken out rapidly, the hemostasis in heart is removed, ligatures LAD again,
From aorta inject 0.25% Evans Blue solution, be put at once -80 DEG C of refrigerators freeze it is spare.Centrifugal blood takes supernatant, puts -20
DEG C refrigerator freezes.After all experimentss strictly LDH, CK biochemical indicator are surveyed by kit operation.Heart after freezing is taken out,
Heart is cut into 2mm small pieces 5-6 pieces from the apex of the heart to heart base portion is parallel.It puts 37 DEG C of 1%TTC (pH7.4) solution medium temperatures and incubates 15min, after
10% formalin is put to fix.Ischemic hazardous area is red, and infarcted region is white, uses image analysis software (Sigma Scan
Program 4) area of ischemic hazardous area (AAR) and infarcted region (IS) is calculated, myocardial infarction area is represented with IS/AAR.
3. result of the test
Influence of 4 ginkgo biloba p.e of table to myocardial ischemia reperfusion in rats infarct size
| Group | Size of animal | (IS/AAR)/% | Inhibiting rate/% |
| Sham-operation group | 10 | 3.71±1.04 | __ |
| Model group | 10 | 63.43±10.21※※ | __ |
| Bilobanone ester dropping pills group | 10 | 38.78±9.99## | 38.86 |
| Ginkgo biloba p.e X0Group | 10 | 52.12±7.74# | 33.60 |
| Ginkgo biloba p.e X1Low dose group | 10 | 28.22±6.69##△§§ | 55.51 |
| Ginkgo biloba p.e X1High dose group | 10 | 22.09±5.56##△§§ | 65.17 |
| Ginkgo biloba p.e X2Group | 10 | 23.48±5.11##△§§ | 62.98 |
Compared with sham-operation group,※※P ＜ 0.01；Compared with model group,##P ＜ 0.01,#P ＜ 0.05；It is dripped with Bilobanoate
Ball is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§§P ＜ 0.01.
As can be seen from Table 4, compared with sham-operation group, infarct size dramatically increases model group, shows myocardial ischemia mould
Type replicates successfully, and compared with model group, and bilobanone ester dropping pills group infarct size is obviously reduced, and further demonstrates that modeling success；
Ginkgo biloba p.e X1Group and ginkgo biloba p.e X2Group can be reduced significantly myocardial infarction area compared with model group, and act on aobvious
Work is better than bilobanone ester dropping pills group and ginkgo biloba p.e X0Group.Prompting the present invention in ginkgo biloba p.e to myocardial ischemia again
Perfusion injury has better protective effect.
Influence (x ± s, n=10) of 5 ginkgo biloba p.e of table to LDH in ischemia-reperfusion rat blood serum and CK activity
| Group | Size of animal | LDH/(U·L-1) | CK/(U·mL-1) |
| Sham-operation group | 10 | 2630.23±210.34 | 40.23±10.34 |
| Model group | 10 | 4553.27±302.24※※ | 90.47±18.46※※ |
| Bilobanone ester dropping pills group | 10 | 3800.25±407.78## | 70.57±20.14# |
| Ginkgo biloba p.e X0Group | 10 | 3980.24±309.17## | 78.45±18.09# |
| Ginkgo biloba p.e X1Low dose group | 10 | 3201.23±505.12##△§ | 61.23±20.09##△§ |
| Ginkgo biloba p.e X1High dose group | 10 | 3091.01±403.12##△§ | 58.78±10.34##△§§ |
| Ginkgo biloba p.e X2Group | 10 | 3112.45±398.09##△§ | 62.00±19.48##△§ |
Compared with sham-operation group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01,；It is dripped with Bilobanoate
Ball is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05,§§P ＜ 0.01.
As can be seen from Table 5, for model group compared with sham-operation group, LDH and CK are significantly raised, show myocardial infarction and ischemia model
Replicate successfully, and compared with model group, it is horizontal that bilobanone ester dropping pills group can significantly reduce LDH and CK, further demonstrate that modeling into
Work(；Ginkgo biloba p.e X1Group and ginkgo biloba p.e X2Group can significantly reduce LDH and CK levels compared with model group, and reduce
Effect is significantly stronger than bilobanone ester dropping pills group and ginkgo biloba p.e X0Group.
11 ginkgo biloba p.e of comparative example causes pituitrin the effect of rats with myocardial ischemia
1. for method and step with embodiment 9, only ginkgo biloba p.e composition is different, is specifically grouped as follows：
2. result of the test
Influence of 6 ginkgo biloba p.e of table to treating myocardial ischemia damage Rat Ecg J points
| Group | Size of animal | J point heights (x ± s, mV) |
| Normal group | 10 | 0.049±0.032 |
| Ischemia model control group | 10 | 0.198±0.012※※ |
| Bilobanone ester dropping pills group | 10 | 0.143±0.015# |
| Ginkgo biloba p.e X0Group | 10 | 0.158±0.042# |
| Ginkgo biloba p.e X3Group | 10 | 0.098±0.034##△§ |
| Ginkgo biloba p.e X4Group | 10 | 0.102±0.027##△§ |
| Ginkgo biloba p.e X5Group | 10 | 0.087±0.045##△§ |
| Ginkgo biloba p.e X6Group | 10 | 0.099±0.036##△§ |
Compared with Normal group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01,；With Bilobanoate
Dripping pill is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05.
Influence of 7 ginkgo biloba p.e of table to treating myocardial ischemia damage rat LDH and CK activity
| Group | Size of animal | LDH/(U·L-1) | CK/(U·L-1) |
| Normal group | 10 | 234.67±99.13 | 378.46±100.34 |
| Ischemia model control group | 10 | 598.12±145.09※※ | 698.45±134.21※※ |
| Bilobanone ester dropping pills group | 10 | 378.44±124.44## | 495.57±134.45## |
| Ginkgo biloba p.e X0Group | 10 | 399.98±123.11## | 487.09±107.45## |
| Ginkgo biloba p.e X3Group | 10 | 245.66±101.89##△§ | 396.45±101.67##△§ |
| Ginkgo biloba p.e X4Group | 10 | 231.34±124.54##△§ | 379.34±123.82##△§ |
| Ginkgo biloba p.e X5Group | 10 | 222.56±119.09##△§ | 402.33±101.55##△§ |
| Ginkgo biloba p.e X6Group | 10 | 212.22±121.07##△§ | 387.89±100.78##△§ |
Compared with Normal group,※※P ＜ 0.01；Compared with model group,##P ＜ 0.01；Compared with bilobanone ester dropping pills,△
P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05.
Influence of 8 ginkgo biloba p.e of table to treating myocardial ischemia damage rat SOD and GSH-Px activity
| Group | Size of animal | SOD/(U·mL-1) | GSH-Px/(U·mL-1) |
| Normal group | 10 | 312.78±34.34 | 410.45±30.09 |
| Ischemia model control group | 10 | 201.12±30.98※※ | 309.89±29.04※※ |
| Bilobanone ester dropping pills group | 10 | 260.61±30.44## | 356.56±31.09# |
| Ginkgo biloba p.e X0Group | 10 | 242.11±32.11# | 354.45±29.08# |
| Ginkgo biloba p.e X3Group | 10 | 300.33±40.24##△§ | 401.11±32.23##△§ |
| Ginkgo biloba p.e X4Group | 10 | 289.26±41.12##△§ | 398.12±25.05##△§ |
| Ginkgo biloba p.e X5Group | 10 | 297.34±40.09##△§ | 399.87±29.06##△§ |
| Ginkgo biloba p.e X6Group | 10 | 288.67±43.12##△§ | 389.67±32.34##△§ |
Compared with Normal group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01；With Bilobanoate
Dripping pill is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05.
Influence of 12 ginkgo biloba p.e of comparative example to rat reperfusion injury
1. for method and step with embodiment 10, only ginkgo biloba p.e composition is different, is specifically grouped as follows：
2. result of the test
Influence of 9 ginkgo biloba p.e of table to myocardial ischemia reperfusion in rats infarct size
| Group | Size of animal | (IS/AAR)/% | Inhibiting rate/% |
| Sham-operation group | 10 | 5.32±1.34 | __ |
| Model group | 10 | 50.78±9.83※※ | __ |
| Bilobanone ester dropping pills group | 10 | 32.01±7.74## | 36.96 |
| Ginkgo biloba p.e X0Group | 10 | 35.01±8.02## | 31.01 |
| Ginkgo biloba p.e X3Group | 10 | 26.34±6.69##△§ | 48.13 |
| Ginkgo biloba p.e X4Group | 10 | 24.46±4.98##△§ | 51.83 |
| Ginkgo biloba p.e X5Group | 10 | 23.51±6.92##△§ | 53.70 |
| Ginkgo biloba p.e X6Group | 10 | 25.55±7.09##△§ | 49.68 |
Compared with sham-operation group,※※P ＜ 0.01；Compared with model group,##P ＜ 0.01；Compared with bilobanone ester dropping pills,△P
＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05.
Influence (x ± s, n=10) of 10 ginkgo biloba p.e of table to LDH in ischemia-reperfusion rat blood serum and CK activity
Compared with sham-operation group,※※P ＜ 0.01；Compared with model group,#P ＜ 0.05,##P ＜ 0.01,；It is dripped with Bilobanoate
Ball is compared,△P ＜ 0.05；With ginkgo biloba extract X0It compares,§P ＜ 0.05.
In addition, the present invention has also carried out the ginkgo biloba p.e for removing other compositions described in above-described embodiment as implemented
The experiment of example 9 and embodiment 10, the results showed that ginkgo biloba p.e of the present invention to pituitrin cause myocardial ischemia and
Rat reperfusion injury is respectively provided with significant protective effect, and effect is significantly stronger than existing ginkgo biloba p.e and Bilobanoate drop
Ball.
Claims (8)
1. a kind of ginkgo biloba p.e, which is characterized in that contain Terpene lactones and ginkgo free Flavonoids in this extract,
In, Terpene lactones include ginkalide A, ginkolide B, ginkalide C and Bilobalide, ginkgolides M, ginkgolides
It is one or more in J；Ginkgo free Flavonoids include Quercetin, Kaempferide, Isorhamnetin and Luteolin, trigrain wheat Huang
It is one or more in ketone, red bayberry Quercetin, apiolin.
2. a kind of preparation method of ginkgo biloba p.e as described in claim 1, includes the following steps：
A. it extracts
A certain amount of ginkgo leaf is weighed, it is 1 to press solid-liquid ratio with the ethanol solution of 20%-70%:5-1:20 ratio carries out alcohol extracting,
Refluxing extraction twice, merges extracting solution and is concentrated into density as 1.1-1.2；
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate, column volume is 1L/kg raw medicinal herbs, is first eluted with water 6 times of volumes, then with 60% ethanol elution
8 times of volumes simultaneously collect alcohol eluen, and eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:0.2-1:2 ratio is extracted, together
The method extraction of sample three times, respectively obtains water phase and ester phase；
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ester is mutually concentrated and dried, and is dissolved with the ethyl alcohol of 20%-70%, volume is 0.01-2L/kg raw medicinal herbs, and adds in activated carbon, is added
Enter the 0.1%-0.6% that amount is raw medicinal herbs, 60-90 DEG C of stirring 1-6h carries out depickling, and same condition carries out secondary depickling, filtrate
Drying obtains terpene lactone after concentration；
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, adds in hydrochloric acid and carries out sour water solution, 60-90 DEG C of reaction temperature, reaction time 2-6h, reaction
After, it is 5-6 to be neutralized to PH with NaOH, and macroreticular resin on neutralizer, column volume is 0.5L/kg raw medicinal herbs, is first washed with water 4 times
Volume, then 8 times of volumes are washed with 60% alcohol, it is dry after collecting alcohol eluen and concentrating, obtain total free Flavonoids.
3. a kind of preparation method of ginkgo biloba p.e as described in claim 1, includes the following steps：
A. it extracts
A certain amount of ginkgo leaf is weighed, it is 1 to press solid-liquid ratio with the ethanol solution of 20%-70%:10-1:15 ratio carries out alcohol
It carries, refluxing extraction twice, merges extracting solution and is concentrated into density as 1.1-1.2；
B. macroporous resin purification and extraction split-phase
By macroreticular resin on concentrate, column volume is 1L/kg raw medicinal herbs, is first eluted with water 6 times of volumes, then with 60% ethanol elution
8 times of volumes simultaneously collect alcohol eluen, and eluent is concentrated into no alcohol, and 1 is pressed with ethyl acetate:1 ratio is extracted, same side
Method extracts three times, merges ester phase, respectively obtains water phase and ester phase；
C. the purifying of terpene lactone and the removing of ginkgoic acid
Ester is mutually concentrated and dried, and is dissolved with 50% ethyl alcohol, and volume is 0.05-0.2L/kg raw medicinal herbs, adds in and adds in activated carbon,
The 0.1%-0.6% for raw medicinal herbs is measured, 80 DEG C of stirring 1-2h carry out depickling, and same condition carries out secondary depickling, filtrate concentration
Drying obtains terpene lactone afterwards；
D. total flavonoids hydrolysis and flavone aglycone purifying
Water phase is concentrated into no ethyl acetate, adds in hydrochloric acid and carries out sour water solution, 80 DEG C of reaction temperature, reaction time 2-6h.Reaction knot
Shu Hou, it is 5-6 to be neutralized to PH with NaOH, macroreticular resin on neutralizer, and column volume is 0.5L/kg raw medicinal herbs.4 times of bodies are first washed with water
Product, then 8 times of volumes are washed with 60% alcohol, it is dry after collecting alcohol eluen and concentrating, obtain total free Flavonoids.
4. the preparation method of ginkgo biloba p.e as claimed in claim 2 or claim 3, which is characterized in that in the step B and D
One kind in D101, AB-8 or DA201 of macroreticular resin.
5. the preparation method of ginkgo biloba p.e as claimed in claim 2 or claim 3, which is characterized in that added in the step D
Hydrochloric acid a concentration of 3-7mol/L, volume be water phase 1/20-1/2.
6. purposes of the ginkgo biloba p.e described in claim 1 in terms of disease therapeuticing medicine is prepared.
7. purposes as claimed in claim 6, which is characterized in that the disease is cardiovascular and cerebrovascular relevant disease.
8. purposes as claimed in claim 7, which is characterized in that the cardiovascular and cerebrovascular relevant disease is angina pectoris or cardiac muscle stalk
Extremely.
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| CN108703981A (en) * | 2018-07-09 | 2018-10-26 | 湖北工程学院 | A method of extracting GINKGO BILOBA EXTRACT from ginkgo leaf |
| CN108703981B (en) * | 2018-07-09 | 2021-04-13 | 湖北工程学院 | Method for extracting ginkgetin from ginkgo leaves |
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