HK1087176B - A method for determination of the total amount of the flavonoids in a ginkgo biloba composition - Google Patents

Description

Method for measuring content of total flavonoids in ginkgo leaf composition

The invention is a divisional application of Chinese patent application with application number 200310118517.2 (application date 3/19/1999, original application number 99803683.8) filed on 12/2003 and named "ginkgo leaf composition and preparation method and application".

Technical Field

The invention relates to a botanical drug preparation, in particular to a ginkgo leaf composition and a preparation method and application thereof.

Ginkgo Biloba (Ginkgo Biloba) is the oldest of the existing seed plants and is the only survivor of the Ginkgo Biloba family dating to 2 million years of early eclipse. In china, ginkgo biloba preparations have been used for more than 5000 years for treatment, i.e. from the earliest origins of the Chinese herbs. Since the sixties, the herbal extracts of ginkgo biloba have been used in many countries for the treatment of cerebrovascular and peripheral vascular diseases, such as germany, france, japan and korea.

Background

The main active ingredient in ginkgo leaf is a flavonoid compound comprising at least 14 different compounds, such as flavonols, flavones, flavanones and biflavones, among which flavonoid glycosides and flavonolsThe glycoside comprises kaempferol, quercetin and isorhamnetin, glucose or rhamnose conjugate, and is folium Ginkgo extract (Tebonin) for therapeutic purpose^®、Tanakan^®、Roekan^®Or "EGb 761"), which have been shown experimentally to be potent antioxidants that scavenge oxygen free radicals and thus avoid cell and tissue damage due to aging, which can severely affect brain function, including memory and attention, see j.pincemal et al, La pressure medical vol.15(1986), 1475-; robak et al, Biochem Pharmacol Vol 37(1988), 837-841 and J.Kleijnen with P.Knipchild Ginkgo biloba (Drug Profiles), the Lancet 340: 1136(1992), and flavonoid glycosides and flavonol glycosides also improve peripheral vascular circulation, processes for preparing ginkgo biloba extracts containing high concentrations of the active ingredient flavonoid glycosides are described in DE-B1767098 and DE-B2117429, which preparations are single-component extracts of ginkgo biloba.

In addition to flavones, another major active ingredient in ginkgo leaves is terpene lactones, including bilobalide A, B, C, J, M and bilobalide, which are terpenes containing lactone structures, see k. nakanishi, Pure and Applied Chemistry, vol.14(1967) 89-113; m. Maruyama et al, Tetrahedron Letters (1967), 299-. It has recently been discovered that, due to their properties of antagonizing Platelet Activating Factor (PAF), ginkgolides A, B, C and M, particularly bilobalide B, are effective in the treatment of platelet activating factor-induced diseases such as asthma, bronchitis, senile dementia, allergy, cardiac dysfunction, rheumatism and the like, as well as other circulatory system diseases, see U.S. patent No. 4,734,280; braquet, Drug of the Future, 12, 643, 1987; v Lamant et al, Biochem Pharmacol Vol.36(1987) 2749-52; becker et al, Biomed Biochim Acta Vol 47(1988) 10-11; braquet et al, J Ethnopharmacol Vol.32(1991)135-9 and B Steinke et al, Planta Med Vol 59(1993)155-60 ginkgolides A and B increase cerebral blood flow to protect the brain, see J.Krieglstein et al, European Journal of pharmaceutical Sciences Vol.3(1995)39-48, U.S. Pat. No. 5,002,965, which describes a method of using ginkgolides to prevent rejection in organ transplantation.

DE-A3338995 and related U.S. Pat. No. 4,571,407 use bilobalide, a sesquiterpene lactone structurally related to bilobalide, see K.Nakanishi et al, R.T.Major et al and K.Weinges et al, J.Am.chem Soc., Vol.93, 1971, 3544-3546 for the treatment of encephalopathy, encephaledema, demyelinating neuropathy and myelopathy, with bilobalide being used before the occlusion of the middle cerebral artery with a significant reduction in the area of the cortex and infarct at the site of the infarct, see J.Kriegstein et al, European Journal of Pharmaceutical Sciences Vol.3(1995)39-48 experiments have shown that bilobalide helps to restore the motor nerve in animals, see C.Bruno et al, Planta 59, 1993, 302-307. In addition, bilobalide has been shown to have properties that inhibit the growth of Pneumocystis carinii in vitro and in vivo experiments, see C.Atzori et al, Antirhizobe Agents Chemother 37(7)1993, 1492-1496. The use of bilobalide in U.S. patent No. 5,264,216 can treat certain infections with the following pathogens: trichomonas vaginalis, escherichia coli, lactobacillus, pneumocystis carinii, staphylococcus aureus and streptococcus. The bilobalide has anti-Pneumocystis carinii infection activity, and is mainly used for treating AIDS complicated infection.

In addition to the above compounds, Ginkgo biloba leaves contain at least 12 alkyl phenolic compounds, including ginkgolic acid (sumac), which is a 6-alkyl salicylic acid, the alkyl group being an alkyl group having 0-3 double bonds from the n-13 carbon to the n-19 carbon, see J.L. Gellermann et al, Phytochemistry, Vol.15(1976), 1959-Asahol 1961 and Analytic.Chem., Vol.40(1968), 739-Asahol 743. Similar to the structure of irritants in poison ivy, ginkgolic acid is a factor of the toxic effects of ginkgo biloba extracts including gastrointestinal side effects, headache, skin irritation and edema, and since the sixties, there have been many reports of allergic reactions after exposure to ginkgo biloba or fruits, see g.a. hill et al, j.am.chem. soc., vol.56(1934), 2736-; l.e.becker et al, j.am.med.assoc., vol.231(1975), 1162-; nakamura, Contact Dermatitis, Vol.12(1985), 281-282; tomb et al, Contact Dermatitis, Vol.19(1988)281-3 and J.P.Lepoittevin et al, Arch Dermatol Res Vol.281(1989), 227-30. Thus, scientists in many countries have endeavored to develop desensitizing materials and methods to combat the hypersensitivity caused by ginkgolic acids (see U.S. patent No. 4,428,965). DE-B1767098 and DE-B2117429 have developed a process for removing alkylphenol compounds using chlorinated aliphatic hydrocarbons such as carbon tetrachloride, in which, however, a considerable amount of the medically valuable ginkgolides and bilobalides is lost. DE-B2117429 developed a technique for removing polyphenolic compounds (proanthocyanidins) with tannin properties, in a process using lead compounds. The problems with these processes are the potential health hazards to operators, potential threats to the environment, and hazardous residues in the drug.

The most commonly used current extract of ginkgo biloba (Tebonin) for therapeutic purposes^®、Tanakan^®、Roekan^®Or "EGb 761") contains 24% flavonoid glycosides and 6% terpene lactones; see K.driau La pressure medical Vol.15(1986), 1455-1457, bilobalides A, B, C and J and bilobalide approximately half of these 6%, the Ginkgo biloba extract usually contains less than 10ppm of alkylphenol compounds at a daily therapeutic dose of 120 mg.

In the nineties, great efforts were made to increase the content of active ingredients in ginkgo biloba extracts and to reduce the concentration of ginkgolic acids, while at the same time the possibility of providing different treatments with specific combinations of active ingredients of ginkgo biloba extracts was also investigated. The combination of bilobalide and flavonoid glycoside can shift the activity of the extract towards anti-PAF effect. In contrast, the combination of bilobalide and flavonoid glycoside is more active in treating encephalopathy, encephaledema, demyelinating neuropathy, myelopathy, and methods for removing alkylphenols without using chlorinated aliphatic hydrocarbons and proanthocyanidins without using lead have also been developed.

U.S. Pat. No. 5,399,348 discloses a process for preparing ginkgo biloba extract by separating alkylphenol compounds without using chlorinated aliphatic hydrocarbon, which comprises first passing through a precipitation filtration process and then performing multi-step liquid-liquid extraction from aliphatic hydrocarbon, using lead compounds or polyamide to remove proanthocyanidins. The method comprises the following steps: extracting folium Ginkgo with an organic solvent such as acetone aqueous solution, one-to-three carbon alcohol aqueous solution or anhydrous methanol, separating most of the organic solvent to obtain an aqueous solution, and diluting with water to solid content (solid content) of 5-25 wt%. The diluted aqueous solution is cooled to precipitate to remove lipophilic components, then treated with ammonium sulfate and extracted with methyl ethyl ketone, acetone or a mixture thereof, the extract is diluted with water and alcohol to obtain a water-alcohol solution, and then treated with a lead compound or an insoluble polyamide. The treated hydroalcoholic solution is finally extracted with an aliphatic or alicyclic hydrocarbon solvent to further remove alkylphenol compounds, and a dry extract is finally obtained.

Alternatively, after extraction with methyl ethyl ketone or a mixture of methyl ethyl ketone and acetone, the extract may be concentrated to a solids content of 50-70%, then diluted with water and alcohol to give a 10% solids aqueous, 50% by weight aqueous alcoholic solution of each of the alcohols, then an aqueous solution of a lead salt, lead acetate, alkali or lead acetate or lead nitrate or lead hydroxide, preferably basic lead acetate, is added to the aqueous alcoholic solution until the colour turns brown to ochre, a precipitate is formed, the aqueous alcoholic solution is separated and removed and extracted with an aliphatic or alicyclic hydrocarbon solvent to further remove alkylphenol compounds, then concentrated to an alcohol concentration of not more than 5%, ammonium sulfate is added to a concentration of 20% by weight, the resulting aqueous alcoholic solution is extracted with a mixture of methyl ethyl ketone and ethanol in a ratio of 9: 1 to 4: 6 to form an organic phase extract, which is concentrated to a solids content of 50-70% and dried, in addition to lead salts, polyamides such as polyamide 6, polyamide 66 or crosslinked polyvinylpyrrolidone (Polyvidon) may also be used.

U.S. Pat. No. 5,399348 shows that the obtained ginkgo biloba leaf preparation contains 20 to 30% flavonoid glycoside, A, B, C, J2.5.5 to 4.5% bilobalide, 2.0 to 4.0% bilobalide, less than 10ppm alkylphenol compounds and less than 10% proanthocyanidin by the above method.

U.S. Pat. No. 5,322,688 developed a process for removing alkylphenol compounds similar to that described above, but unlike the removal of proanthocyanidins with lead, U.S. Pat. No. 5,322,688 employed an extraction process using a water-immiscible 4 or 5 carbon alcohol such as n-butanol. The method is characterized in that the ginkgo leaves are extracted by acetone aqueous solution, alcohol aqueous solution with one to three carbons or absolute methanol, most of organic solvent in extracting solution is removed by evaporation or distillation, and the obtained aqueous solution is diluted by water to the solid content of 5-25%. Cooling the diluted water solution, precipitating to remove water-insoluble lipophilic components, treating the water solution with 10-30% ammonium sulfate, extracting with a mixture of methyl ethyl ketone or methyl ethyl ketone acetone, extracting an extract with butanol or pentanol, diluting the butanol or pentanol extract with water and alcohol, extracting the obtained water-alcohol solution with an aliphatic hydrocarbon or alicyclic hydrocarbon solvent to further remove alkylphenol compounds, concentrating the water-phase solution, and drying the obtained concentrate to obtain the extract.

Alternatively, after the above extraction with methyl ethyl ketone or a mixture of methyl ethyl ketone and acetone, the extract may be concentrated to a solid content of 50 to 70% and then diluted with water to a solid content of 10% by weight, the water concentrate being C which is immiscible with water₄Or C₅Alcohol extraction, alcohol layer concentration to solid content of 50-70%, diluting the concentrated solution with water and alcohol to obtain an alcohol aqueous solution containing 5-20% of dry extract and 20-60% of alcohol, extracting the solution with aliphatic hydrocarbon or alicyclic hydrocarbon solvent to further remove alkylphenol compounds, finally, concentrating the aqueous phase concentrate, and drying to obtain dry extract.

U.S. Pat. No. 5,322,688 proposes that the folium Ginkgo preparation obtained by the above method contains 20-30% flavonoid glycoside, A, B, C% and J2.5-4.5% bilobalide, 2.0-4.0% bilobalide, and proanthocyanidin with alkylphenol compounds less than 10ppm and less than 10%.

U.S. Pat. No. 5,389,370 also employs the removal of alkylphenol compounds and proanthocyanidins described in U.S. Pat. No. 5,322,688, but provides a method for preparing ginkgo biloba extract containing high concentrations of active ingredients and combinations thereof. The process of us patent No. 5,389,370 is characterized by: extracting folium Ginkgo containing at least 1.4% flavonoid glycoside with acetone water solution, alcohol water solution containing at most three carbon atoms or anhydrous methanol, separating to remove most organic solvent until the concentration is not more than 10%, diluting the obtained concentrated aqueous solution with water to solid content of 15-20% (by weight), standing, cooling until precipitate is formed, these precipitates are composed of lipophilic components which are not readily soluble in water, are removed by filtration, the remaining aqueous solution is subjected to a multi-stage extraction with formic esters or acetic esters such as ethyl acetate or a mixture of ethyl acetate and an aliphatic or alicyclic hydrocarbon, the dissolved esters in the aqueous phase can be completely removed by distillation, the remaining solution is extracted with a C-4 or C-5 alcohol which is not soluble in water, the alcohol phase is washed with water and then concentrated, the residual solvent is completely removed by azeotropic distillation, and the residue is diluted with 40% by weight of an aqueous alcohol solution to give a diluted residue.

In the above process, the extract may be subjected to activated carbon or silica gel column chromatography to remove the extraction partner in the ethyl acetate or ethyl acetate/hydrocarbon extract. Furthermore, the extract from the above process, which is obtained by ethyl acetate or ethyl acetate/hydrocarbon mixture, may be first treated with activated carbon to remove accompanying impurities, and then the bilobalide is crystallized out, and pure bilobalide and the remaining bilobalide are separated from the mother liquor by column chromatography.

The diluted residue obtained in the last step of the above process can be further extracted with aliphatic or alicyclic hydrocarbon solvent to reduce the content of alkylphenol compounds, and the aqueous phase is concentrated and evaporated to obtain a dry extract.

U.S. Pat. No. 5,389,370 reports that the ginkgo leaf preparation obtained by the method contains 40-60% of flavonoid glycoside; 5.5-8% of ginkgolide A, B, C and J, and 5-7% of bilobalide, or 5.5-8% of ginkgolide and less than 0.1% of bilobalide, or 5-7% of bilobalide and less than 0.1% of bilobalide; 0-10% proanthocyanidin and an alkylphenol compound at a maximum concentration of 10ppm, preferably at a level of less than 1 ppm.

U.S. Pat. No. 5,637,302 relates to a process for preparing an extract of ginkgo biloba leaves, wherein crude extract of ginkgo biloba leaves is extracted with a solvent consisting of n-butanol and toluene, thereby avoiding the use of chlorinated aliphatic hydrocarbons and lead compounds, and further, the use of this process also solves the problem of using a large number of different solvents which are miscible with each other in other inventions. The process of U.S. Pat. No. 5,737,302 is characterized in that ginkgo leaves are extracted with an aqueous solvent of acetone aqueous solution or methanol and/or ethanol and aqueous solution, these aqueous extracts are directly extracted with n-hexane or n-heptane or a toluene/butanol mixture to remove inactive lipophilic substances such as alkylphenols and polyphenols, the defatted solution is concentrated to a volume corresponding to the weight of the crude drug, and then put in a refrigerator for 24 hours, followed by centrifugation to obtain a semi-crystalline substance composed of dimeric flavonoids, and the aqueous phase is subjected to countercurrent extraction with toluene/butanol at a volume ratio of 1: 2 to 1: 4. The toluene-butanol phase is washed by water in countercurrent flow, concentrated into an extract sample and washed by water or a water-alcohol mixture to further remove residual trace toluene and butanol, and finally dried.

In addition, the aqueous solution after degreasing still contains a proportion of dimeric flavones, which can be passed through an adsorption resin such as an aromatic polymer resin, which has a significant activity on phenols and can readily adsorb many active ingredients, the adsorbed active ingredients being adsorbed by an organic solvent such as a lower alcohol (C)₁-C₄) Or the water-miscible ketone is re-eluted from the resin.

U.S. Pat. No. 5,637,302 shows that the above method can be used to obtain a ginkgo biloba extract preparation containing 22-26% flavonoid glycoside, 2.5-4.5% ginkgolide, 2.5-4.5% bilobalide, containing almost no alkylphenol compounds and having proanthocyanidin less than 10%.

Disclosure of Invention

The object of the present invention is to further identify each flavonoid component, to provide a more refined extract of ginkgo biloba, to determine its content and to adjust the ratio between them. These single components include flavonols, flavones, flavanols and flavonol glycosides. The content of the product meets the medicament test requirements and component reproducibility established by the state no matter what the different components in the ginkgo leaf raw material are. The more defined ginkgo extract of this composition aggregates the active ingredients and reduces the unknown ingredients. Has more standard production process and quality control standard. The invention improves the safety of the prepared medicine, and enhances the medication confidence of doctors and patients and the repeatability of medicine screening.

Another objective of the invention is to provide a high-content ginkgo extract, the content indexes are respectively 44-78% of flavonoids; 2.5 to 10 percent of ginkgolide; 2.5 to 10 percent of bilobalide. High levels of ginkgo extracts are a requirement of many national pharmaceutical standards, but conventional extracts are difficult to meet since these terms apply only to purified compounds. It has not been possible to prepare such high content of ginkgo extract so far.

Another advantage of high extract content is the reduction of daily dosage and the reduction of formulation size. The advantages are even more pronounced because most patients taking ginkgo preparations are elderly.

Still another advantage is that the high content of extract greatly removes inactive ingredients, which increases the safety of administration, and the more simplified active ingredients make the testing of the main ingredients and the determination of impurities more convenient. The purified Ginkgo biloba extract can be used for inhibiting foreign body rejection reaction during organ transplantation.

It is also an object of the invention to remove ginkgolic acids and eliminate the occurrence of allergic reactions.

Since the present medicinal ginkgo extracts are mainly used for treating poor circulation in the brain and peripheral arteries, it is also an object of the present invention to provide a method for treating angina pectoris caused by coronary heart disease.

The ginkgo extract provided by the invention contains 44-78% of flavonoids, 2.5-10% of ginkgolides, 2.5-10% of bilobalide and 0.1-5 ppm of ginkgoic acid.

The invention relates to a composition extracted from ginkgo biloba leaves, in particular comprising different compositions containing new active ingredients and mixtures thereof. Provides a method for preparing the same extract, a method for identifying and measuring the content of the same extract, and also provides a medicament containing the active ingredients and a mixture and a method for treating angina caused by coronary heart disease by using the medicament.

The invention provides a composition, which comprises about 44-78% of flavonoids, 2.5-10% of ginkgolides (comprising A, B, C and J or a mixture thereof), 2.5-10% of bilobalide and 0.1-5 ppm of ginkgolic acid.

The invention provides a composition, which contains about 44-78% of flavonoids (including flavonols, flavanols and flavonoid glycosides), 2.5-10% of ginkgolides (including ginkgolides A, B, C and J or a mixture thereof), 2.5-10% of bilobalide and 0.1-5 ppm of ginkgolic acid.

The invention provides a composition, which contains about 44-78% of flavonoids (wherein the flavonoid glycoside is 20-75%), 2.5-10% of ginkgolides (comprising A, B, C and J or a mixture thereof), 2.5-10% of bilobalide and 0.1-5 ppm of ginkgolic acid.

The invention provides a composition, which contains about 44-78% of flavonoids (wherein the ratio of flavonoid glycoside to flavonol is 1-30: 1), 2.5-10% of ginkgolides (comprising ginkgolides A, B, C and J or a mixture thereof), 2.5-10% of bilobalide and 0.1-5 ppm of ginkgolic acid.

The invention provides a composition comprising about 44-78% flavonoids (containing flavonoid glycosides), 5-20% lactones (consisting of 2.5-10% of ginkgolides A, B, C and J, either alone or in combination, and 2.5-10% of bilobalide), wherein the ratio of flavonoid glycoside to lactones is about 3.5-4.5: 1, and further comprises ginkgolic acid 0.1-5 ppm.

The invention provides a composition, wherein flavonoid compounds containing flavonoid glycosides are not less than 44%, the ginkgolides containing single components of ginkgolides A, B, C and J or a mixture of the ginkgolides are not less than 6%, and the ginkgolic acid content is 0.1-5 ppm.

The ginkgolic acid content of the composition provided by the invention is about 0.1-0.5 ppm.

The composition of the composition provided by the invention is extracted from ginkgo leaves.

The composition of the composition provided by the invention is extracted from ginkgo leaves obtained from artificially cultured plants.

The invention provides a method for obtaining a ginkgo composition, which comprises the following steps:

(a) obtaining dry ginkgo leaves

(b) Pulverizing leaves into small pieces

(c) Extracting pulverized leaves with water, C1-C3 aliphatic alcohol, acetone and their mixture under reflux, and extracting flavonoids and terpene lactones to obtain extractive solution and residue.

(d) Separating the residue from the extractive solution

(e) The extract is concentrated at 60 ℃ to a density of about 1.2 to 1.25.

(f) Treating the concentrate of step (e) with at least two resins under conditions effective to bind the flavonoid and the lactone.

(g) Eluting the chromatographic column combined with flavone and lactone to obtain the ginkgo extract containing flavone and lactone.

The invention provides a chromatography method, namely a chromatography method for carrying out column chromatography in a column.

Resins useful in the methods of chromatography provided herein include, but are not limited to, porous polymers, silica gel, alumina, polyamides, activated carbon, cellulose, and glucose gel.

The chromatography provided by the invention is characterized in that the column is eluted by water, aliphatic alcohol with 1-3 carbon atoms, acetone and a mixture or ester (methyl ester or ethyl ester) thereof.

The invention provides a method for identifying and analyzing flavone in a ginkgo composition, which comprises the following steps:

(a) the composition was dissolved in methanol to prepare a test solution.

(b) Preparing a standard solution: refluxing standard folium Ginkgo in 60% ethanol water solution, filtering, evaporating ethanol, concentrating the filtrate, and extracting the concentrated water solution with petroleum ether, ethyl acetate and n-butanol respectively; concentrating the n-butanol extractive solution to dryness, and dissolving in methanol.

(c) Analysis was performed according to the Thin Layer Chromatography (TLC) method: each of the above-mentioned solutions was spotted on the same thin layer silica gel plate, developed with a mixture of ethyl acetate, formic acid, acetic acid and water, dried in air, sprayed with an ethanol solution of 1% aluminum chloride, and then observed with 365nm ultraviolet light, and 8 yellow spots were seen at the designated positions on chromatograms of the standard sample and the test sample.

The invention provides a method for identifying and analyzing terpene lactones in a ginkgo composition, which comprises the following steps:

(a) preparing a test solution: refluxing the ginkgo composition with ethyl acetate, then filtering, concentrating the filtrate to dryness, and dissolving in methanol.

(b) Preparing a standard solution: five standard samples of ginkgolide A, B, C, J and bilobalide were dissolved in methanol to make five standard solutions.

(c) Analysis according to TLC method: under the same GF₂₅₄Spotting the above solutions on thin layer chromatography silica gel plate, respectively, by mixing ethyl acetate, toluene, acetone and cyclohexaneThe solvent spreads the sheet, after spreading, it is dried in air, heated, and observed under a 254nm ultraviolet lamp, and the standard and test samples appear as spots of the same color at the same position.

The invention provides a method for identifying and analytically checking ginkgolic acid in a ginkgo composition, which comprises the following steps:

(a) preparing a test solution, refluxing the ginkgo biloba composition with n-hexane, filtering, concentrating the filtrate to dryness and dissolving in ethyl acetate.

(b) Preparation of standard sample solutions: dissolving ginkgolic acid standard in ethyl acetate.

(c) Analysis was performed by thin layer chromatography: under the same GF₂₅₄And (3) spotting the solution on a thin-layer silica gel plate, developing the thin plate by using a mixed solvent of n-hexane, ethyl acetate and acetic acid, drying in air, and observing under 315 and 368nm ultraviolet light, wherein the absorption of an analysis sample solution is smaller than that of a standard solution.

The invention provides a method for determining total flavonoids in a ginkgo composition, which comprises the following steps:

(a) preparing a standard solution: and quantitatively dissolving the dried rutin in an ethanol water solution to obtain a standard solution.

(b) Drawing a standard curve: different amounts of standard solution were pipetted into the flasks, each flask was graduated with water, acetic acid-sodium acetate buffer (pH 4.5), 0.1M sodium chloride and 70% aqueous ethanol, and a standard curve was plotted according to absorption at 270 nm.

(c) The samples were analyzed spectrophotometrically: dissolving a composition sample by using 70% ethanol, sucking the solution into a volumetric flask, preparing a sample solution by adopting the method, measuring the absorption of the sample at 270nm according to a standard curve to obtain the concentration of the sample solution, wherein the content of the total flavone calculated from a rutin dried product is 85-115% of the labeled amount.

The invention provides a method for measuring flavonoid glycoside in a ginkgo composition, which comprises the following steps:

(a) preparing a test solution: the sample composition was dissolved with methanol and 25% hydrochloric acid sugar, refluxed, salts and insoluble matter removed, left to cool and transferred to a volumetric flask, the flask was washed with methanol, and the washings were poured into the volumetric flask and diluted to the mark with methanol.

(b) Preparation of standard solution: dissolving quercetin, kaempferol and isorhamnetin in the same volumetric flask, and diluting with methanol to scale.

(c) Measuring the content by high pressure liquid phase method, calculating the relative peak area of quercetin, kaempferol and isorhamnetin, and calculating the content of total flavonoid glycoside by the following formula: the amount of quercetin × 2.50+ the amount of kaempferol × 2.63+ the amount of isorhamnetin × 2.36.

The invention provides a method for measuring the content of terpene dilactone in a ginkgo extract, which comprises the following steps:

(a) preparation of sample solution: the composition is refluxed with acetone, filtered, the filtrate is concentrated, the residue is dissolved in methyl acetate, extracted with water and the aqueous layer is extracted once more with methyl acetate. The two methyl acetate layers were combined and evaporated to dryness before being dissolved in methanol.

(b) Preparation of standard solution: lactone A, B. C. J and bilobalide are dissolved in the same volumetric flask with methanol.

(C) And (3) testing: the test solution and the standard solution are detected by High Pressure Liquid Chromatography (HPLC), A, B, C, J and bilobalide content are calculated according to external standard method, and total lactone content can be obtained by summation.

The invention provides a method for testing the content of lactone in a ginkgo composition by using ginkgolide A, B, C, J and bilobalide monomers as standards.

The invention provides a method for testing the ginkgolic acid content in a ginkgolic composition by using a ginkgolic acid monomer as a standard substance.

The invention provides a method for measuring flavonoid compounds by using a rutin monomer as a standard substance.

The invention provides a method for measuring flavonol and glycoside thereof by using quercetin, kaempferol and isorhamnetin monomers as standard substances.

The composition provided by the invention can be used as a food additive or added into a beverage.

The above compositions provided by the present invention may be used in creams, ointments or as starting materials for the formulation of these.

The invention provides an oral preparation containing the components.

The oral dosage form comprises: pills, capsules, granules, tablets or suspensions.

The invention provides an injection preparation, which can be used by different routes, such as intravenous injection, intramuscular injection, subcutaneous injection and intraperitoneal injection.

The invention provides a cosmetic using the components.

The present invention provides a pharmaceutical composition prepared by the above method, which comprises the above-mentioned effective amount of the above-mentioned composition and a pharmaceutically acceptable carrier. The invention also provides a preparation method of the medicinal composition.

By an acceptable pharmaceutical carrier according to the invention is meant any standard pharmaceutical carrier. Examples of such carriers are well known and include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline, phosphate buffered saline containing polysorbate 80, aqueous oil/water suspensions and various types of wetting agents, and other carriers including sterile solutions, tablets, coated tablets and capsules.

Typical carriers include excipients such as: starch, milk, sugar, specialty clays, gelatin, stearic acid or salts (including magnesium stearate, calcium), talc, vegetable fats or oils, gums, glycols, or other known excipients. The carrier may also include flavorings and colors or other additives. The compositions containing the carrier are formulated according to conventional methods.

The present invention provides a method for treating angina pectoris of various types and degrees caused by coronary heart disease by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for ameliorating ischemia-induced tachycardia by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method of alleviating angina pectoris by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for reducing the amount of glyceryl trinitrate used by administering to a patient an effective amount of the above-described pharmaceutical composition.

The present invention provides a method for reducing palpitations by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for lowering cholesterol and triglycerides in the blood of a hyperlipidemic patient by administering to the patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for reducing platelet aggregation in blood by administering to a patient an effective amount of the above-described pharmaceutical composition.

The present invention provides methods for increasing exercise tolerance, extending exercise duration, extending the interval between exercise initiation and angina pectoris onset and the 1mm reduction in the ST component by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for treating impotence by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method of treating psoriasis by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method of treating pigmentation by administering to a patient an effective amount of the above pharmaceutical composition.

The present invention provides a method for treating amnesia, AIDS, alzheimer's disease, angina, arteriosclerosis, arthritis, asthma, atherosclerosis, autism, enuresis, cerebral trauma, cardiac rhythm disorders, chilblain, wind cold, coronary heart disease, deafness, dementia, depression, diabetic vasoconstriction accompanying gangrene and angina, dizziness, blurred vision, memory decline, fatigue, filariasis, headache, hyperlipidemia, hypertension, intermittent claudication, renal dysfunction, leg spasm, myocardial infarction, parkinson's disease, blood circulation disorders, embolism syndrome, raynaud's disease, rheumatism, aging, chest distress, tinnitus, tuberculosis, varicose veins, vertigo by administering to a patient the above effective amount of the pharmaceutical composition.

The above pharmaceutical compositions are used for the treatment of diseases, since they have the following effects: analgesia, asthma relief, inflammation relief, atherosclerosis prevention, antibiosis, anticancer, anticoagulation, antidiabetic, hyperlipidemia reduction, hypertension reduction, antinuclear radiation resistance, antiplatelet aggregation, antioxidation, antithrombotic, antitubercular, antitussive, bronchodilator, telangiectasia, capillary protection, cerebral circulation stimulant, cerebral vasodilator, attention focusing, memory improvement, hearing improvement, peripheral circulation improvement, dopamine increase, epinephrine, norepinephrine, nerve conduction regulator, cervical atherosclerosis prevention and blood vessel stretching agent, etc.

Detailed Description

The following examples will further aid the understanding of the invention, and one of ordinary skill in the art will indeed recognize that the methods and results described above are merely illustrative of the invention. The following description will be made in more detail.

The following examples will aid in the understanding of the present invention and, further, those skilled in the art will recognize the above methods and results.

Examples 1, 1,

The invention provides a method for preparing a ginkgo composition, which comprises the following steps:

(a) extraction: the ginkgo green leaves are collected from late summer to early autumn, the content of effective treatment components is high and the moisture content is lower than 8%, the ginkgo green leaves are crushed into fragments smaller than 4mm in a crusher after being dried, then the ginkgo green leaves are refluxed twice under normal pressure by using 60% aqueous ethanol with the volume being 12 times that of the ginkgo green leaves, each time is 3 hours, the ginkgo green leaves are refluxed for 0.5 hour for the third time by using water with the volume being 10 times that of the ginkgo green leaves after solid residues are removed by filtration, and the three filtrates are combined.

(b) Concentration: and c, concentrating the filtrate obtained in the step a under reduced pressure to obtain a viscous extract without alcohol (the density d is 1.2-1.25, and the temperature t is 60 ℃).

(c) And (3) precipitation: dissolving the viscous extract of step (b) with 2 times of hot water, cooling to form precipitate, and filtering to remove precipitate.

(d) Column chromatography: loading the filtrate of step (c) onto macroporous resin column (XAD-4) to make the ratio of resin to leaves 1: 1, eluting with 2 times of pure water, 6%, 18%, and 30% aqueous ethanol, and eluting with 65% ethanol until the color of the fraction becomes light. Combining 18% and 30% fractions, vacuum concentrating to small volume without alcohol, loading the concentrate on polyamide column with polyamide to crude leaf ratio of 1: 3, eluting with pure water and 95% ethanol respectively, combining 95% fraction and 65% fraction of macroporous resin column, and vacuum concentrating to alcohol-free fraction.

(e) Removing ginkgolic acid: the concentrate from step (d) was extracted three times with two thirds volume of cyclohexane and the aqueous fraction was concentrated under reduced pressure.

(f) And (3) drying: adjusting the density of the concentrated extract to 1.05, and then carrying out spray drying, wherein the temperature of a spray drying chamber is controlled to be 140-160 ℃.

The final product is brown yellow powder, and has special fragrance and slight bitter taste.

The column chromatography is characterized by secondary treatment, i.e. the extract is firstly put into a macroporous resin column (XAD-4) with the ratio of the column to the leaves being 1: 1, and then is put into a polyamide column with the ratio of the column to the leaves being 1: 3.

Specifically, the removal of ginkgolic acids is characterized by secondary treatment, namely, the extract is subjected to macroporous resin column (XAD-4) chromatography and then is treated with cyclohexane for three times.

Examples 2,

The invention provides a method for preparing a ginkgo composition, which comprises the following steps:

(a) extraction: collecting green leaves of semen Ginkgo in late summer and early autumn, drying, and pulverizing into leaves with size less than 4 mm. And then refluxing for 2 times by using a solvent system in an amount which is 12 times that of the filtrate, wherein the solvent system can adopt water, water-ethanol and acetone-water, filtering to remove solids or residues, and combining the two filtrates.

(b) Concentration: concentrating the filtrate obtained in the step (a) into an extract without organic solvent in vacuum, (d is 1.2-1.25, t is 60 ℃)

(c) And (3) precipitation: dissolving the extract obtained in the step (b) in 2 times of boiling water, cooling to about 12 ℃, forming a precipitate, and filtering and separating.

(d) Column chromatography: and (c) putting the extract obtained in the step (c) on a certain resin column, and eluting with water or a mixed solvent of water and an organic solvent, wherein the mixed solvent can be alcohol with 1-3 carbon atoms and acetone. Concentrating the eluted fraction, loading the concentrated fraction onto another resin column, eluting with one or two solvent systems selected from water, C1-C3 alcohol, ketone and ester to obtain a fraction containing high content of lactone and flavonoid compounds, and concentrating the fraction under reduced pressure to remove aqueous alcohol.

(e) Removing ginkgolic acid: extracting the concentrated solution obtained in the step (d) with two thirds of saturated or unsaturated alkane for three times, and partially vacuum-concentrating the water.

(f) And (3) drying: adjusting the density of the concentrated extract to 1.02-1.10 (40 ℃), and then performing spray drying, wherein the room temperature of the spray drying is controlled at 140-160 ℃.

Examples 3,

The invention provides a method for preparing a ginkgo composition, which comprises the following steps: refluxing 100kg of dried ginkgo leaf powder with 1000kg of 50% ethanol solution for 2 hours, filtering, refluxing the filtered residue with 1000kg of 50% aqueous ethanol for 2 hours, filtering, combining the two filtrates, vacuum-concentrating at 60 ℃ until no alcohol exists (d is 1.2), dissolving the concentrated solution in 300kg of water, standing for 48 hours, and filtering. Passing the filtrate through a mixing column of macroporous resin and polyamide, eluting with 100kg of water, 100kg of 30% ethanol solution, 100kg of 60% ethanol solution and 100kg of 90% ethanol solution respectively, combining the 30%, 60% and 90% ethanol water solution eluates, concentrating the combined solution in vacuum to about 70kg of extract, extracting the extract with n-hexane for three times, and concentrating and drying the water phase in vacuum. 1.5kg of the final product contained less than 5% water, 47.2% flavonoids with about 24.8% flavonoid glycosides, 6.3% lactones and less than 5ppm ginkgolic acids.

Examples 4,

Starting from the column chromatography of example 3: fractions from the column eluted with 30% aqueous ethanol were concentrated under reduced pressure to remove ethanol, and the concentrate was extracted three times with two-thirds volume of ethyl acetate each time. Evaporating ethyl acetate phase at 45 deg.C under reduced pressure, dissolving the residue with 50% ethanol water solution under heating, and standing for 24 hr to obtain bilobalide. After separation and crystallization, mother liquor is evaporated under reduced pressure, and is subjected to layer column separation by using 1.5 times of active carbon and silica gel respectively, and all parts of bilobalide and ginkgolide are collected respectively; concentrating to dry, and mixing with 10g of bilobalide crystal (A) with total terpene lactone content not less than 80%.

The 60% aqueous ethanol fraction and the 70% aqueous ethanol fraction eluted from the column in example 3 were pooled and collected. Concentrating under reduced pressure until no alcohol is obtained. The concentrate was applied to a polyamide column and eluted with 10%, 20%, 75% and 90% aqueous ethanol solutions, respectively. Concentrating 75% fraction to dryness, dissolving the powder with anhydrous ethanol, loading on polyamide column, eluting with 100% ethanol, 70% ethanol water solution and 20% ethanol water solution respectively, collecting 70% ethanol water solution, mixing with 2kg silica gel, concentrating the mixture to dryness, and eluting with ethyl acetate and ethanol respectively. The ethanol fraction was concentrated to dryness to give (B)90 g. The content of total flavone is not less than 80%.

The high content lactone (A) and flavone (B) extracts are mixed according to different proportions to obtain final products with different contents, for example 10g (A) and 90g (B), 100g (A + B) is obtained, the mixture contains 77% of flavonoid compounds and 8% of lactone, 10g (A) and 70g (B) are mixed to obtain 80g (A + B), and then the product contains 70% of flavone and 10% of lactone.

Examples 5,

Methods for identifying and quantitatively analyzing flavone, lactone and ginkgolic acid, and determining flavone, lactone and terpene lactone content are provided.

The invention provides a method for identifying and quantitatively analyzing flavone in a ginkgo leaf composition, which comprises the following steps:

(a) preparing a test solution: 0.1g of the composition was dissolved in 1ml of methanol.

(b) Preparing a standard solution: refluxing 1g of standard folium Ginkgo with 10ml of 60% ethanol for 2 hr, filtering, concentrating the filtrate, and evaporating ethanol; the n-butanol fraction was extracted with 10ml of petroleum ether, ethyl acetate, n-butanol, respectively, concentrated to dryness and dissolved in 1ml of methanol.

(c) And (3) measuring a test solution: after 10. mu.l of each of the above solutions was spotted on the same silica gel sheet, developed with a mixed solvent of ethyl acetate, formic acid, acetic acid and water (100: 11: 26), the solvent was blown off by air drying, sprayed with a 1% aluminum chloride ethanol solution, observed under a 365nm ultraviolet lamp, and 8 yellow spots were observed at the designated positions on chromatograms of the standard sample and the test sample.

The invention provides a method for identifying and quantitatively analyzing lactone in a ginkgo composition, which comprises the following steps:

(a) preparing a test solution: reflux 0.5g of the composition with 20ml of ethyl acetate for 30min, filter, concentrate the filtrate to dryness: dissolved in 2ml of methanol.

(b) Preparing standard solutions, respectively taking standard products of ginkgolide A, B, C, J and bilobalide B, and dissolving in methanol to obtain 5 standard solution of 0.5mg/1 ml.

(c) Testing a test solution: sucking 10 μ l of each solution, dropping on the same silica gel GF254 plate, developing with the mixed solvent of ethyl acetate, toluene, acetone and cyclohexane (4: 3: 2: 1), blowing the solvent in air, heating at 150 deg.C for 1 hr, and observing under the wavelength of 254nm to obtain the chromatographic spots of the same color on the designated positions of the sample and standard sample.

The invention provides a method for identifying and quantitatively analyzing ginkgolic acid in a ginkgo mixture, which comprises the following steps:

(a) preparing a test solution: 4g of the composition are refluxed with 100ml of n-hexane for 2 hours, filtered, the filtrate is concentrated to dryness and dissolved in 1ml of ethyl acetate.

(b) Preparing a standard solution: dissolving ginkgolic acid standard substance in ethyl acetate to make the solution contain 0.04mg standard substance per ml,

(c) testing a test solution: sucking 10 μ l of the above solutions, respectively dropping on the same silica gel GF254 thin plate, developing with mixed solvent of n-hexane, ethyl acetate and acetic acid (80: 15: 5), blow-drying the solvent in air, and observing under ultraviolet lamp at 315nm and 368nm to obtain the sample solution with absorption lower than that of standard solution (0.0005%).

The invention provides a method for analyzing the total content of flavonoid compounds in a ginkgo leaf composition in a certain amount, which comprises the following steps:

(a) preparing a standard solution: 20mg of dried rutin was dissolved in 70ml of 70% aqueous ethanol.

(b) Drawing a standard curve: 0.2, 0.4, 0.6, 0.8, 1.0, 1.2ml of the standard solution was pipetted into a 10ml volumetric flask, 3ml of water, 2ml of a buffer solution of acetic acid-sodium acetate (pH 4.5), 2ml of 0.1M aluminum chloride were added, and then 10ml of the solution was added with 70% aqueous ethanol, and a standard curve was drawn at 270nm based on the absorbance of each sample of the standard solution.

(c) The test solution is measured by spectrophotometry: 25mg of the ginkgo composition was dissolved in 70% aqueous ethanol in a 50ml volumetric flask. Sucking 0.5ml of the solution, adding the solution into a 10ml volumetric flask, preparing a test solution according to the method, measuring the absorption at 270nm, obtaining the concentration of the test solution from a standard curve, and counting the total flavone as rutin to be between 85 and 115 percent of the marked amount under the anhydrous condition.

The invention provides a method for measuring flavonoid glycoside content, which comprises the following steps:

(a) preparing a test solution: dissolving 75mg of the ginkgo biloba leaf composition with 20ml of methanol and 5ml of 25% hydrochloric acid and refluxing for 60min, after separation, standing to cool and immediately transferring to a 50ml volumetric flask, washing the flask with 5ml of methanol 3 times, incorporating the washing solution into the volumetric flask and diluting to the mark with methanol.

(b) Preparing a standard solution: 4.2mg of quercetin, 6.0mg of kaempferol, 1.2mg of isorhamnetin were dissolved in a 50ml volumetric flask with methanol and diluted with methanol. Each 1ml of the standard solution contains 24 μ g of isorhamnetin, 84 μ g of quercetin and 120 μ g of kaempferol.

(c) And (3) measuring a test solution: calculating relative peak area of HPLC to determine the content of kaempferol, quercetin and isorhamnetin. The total flavonoid glycoside content is equal to the quercetin content × 2.50+ the amount of kaempferol × 2.63+ the amount of isorhamnetin × 2.36.

The invention provides a method for measuring terpene lactone content, which comprises the following steps:

(a) preparing a test solution: 100mg of the composition are refluxed with 60ml of acetone for 2 hours, the filtrate is concentrated after filtration, the residue is dissolved again in 20ml of methyl acetate, extracted with 10ml of water, the aqueous layer is extracted again with 20ml of methyl acetate, the two methyl acetate layers are combined, concentrated to dryness and dissolved in 5ml of methanol.

(b) Preparing a standard solution: 30mg each of ginkgolide A, B, C, J and bilobalide (BB) were dissolved in methanol in 50ml volumetric flasks.

(c) And (3) measuring a test solution: the sample amount of the standard sample and the sample amount of the sample to be tested are respectively 15 mu l, the liquid chromatogram of the standard sample and the sample to be tested are obtained, the content of each of Ginkgolides A (GA), B, C, J and bilobalide (BB) can be calculated by an external standard method, and then the total content of the lactones is calculated, wherein the total content of the terpene lactones in the anhydrous extract is about 5-20%.

The above process is particularly carried out with a highly purified chemical structure of C₂₇H₃₀O₁₆The rutin monomer is used as a standard substance to determine the content of the ginkgo total flavonoid compounds.

Specifically, the method uses highly purified monomers of quercetin, kaempferol and isorhamnetin with definite chemical structures as standard substances to determine the content of flavonol glycoside and flavonol glycoside in the ginkgo leaf composition.

Specifically, the method uses highly purified GA, GB, GC, GJ and BB with definite chemical structures as standard substances to identify lactone and determine the content of the lactone.

The method specifically uses highly purified ginkgolic acid with a definite chemical structure as a standard substance to identify ginkgolic acid in the ginkgo biloba composition.

Examples 6,

Coated tablets, each tablet containing

Ginkgo extract 40.00mg

Lactose 100.00mg

Starch 40.00mg

Microcrystalline cellulose 36.00mg

Hydroxypropyl methylcellulose 30.00mg

Magnesium stearate 1.50mg

Opadry (OPADRY) (for coating) 7.50mg

The total amount of the coated tablet is about 255.00mg

Examples 7,

Granules:

composition of each bag

Ginkgo extract 40mg

Sucrose 420mg

Dextrin 480mg

Starch 20mg

Hydroxypropyl cellulose 20mg

Sweet glucoside 20mg

The total amount of the granules is 1000mg

Example 8 injection solution

1 the ampoule comprises

Ginkgo extract 40mg

Mannitol 100mg

Nicotinamide 100mg

Arginine monohydrochloride 50mg

Disodium ethylenediaminetetraacetate (EDTA-2Na) 4.00mg

Example 9 beverage

500ml of the beverage comprises:

ginkgo biloba extract 2.0g

500ml of 38% ethanol

Proper amount of perfume

Experimental data

The ginkgo extract described in the present invention can be formulated in a conventional manner, for example, into oral liquid, coated tablet, tablet or injection. The preparation of the invention can be used for treating cerebrovascular and peripheral vascular circulatory disorders, in particular angina pectoris caused by coronary artery disease, clinical tests in China show that 243 cases in total prove that the ginkgo biloba extract is safe and effective in treating angina pectoris caused by coronary heart disease, and the detailed conditions of the clinical tests are summarized as follows:

participating in hospital

The 4 hospitals participated in this clinical trial, including:

the university of Beijing traditional Chinese medicine is affiliated with the Dongguangmen hospital;

shanghai medical university affiliated Huashan Hospital;

shanghai second medical university affiliated ninth national hospital;

shanghai medical university affiliated Longhua Hospital.

Patient selection criteria:

clinical trials were conducted from 11 months to 7 months in 1995 to 1996, and 243 patients with angina pectoris due to coronary artery disease were enrolled.

Criteria for selecting patients are as follows:

and (3) inclusion standard: the patient was diagnosed with angina pectoris and chest pain occurred more than 2 times per week. The results of ischemia changes or exercise tests on the electrocardiogram were positive.

Exclusion criteria: (1) patients with active myocardial defects or other cardiopulmonary functional diseases, severe neurosis, climacteric syndrome and chest pain caused by cervical spondylosis. (2) Patients with angina pectoris and hypertension (BP > 24/14.87 Kpa). (3) Patients with severe cardiopulmonary failure, severe arrhythmia, liver and kidney dysfunction and hematopoietic dysfunction. (4) Lactating women and patients with allergic constitution. (5) The age is less than 18 years old or greater than 70 years old.

Deleting data standard: patients who do not take medicine according to the prescription and patients who lack information and cannot determine the clinical test result.

The investigators randomized 153 patients to the Ginkgo biloba group and 90 patients to the control group. The ginkgo group was randomly grouped again, 123 belonged to the test group, and 30 belonged to the open group. The basic data for 243 patients treated in 4 hospitals are tables 1-5, and the results show that the sex, age, onset time, type and severity of angina pectoris and complications in three groups all show significant differences, with a hospitalization rate of 72.4%.

Age mean and gender distribution P > 0.05 in Table 13 groups

Group of	Number of	Age (X + -SD)	Number of cases			Percentage of
								For male	Woman	Total number of	For male	Woman
			Test set	123	62.1±5.8	76	47						123	62	38
Control group	90	62.8±5.8						61	29	90	68	32
			Open group	30	62.7±7.2	17	13						30	57	43

Duration of disease P > 0.05 in Table 23 group

Group of	Number of cases	1 month &	6 month ^ e	1 year &	E3 year E	E5 year ^ e	E10 year E
								Test set	123	6	8	26	28	24	32
Control group	90	6	8	14	15	33	14
								Open group	30	1	1	3	8	8	7

TABLE 33 groups of angina pectoris severity P > 0.05

Group of	Number of cases	Low degree of	Degree of moderate	High degree of
					Test set	123	24	84	15
Control group	90	24	53	13
					Open group	30	6	16	8

TABLE 43 group of angina pectoris type P > 0.05

Group of	Number of cases	Tired type	Self-generating type	Hybrid type
					Test set	123	96	8	19
Control group	90	67	5	18
					Open group	30	23	3	4

TABLE 53 complications of angina pectoris > 0.05

Group of	Number of cases	Hypertension (hypertension)	Diabetes mellitus	Hyperlipidemia
					Test set	123	34	4	26
Control group	90	25	3	23
					Open group	30	9	0	14

Diagnostic criteria:

1. diagnostic criteria for angina pectoris severity

The diagnostic criteria for the severity of angina in clinical trials were the reference: in 1979, the research conference on coronary heart disease angina pectoris and electrocardiogram curative effect evaluation standards of coronary heart disease angina pectoris and arrhythmia by combining western medicines is detailed as follows:

(1) mild: typical angina attacks last several minutes, 1-3 attacks 1 day or 2-3 attacks per week, and the attacks are not severe, but sometimes require nitroglycerin to be taken by the patient.

(2) Medium: angina attacks more than 3 times a day, lasting from minutes to 10 minutes each time, are more painful than mild attacks and patients usually require nitroglycerin.

(3) Severe: angina pectoris attacks frequently, lasts for a long time and is serious every day, daily life is affected, and patients often take nitroglycerin.

2. Diagnostic criteria for angina pectoris type:

the diagnostic criteria for angina pectoris type in clinical trials were "clinical diagnostic criteria for ischemic heart disease" made by the International society for cardiology and the United Commission of the world health organization, and the diagnostic criteria are detailed below:

(1) exertional angina pectoris: the pain is rapidly relieved by rest or administration of nitroglycerin due to transient chest pain episodes caused by excessive fatigue or other increased myocardial oxygen demand.

(2) Spontaneous angina pectoris: chest pain episodes are not significantly associated with any condition that increases myocardial oxygen consumption, and pain persists longer and more severely than exertional angina, and is generally not easily alleviated by nitroglycerin.

(3) Mixed angina pectoris: spontaneous angina pectoris may be associated with exertional angina, and is called mixed angina.

Designing a clinical test scheme:

the clinical trial was carried out as follows: 243 patients with angina pectoris caused by coronary artery disease were enrolled in the trial. The investigators used 153 patients as ginkgo trials, 90 patients as controls, and 123 patients were randomized in the ginkgo trials to test and 30 patients as open tests, and the 213 cases in the test and control groups were performed by double-blind, amplified parallel controls and open treatments. The drug used in the ginkgo group is a preparation of the ginkgo extract of the invention. 1g of the preparation contained 40mg of ginkgo biloba extract, 480mg of dextrin, 420mg of sucrose, 20mg of starch, 20mg of low-substituted hydroxypropylcellulose and 20mg of stevioside. The preparation is prepared into granule and then packed into small bag, and 1g of the preparation is taken by patients in clinical test for 3 times per day for 6 weeks.

The drug used in the control group was Bailuoda (Brainway)^®) Produced by Shanghai Xinyi pharmaceutical factory, No. 70, located in the North road of Sichuan, Shanghai, China. In clinical trial, patients take 1 tablet of Bailuda every time, 3 times daily. As a positive control, the treatment cycle was the same for 6 weeks.

Because the two medicament forms are different (granules and capsules), blank placebo with different medicament forms is added in each group, namely the blank capsules (placebo) are added in the treatment group except for the ginkgo granules, and the blank granules (placebo) are added in the control group except for the tarda. The packaging, appearance of each placebo was identical to the corresponding drug.

The long-acting coronary heart disease medicine is stopped taking from one week before clinical test. Patients with mild hypertension are allowed to take their usual medications. Nitroglycerin may be administered temporarily if the patient cannot tolerate the onset of angina pectoris.

Criteria for evaluating the efficacy of a drug: the evaluation is carried out according to the part of clinical trial guidelines for new traditional Chinese medicines issued by the ministry of health of China in 1993 on the treatment of patients with coronary heart disease and angina pectoris, and the research conference of treating coronary heart disease and angina pectoris and arrhythmia in 1979 by combining western medicines and the evaluation criteria of the curative effect of coronary heart disease and angina pectoris and electrocardiogram. The criteria are detailed below:

for patients with mild angina pectoris: (1) the effect is shown: the disappearance or substantial disappearance of chest pain (2) is effective: a marked reduction in the onset of chest pain (3) ineffective: no improvement in chest pain symptoms (4) worsening: the chest pain is aggravated and reaches the standard degree above "moderate".

For patients with moderate angina pectoris: (1) the effect is shown: disappearance of chest pain symptoms (2) was effective: reduction of chest pain or achievement of mild criteria (3) is ineffective: no improvement was observed (4) worsening: chest pain worsens to a "heavier" level.

For patients with severe angina pectoris: (1) the effect is shown: the chest pain disappeared or was reduced to the "mild" standard (2) and was effective: the reduction of chest pain to "moderate" criteria (3) is not effective: chest pain symptoms worsen as before treatment (4): chest pain symptoms worsen (or reach "severe criteria").

The electrocardiogram curative effect evaluation standard is as follows:

(1) the effect is shown: the electrocardiogram is recovered to be approximately normal or reaches normal electrocardiogram "

(2) The method has the following advantages: the decreased S-T segment increases by > 0.05mv but not normal, the T wave change decreases, or the T wave change goes from flat to upward, the atrioventricular block or the intraventricular conduction block improves.

(3) And (4) invalidation: no improvement was observed in the electrocardiogram.

(4) Weighting: the S-T section is reduced and aggravated after treatment, T wave is deepened or the T wave is changed from flat to inverted, and ectopic heart rhythm and arrhythmia occur.

Calculating the stop rate of nitroglycerin:

the clinical test conclusion is as follows:

in clinical trials, no significant side effects were observed in the ginkgo and control groups, and only 3 patients had gastric upset during treatment, with one patient having mild vomiting and remission without treatment.

The results of random control, double blind and open clinical trial data show that the ginkgo biloba extract preparation is safe and effective in treating coronary heart disease and angina pectoris. The ginkgo biloba preparation shows a strong biological activity, and compared with the existing traditional Chinese medicine preparation for treating angina pectoris, the ginkgo biloba preparation is superior to the existing preparation in one or more aspects of safety and effectiveness, and can reduce the use of nitroglycerin.

1. The effective components of the ginkgo preparation are effective in treating coronary heart disease and angina pectoris, and the curative effect is better than that of a contrast medicament.

TABLE 6 Effect of Ginkgo biloba extract on angina pectoris patients

Group of	Number of cases	Show effect	Is effective	Invalidation	Weighting device	Total effective rate
							Test set	123	47(38.2)	66(53.7)	10(8.1)	0	91.9％**
Control group	90	14(15.6)	56(62.2)	20(22.2)	0	77.8％
							Open group	30	13(43.3)	15(50)	2(6.7)	0	93.3％*

^**P＜0.01，^*P is less than 0.05, compared with the control group.

2. The effective components of the ginkgo preparation are effective in improving ischemic electrocardiogram, and the effective rate exceeds that of a control group.

TABLE 7 Effect of Ginkgo biloba extract as effective component for improving ischemic electrocardiogram

Group of	Number of cases	Show effect	Is effective	Invalidation	Weighting device	Total effective rate
							Test set	117	22(18.8)	50(53.7)	44(37.6)	1	61.5％
Control group	86	7(8.1)	39(45.3)	39(45.3)	1	53.5％
							Open group	30	6(20)	14(46.6)	10(33.3)	0	66.7％

3. The effective components of the ginkgo biloba preparation are effective in relieving angina pectoris, and the effect of reducing the use of nitroglycerin is stronger than that of a control group.

TABLE 8 reduction of nitroglycerin by the effective components of Ginkgo biloba extract

Group of	Number of cases	Number of cases taken before treatment	Number of therapeutic effect treatment cases (%)	Decrement amount (%)	Rate of reduction
						Test set	123	111	63(56.8)	33(29.7)	86.5％*
Control group	90	82	28(34.1)	16(19.5)	53.7％
						Open group	30	27	18(66.7)	7(25.8)	92.5％*

^*P is less than 0.01, and is compared with a control group

4. The effective components of the ginkgo preparation can effectively relieve palpitation, and the effective rate exceeds that of a control group.

TABLE 9 improvement of angina pectoris symptoms by effective components of Ginkgo biloba extract

Group of	Item	Palpitations	Chest pain
				Test set	Effective rate (%) symptom disappearance (%)	11070(63.6)26(20.9)*	10765(63.1)24(33)
Control group	Effective rate (%) symptom disappearance (%)	2334(44.7)11(14.5)	6731(46.3)22(32.8)
				Open group	Effective rate (%) symptom disappearance (%)	206(30)13(65)*	2913(44.8)15(51.2)

^*P is less than 0.01 and is compared with a control group

5. The effective components of the ginkgo preparation can effectively reduce the cholesterol and triglyceride levels of patients with dyslipidemia.

TABLE 10 effects of effective components of Ginkgo biloba extract on blood lipid of dyslipidemia patients

Item	Number of cases	Before administration	After administration
				Cholesterol	56	6.57±0.93	6.01±1.25*
Triglycerides	94	2.46±0.85	2.31±0.79*
				Low density lipoprotein	75	4.15±0.64	3.81±1.11

^*P is less than 0.05, and the ratio of P to P before administration

6. The effective components of the ginkgo preparation can reduce the specific gravity of medium-density and low-density whole blood and inhibit platelet aggregation, and the difference between the effective components before and after administration is obvious.

TABLE 11 Effect of active ingredients of Ginkgo biloba extract on hemogram change of patients

Item	Testing			Control group
							Number of cases	Before administration	After administration	Number of cases	Before administration	After administration
	High blood weight to low blood weight	603063	5.97±1.528.07±1.518.85±3.10	5.96±1.396.86±0.90**8.28±2.51*	303030	6.19±1.207.76±0.848.46±3.13							6.29±1.057.04±0.59**8.25±2.56
Specific gravity platelet aggregation rate of serum							9330	1.74±0.3075±16.4	1.73±0.1757.1±15.5	3030	1.81±0.1772.3±15.4	1.74±0.1662.6±14.9

^*P＜0.05，^**P < 0.01 to pre-dose ratio.

7. The effective components of the ginkgo preparation have the effects of improving exercise tolerance, prolonging exercise duration, prolonging the interval time between exercise initiation and angina pectoris attack, and reducing the interval time from the exercise initiation to ST segment by 1 mm. And the effect is stronger than that of the control drug.

TABLE 12 Effect of the active ingredients of Ginkgo biloba extract on the kinetic energy of patients

Item	Test set (n ═ 21)		Control group (n ═ 20)
					Before administration	After administration	Before administration	After administration
	Total exercise time (S)	405±141	550±125**	428±136					497±144**^
Total exercise tolerance (mets)					7.93±2.25	10.23±1.97**	8.13±2.40	9.44±2.20*
	ST segment 1mm motion (mets)	267±94	344±120**	303±114					305±130
Movement starts until ST drops by 1mm interval (mets)					5.88±1.40	7.09±1.75**	6.30±2	6.31±2.15^^
	Exercise began to the onset of angina pectoris (S)	390±138	523±119**	399±131					480±132**
ST recovery time (S)					574±149	585±134	608±132	595±128
	ST segment reduction at moving peak	1.60±0.37	1.63±0.45	1.56±0.48					1.58±0.44

^*P＜0.05 ^**P < 0.01 to predose ratio

The ratio of ^ P less than 0.05 ^ P less than 0.01 to the control group

8. The effective components of the ginkgo preparation can be used for angina pectoris of various types and different degrees (tables 13 and 14), and have no influence on blood pressure and heart rate (table 15) and liver and kidney functions.

TABLE 13 Effect of Ginkgo biloba extract on angina pectoris of 3 degrees

Severity of angina pectoris	Number of cases	Show effect	Is effective	Invalidation
					Mild degree of	55	23	33	1
Of moderate degree	80	33	46	1
					Severe degree	18	3	7	8

TABLE 14 Effect of Ginkgo biloba extract on 3 types of angina pectoris

Type of angina pectoris	Number of cases	Show effect	Is effective	Invalidation
					Tired type	119	48	65	6
Self-generating type	25	6	16	3
					Hybrid type	9	5	3	1

TABLE 153 blood pressure and Heart Rate Change tables before and after drug administration (X + -SD)

Item	TestingGroup/open group			Control group
							Number of cases	Before administration	After administration	Number of cases	Before administration	After administration
	SBP (KPa) systolic blood pressure	153	19.1±2.4	18.7±2.1	90	19.2±2.2							19±1.8
DBP (Kpa) diastolic blood pressure							153	11.4±1.1	11.3±1.1	90	11.5±1	11.4±0.9
	(HR) Heart Rate	153	76.9±8.9	75.9±7.5	90	75.6±6.5							76.1±7.4

TABLE 16 Effect of active ingredients of Ginkgo biloba extract on liver and kidney function of patients

Item	Test set			Control group
							Number of cases	Before administration	After administration	Number of cases	Before administration	After administration
	Gluaprolactam transferase	123	16.8±6.6	14.8±6.8	60	13.6±5.9							14.3±6
Urea nitrogen							123	5.81±2.07	5.82±3.08	60	5.75±1.34	5.53±1.09
	Creatinine	153	81.8±24.1	78.2±20.3	90	84.7±18.3							84±20.4

As can be seen from the above description, the present invention provides a novel composition of ginkgo biloba extract, a method for preparing the composition, and a method for detecting various components in the composition. The composition can be used as food additive or added into beverage, and also can be added into cream, ointment or raw materials for preparing them. The composition can also be made into oral preparation, injection or cosmetic. The composition of the present invention can be combined with a medicinal carrier to prepare a medicinal composition, so that the medicinal composition can treat angina pectoris, ischemic heart disease and palpitation of different degrees and different types caused by coronary heart disease, reduce platelet aggregation, improve exercise tolerance and prolong exercise endurance, and treat impotence, psoriasis, pigmentation and other diseases.

Claims (2)

1. A method for determining the content of total flavonoids in a ginkgo leaf composition comprises the following steps:

a) dissolving dried rutin in 70% ethanol to obtain standard solution;

b) transferring different amounts of standard solutions into volumetric flasks, adding 3ml of water, 2ml of acetic acid-sodium acetate buffer solution with pH 4.5 and 2ml of 0.1M aluminum chloride into each volumetric flask, adding the solution to 10ml with 70% ethanol, and drawing a standard curve with an absorption trace point at 270nm of each sample;

c) the test was carried out according to spectrophotometry, the composition being dissolved in 70% ethanol and a certain amount of the solution being transferred in a volumetric flask, the rest being as above: and (3) measuring the absorption at 270nm, obtaining the concentration of the sample according to a standard curve, and taking the total flavone content in an anhydrous state as 85-115% of the labeled amount in terms of rutin.

2. The method according to claim 1, wherein said rutin monomer is used as a standard to determine the total flavonoid content of the ginkgo biloba leaf composition.