Ginkgo biloba extract for promoting blood flow and method of making same

Abstract

The application belongs to the technical field of medicine, and particularly relates to a ginkgo biloba extract for promoting blood flow and a preparation method thereof. The preparation method of the ginkgo biloba extract comprises the following steps: (1) first, ginkgo biloba leaves are crushed, supercritical CO2 extraction is carried out, and an extract is collected to obtain a crude extract; (2) then, the crude extract, an enzyme preparation and water are mixed and left to stand to obtain an enzymatic hydrolysate; and (3) finally, the enzymatic hydrolysate is subjected to X-5 macroporous resin adsorption and elution, and dried to obtain the ginkgo biloba extract. The enzyme preparation is a mixture of beta-glucosidase, amino acid oxidase and alpha-amylase. The enzyme preparation of beta-glucosidase, amino acid oxidase and alpha-amylase in a specific ratio is mixed with the crude extract, the flavonoid glycoside content in the ginkgo biloba extract is improved, and the flavonoid glycoside includes quercetin, kaempferol and isorhamnetin in a specific ratio, and the blood rheology can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a ginkgo leaf extract that promotes blood flow and its preparation method. Background Technology

[0002] Ginkgo biloba is one of the oldest plants on Earth, possessing unique pharmacological and therapeutic value. Ginkgo leaf extract contains flavonoids, terpene lactones, polyphenols, organic acids, polyisoamyl alcohol, and various trace elements, exhibiting antioxidant, anti-platelet-activating factor (PAF) activity, lipid-lowering, and blood sugar-lowering effects. It also improves cardiovascular and cerebral blood circulation and has clinical effects on the central nervous, digestive, and respiratory systems. Ginkgolides and ginkgo flavonoids are the main active components of ginkgo leaf extract. Ginkgolides are diterpenoid ester compounds, including ginkgolides A, B, C, J, and M (GKA, GKB, GKC, GKJ, GKM). Clinical and pharmacological studies have shown that ginkgo flavonoids can dilate microvessels and increase blood flow to the skin. Ginkgolides have a specific antagonistic effect on PAF, inhibiting inflammation and pruritus caused by PAF. Ginkgo biloba extract has the effects of enhancing cardiac function, improving microcirculation, reducing blood viscosity, antagonizing platelet-activating factor, scavenging free radicals in the body and significantly dissolving fibrinogen, and enhancing the heart's tolerance to ischemia.

[0003] Reported processing technologies for Ginkgo biloba extract include: enzymatic method, hot maceration method, microwave extraction method, supercritical carbon dioxide extraction method, and water or organic solvent method.

[0004] Chinese invention patent application CN106176838A discloses a method for producing ginkgo leaf extract. The method includes the following steps: pulverizing the raw material and then packing it into a chromatography column with a diameter-to-height ratio of 1:3-5; extracting the extract with ethyl acetate by countercurrent flow from the bottom to the top of the chromatography column under pressure to obtain an extract; concentrating the extract under vacuum; extracting the concentrate with 4-6 times the weight of distilled water relative to the raw material, adding 2-4% diatomaceous earth by weight of the raw material, filtering to obtain a filtrate; loading the filtrate onto a macroporous adsorption resin, with a resin volume to raw material weight (L:kg) ratio of 1:3-5, then eluting with 60-70% ethanol (3 BV) to obtain an eluent; adding a mixture of activated carbon, gelatin, and diatomaceous earth, stirring thoroughly, cooling, centrifuging, filtering, concentrating, and drying to obtain the finished product. The ginkgo leaf extract produced by this invention contains ≥30% flavonol glycosides, ≥9% ginkgolides, and ≤1 ppm ginkgolic acid.

[0005] Another Chinese invention patent application CN106038619A discloses a ginkgo leaf extract and its preparation method, including the following steps: (1) Preparation of ginkgo leaf powder: remove impurities from ginkgo leaves and dry them, then crush the dried ginkgo leaves and sieve them; (2) Supercritical CO2 extraction: add the ginkgo leaf powder obtained in step (1) into a CO2 supercritical extractor, add anhydrous ethanol as an entrainer, and perform supercritical extraction. After extraction, collect the extract to obtain a crude ginkgo leaf extract solution; (3) Pretreatment of crude ginkgo leaf extract: filter the crude ginkgo leaf extract solution, concentrate it, dilute it with ethanol solution, cool it at 3-5℃ for 10-14 hours, filter it again, and prepare the column liquid; (4) Purification with macroporous resin: pass the column liquid through an AB-8 resin column, first elute with pure water, then elute the resin column with ethanol solution, and collect the eluent; (5) Drying the ginkgo leaf extract into powder: concentrate the eluent by rotary evaporation and vacuum dry it to obtain the ginkgo leaf extract powder product.

[0006] However, the main problems with the above technologies are: high production costs, significant environmental impact, low content of extracted effective components, unreasonable process routes, and difficulty in achieving large-scale industrial production.

[0007] Therefore, there is an urgent need to provide a preparation process for ginkgo leaf extract that has a high content of effective ingredients, is environmentally friendly, and is suitable for industrial production. Summary of the Invention

[0008] This invention addresses the problems existing in the prior art by providing a ginkgo leaf extract that promotes blood flow and its preparation method.

[0009] A method for preparing a ginkgo leaf extract that promotes blood flow includes the following steps:

[0010] (1) First, crush the ginkgo leaves, extract them by supercritical CO2, collect the extract, and obtain crude ginkgo leaf extract;

[0011] (2) Mix the crude extract of ginkgo leaves, enzyme preparation and water, let stand, and obtain the enzymatic hydrolysate;

[0012] (3) Finally, the enzymatic hydrolysate is adsorbed and eluted with X-5 macroporous resin, and then dried to obtain the final product.

[0013] The enzyme preparation mentioned in step (2) is a mixture of β-glucosidase, amino acid oxidase and α-amylase.

[0014] Preferably, the entrainer for supercritical CO2 extraction in step (1) is an aqueous ethanol solution with a volume fraction of 30-40%, the extraction pressure is 15-25 MPa, the extraction temperature is 40-50℃, and the extraction time is 2-3 h.

[0015] Preferably, the mass-to-volume ratio of the ginkgo leaf to the entrainer is 1 g: 10-20 mL.

[0016] Preferably, the enzyme preparation in step (2) is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 1-3:1-3:1-2, and the standing time is 6-8 h and the standing temperature is 30-50℃.

[0017] Preferably, the mass-to-volume ratio of the crude ginkgo leaf extract, enzyme preparation and water in step (2) is 1 g: 0.001-0.005 g: 2-6 mL.

[0018] Preferably, the adsorption elution in step (3) includes eluting with 1-3 column volumes of water first, and then eluting with 3-6 column volumes of ethanol solution with a volume fraction of 45-55%, collecting the ethanol eluent, and the elution flow rate is 1-3 mL / min.

[0019] Preferably, in step (3), the solid content needs to be concentrated under reduced pressure to 60-70% before drying, the drying temperature is 80-90℃, and the drying time is 12-24 h.

[0020] The present invention also provides a ginkgo leaf extract prepared by the above preparation method, wherein, by mass percentage, the ginkgo leaf extract contains ≥24% total flavonol glycosides, 6-8% total terpene lactones, and less than 1 ppm total ginkgolic acid.

[0021] Preferably, the peak area ratio of quercetin, kaempferol and isorhamnetin in the total flavonol glycosides is 0.9-1.1:1:0.5-0.8.

[0022] The present invention also provides the application of the ginkgo leaf extract prepared by the above preparation method in the preparation of drugs that promote blood flow.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In the preparation process of the extract, the present invention selects an ethanol aqueous solution with a volume fraction of 30-40% as the entrainer for supercritical CO2 extraction, which greatly improves the extraction yield of flavonol glycosides in Ginkgo biloba extract and reduces production costs.

[0025] (2) The present invention uses enzyme preparations of β-glucosidase, amino acid oxidase and α-amylase in a specific ratio to increase the content of flavonol glycosides in Ginkgo biloba extract and obtain Ginkgo biloba extract with a specific ratio of quercetin, kaempferol and isorhamnetin.

[0026] (3) The present invention uses X-5 macroporous resin to purify and separate the enzymatic hydrolysate of ginkgo leaves, which effectively increases the separation efficiency of the effective components in the enzymatic hydrolysate and improves the extraction efficiency of flavonol glycosides in ginkgo leaf extract.

[0027] (4) The present invention provides a ginkgo leaf extract that promotes blood flow. The main components of the extract are quercetin, kaempferol and isorhamnetin. By using a specific ratio of the three, the effect of significantly improving blood rheology can be achieved. Detailed Implementation

[0028] It is worth noting that all raw materials used in this invention are commercially available products. Ethanol is analytical grade and was purchased from Aladdin; n-hexane is analytical grade and was purchased from Aladdin; β-glucosidase, CAS number 9001-22-3, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; amino acid oxidase is D-amino acid oxidase, CAS number 9000-88-8, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; α-amylase, CAS number 9000-90-2, was purchased from Aladdin.

[0029] Example 1

[0030] A method for preparing a ginkgo leaf extract that promotes blood flow, comprising the following steps:

[0031] (1) First, crush and grind the ginkgo leaves, and pass them through an 80-mesh sieve to obtain ginkgo leaf powder.

[0032] (2) Ginkgo leaf powder and entrainer were mixed at a mass-volume ratio of 1 g: 15 mL (entrainer was an ethanol aqueous solution with a volume fraction of 40%) and added to a CO2 supercritical extractor. Supercritical extraction was carried out at an extraction pressure of 20 MPa and an extraction temperature of 45℃ for 2.5 h. The CO2 flow rate was 20 L / h. After extraction, crude extract of ginkgo leaves was obtained.

[0033] (3) Mix the crude extract of ginkgo leaves, enzyme preparation and water in a mass-volume ratio of 1 g: 0.003 g: 3 mL and let stand at 40℃ for 7 h to obtain the enzymatic hydrolysate. The enzyme preparation is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 2:2:1.5.

[0034] (4) Finally, the enzymatic hydrolysate was first adsorbed by X-5 macroporous adsorption resin, then eluted with 2 column volumes of purified water, and then eluted with 4 column volumes of 50% ethanol solution. The ethanol eluent was collected at a flow rate of 2 mL / min, concentrated under reduced pressure to a solid content of 70%, and dried at 85℃ for 18 h to obtain Ginkgo biloba extract.

[0035] Example 2

[0036] A method for preparing a ginkgo leaf extract that promotes blood flow, comprising the following steps:

[0037] (1) First, crush and grind the ginkgo leaves, and pass them through an 80-mesh sieve to obtain ginkgo leaf powder.

[0038] (2) Ginkgo leaf powder and entrainer were mixed at a mass-volume ratio of 1 g: 10 mL (entrainer is an aqueous ethanol solution with a volume fraction of 30%) and added to a CO2 supercritical extractor. Supercritical extraction was carried out at an extraction pressure of 15 MPa and an extraction temperature of 40℃ for 2 h. The CO2 flow rate was 15 L / h. After extraction, crude extract of ginkgo leaves was obtained.

[0039] (3) Mix the crude extract of ginkgo leaves, enzyme preparation and water in a mass-volume ratio of 1 g: 0.001 g: 2 mL and let stand at 30℃ for 8 h to obtain the enzymatic hydrolysate. The enzyme preparation is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 1:1:1.

[0040] (4) Finally, the enzymatic hydrolysate was first adsorbed by X-5 macroporous adsorption resin, then eluted with 3 column volumes of purified water, and then eluted with 3 column volumes of 45% ethanol solution. The ethanol eluent was collected at a flow rate of 2 mL / min, concentrated under reduced pressure to a solid content of 60%, and dried at 80℃ for 24 h to obtain Ginkgo biloba extract.

[0041] Example 3

[0042] A ginkgo leaf extract that promotes blood flow, prepared through the following steps:

[0043] (1) First, crush and grind the ginkgo leaves, and pass them through an 80-mesh sieve to obtain ginkgo leaf powder.

[0044] (2) Ginkgo leaf powder and entrainer were mixed at a mass-volume ratio of 1 g: 20 mL (entrainer is an ethanol aqueous solution with a volume fraction of 30%) and added to a CO2 supercritical extractor. Supercritical extraction was carried out at an extraction pressure of 25 MPa and an extraction temperature of 50℃ for 2 h. The CO2 flow rate was 20 L / h. After extraction, crude extract of ginkgo leaves was obtained.

[0045] (3) Then mix the crude extract of ginkgo leaves, enzyme preparation and water in a mass-volume ratio of 1 g: 0.005 g: 6 mL, and let it stand at 50℃ for 6 h to obtain the enzymatic hydrolysate. The enzyme preparation is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 3:3:2.

[0046] (4) Finally, the enzymatic hydrolysate was first adsorbed by X-5 macroporous adsorption resin, then eluted with 1 column volume of purified water, and then eluted with 6 column volumes of 50% ethanol solution. The ethanol eluent was collected at a flow rate of 2 mL / min, concentrated under reduced pressure to a solid content of 70%, and dried at 90℃ for 12 h to obtain Ginkgo biloba extract.

[0047] Comparative Example 1

[0048] A method for preparing a Ginkgo biloba extract that promotes blood flow differs from Example 1 only in that the entrainer for supercritical CO2 extraction in step (2) is anhydrous ethanol solution.

[0049] Comparative Example 2

[0050] A method for preparing a ginkgo leaf extract that promotes blood flow differs from Example 1 only in that the enzyme preparation in step (3) is a mixture of β-glucosidase and α-amylase in a mass ratio of 1:1.

[0051] Comparative Example 3

[0052] A method for preparing a ginkgo leaf extract that promotes blood flow differs from Example 1 only in that the enzyme preparation in step (3) is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 5:4:1.

[0053] Comparative Example 4

[0054] A method for preparing a Ginkgo biloba extract that promotes blood flow differs from Example 1 only in that X-5 macroporous resin is replaced with D101 macroporous resin in step (4).

[0055] Content determination

[0056] According to the determination method of Ginkgo biloba extract in the 2020 edition of the Chinese Pharmacopoeia, the contents of terpene lactones and total ginkgolic acid in the Ginkgo biloba extracts prepared in Examples 1-3 and Comparative Examples 1-4 were determined. The total ginkgolic acid content was calculated as heptadecanenoic acid C17:1. Quercetin was used as a reference standard to determine the contents of total flavonol glycosides. The peak area ratios of quercetin, kaempferol and isorhamnetin in the total flavonol glycosides were further determined. The results are shown in Table 1 below.

[0057] Table 1. Determination of the content of Ginkgo biloba extract prepared in Examples 1-3 and Comparative Examples 1-4

[0058]

[0059] Experiment 1: Effects of the Ginkgo biloba extract of this invention on blood rheology in hyperlipidemic rats.

[0060] 1. Test samples: Ginkgo biloba extracts prepared in Examples 1-3 and Comparative Examples 1-4 of the present invention.

[0061] 2. Experimental animals: 90 ordinary-grade SD rats, male, weighing 200 ± 20 g. Provided by Shanghai Xipu-Bikai Laboratory Animal Co., Ltd., license number: SCXK (Shanghai) 2008-0016.

[0062] 3. Instruments and reagents: LIANG-100 type blood rheology analyzer; LIANG-100 type erythrocyte electrophoresis analyzer; DXC-300A type erythrocyte deformability analyzer; XJ-F type platelet aggregometer; TDL-5 cell separator for measurement; 0.1% hydrochloric acid adrenaline, anesthetic chloral hydrate, anticoagulant sodium citrate.

[0063] 4. Experimental method: The rats were randomly divided into 9 groups, namely the control group, the model group, Examples 1-3 and Comparative Examples 1-4 groups, with 10 rats in each group. The groups of Examples 1-3 and Comparative Examples 1-4 were given the corresponding products by gavage (dosage 100 mg / kg·BW), and the model group and the control group were given an equal amount of normal saline by gavage for 7 consecutive days. Except for the control group, on the 7th day, 2 hours after administration, 0.1% hydrochloric acid adrenaline injection (0.5 mL / kg) was subcutaneously injected into the other groups, twice in total, with an interval of 5 hours; 2 hours after the first injection, the rats were immersed in ice water for 5 minutes to make an acute blood stasis model. All animals were fasted but not water-deprived. 18 hours later, the rats were anesthetized with chloral hydrate at a mass concentration of 0.1 g / mL, and blood was collected by carotid artery intubation. 5 mL of blood was collected per rat, anticoagulated with sodium citrate at a mass concentration of 0.038 g / mL in a ratio of 1:9, and the indexes of whole blood (high shear, low shear) viscosity, plasma viscosity and hematocrit were measured.

[0064] 5. Experimental results: As shown in Table 2, compared with the control group, the whole blood (high shear, low shear) viscosity, plasma viscosity and hematocrit in the model group were significantly increased ( P <0.05), indicating that the model was established. The Ginkgo biloba extracts prepared in the present invention could significantly reduce the whole blood (high shear, low shear) viscosity, plasma viscosity and hematocrit in hyperlipidemic rats compared with the model group ( P <0.05). From the experimental data of the examples and comparative examples, it can be seen that through the specific ratio of quercetin, kaempferol and isorhamnetin, the effect of significantly improving hemorheology can be achieved. Among the total flavonol glycosides of the Ginkgo biloba extract prepared in Example 1, when the peak area ratio of quercetin, kaempferol and isorhamnetin was 1:1:0.8, the effect of improving hemorheology was the best ( P <0.01).

[0065] Table 2 Changes in hemorheology of each group

[0066]

[0067] Note: Compared with the model group, express P <0.05, express P <0.01; compared with the control group, # express P <0.05.

[0068] Experiment 2: Effects of the Ginkgo biloba extract of this invention on the hemorheology of elderly patients with ischemic cerebrovascular disease.

[0069] 1. Test sample: Ginkgo biloba extract prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0070] 2. Subjects: Cases were selected according to the diagnostic reference criteria of the "Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2018". A total of 50 elderly patients with ischemic cerebrovascular disease were included, including 27 males and 23 females, aged 66-89 years, with a disease duration of 5.8±3.8 years. Before treatment, all patients had abnormal blood rheology.

[0071] 3. Experimental Methods: Fifty elderly patients with ischemic cerebrovascular disease were randomly divided into 5 groups of 10 patients each. Patients received basic antihypertensive, lipid-lowering, and antithrombotic drug treatment for 7 days. In addition to the basic treatment, 50 mg of Ginkgo biloba extract prepared in Example 1 and Comparative Examples 1-4 of this invention was dissolved in 250 mL of 0.9% sodium chloride injection (purchased from Sichuan Pacific Pharmaceutical Co., Ltd.) and administered via slow intravenous drip once daily for 7 consecutive days. Whole blood high-shear viscosity, whole blood low-shear viscosity, and plasma viscosity were measured using a Tianhai fully automated blood rheology analyzer (MVIS-2035). Hematocrit and reticulocyte counts were measured using a fully automated hematology analyzer. Data were processed using SPSS 23.0 statistical software. P <0.05 indicates a statistically significant difference.

[0072] 4. Experimental Results: As shown in Table 3, after taking the Ginkgo biloba extract prepared in Example 1 and Comparative Examples 1-4, the whole blood high-shear viscosity, whole blood low-shear viscosity, plasma viscosity, and reticulocyte count were all lower than before treatment. Among them, the differences in all data in Example 1 were statistically significant. P <0.05), indicating that the Ginkgo biloba extract prepared in this invention has the effect of reducing blood viscosity and improving blood rheology in elderly patients with ischemic cerebrovascular disease.

[0073] Table 3 Comparison of hemorheology before and after treatment

[0074]

[0075] Note: Indicates a comparison with before treatment. P <0.05.

[0076] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a ginkgo biloba leaf extract for promoting blood flow, characterized by, Includes the following steps: (1) First, crush the ginkgo leaves, extract them by supercritical CO2, collect the extract, and obtain crude ginkgo leaf extract; (2) Mix the crude extract of ginkgo leaves, enzyme preparation and water, let stand, and obtain the enzymatic hydrolysate; (3) Finally, the enzymatic hydrolysate is adsorbed and eluted by X-5 macroporous resin and dried to obtain the final product; In step (1), the entrainer for supercritical CO2 extraction is an aqueous ethanol solution with a volume fraction of 30-40%, and in step (2), the enzyme preparation is a mixture of β-glucosidase, amino acid oxidase and α-amylase in a mass ratio of 1-3:1-3:1-2.

2. The preparation method according to claim 1, characterized in that, The extraction pressure in step (1) is 15-25 MPa, the extraction temperature is 40-50℃, and the extraction time is 2-3 h.

3. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the ginkgo leaf to the entrainer is 1 g: 10-20 mL.

4. The preparation method according to claim 1, characterized in that, The standing time in step (2) is 6-8 hours, and the standing temperature is 30-50℃.

5. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the crude extract of ginkgo leaves, enzyme preparation and water in step (2) is 1 g: 0.001-0.005 g: 2-6 mL.

6. The preparation method according to claim 1, characterized in that, The adsorption elution in step (3) includes eluting with 1-3 column volumes of water, followed by elution with 3-6 column volumes of ethanol solution with a volume fraction of 45-55%, and collecting the ethanol eluent. The elution flow rate is 1-3 mL / min.

7. The preparation method according to claim 1, characterized in that, Before drying in step (3), the solid content needs to be concentrated under reduced pressure to 60-70%, the drying temperature is 80-90℃, and the drying time is 12-24 h.

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