CN109394800B - Ginkgo leaf extract, injection containing the same and quality control method thereof - Google Patents

Ginkgo leaf extract, injection containing the same and quality control method thereof Download PDF

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CN109394800B
CN109394800B CN201811357726.5A CN201811357726A CN109394800B CN 109394800 B CN109394800 B CN 109394800B CN 201811357726 A CN201811357726 A CN 201811357726A CN 109394800 B CN109394800 B CN 109394800B
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ginkgo
leaf extract
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ginkgo biloba
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胡金
劳芳
董官波
王正彬
孔令伟
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Deng Shengju
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Deng Shengju
Honghe Pharmaceutical Co ltd
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Abstract

The invention relates to a ginkgo biloba extract, an ligustrum lucidum sodium chloride injection containing the extract and a quality control method of the ginkgo biloba extract and the ligustrum lucidum sodium chloride injection. The ginkgo leaf extract contains 24.0-37.0% of total flavonol glycosides and 6.0-12.0% of terpene lactones according to dry product. The ligustrazine phosphate and the ginkgo leaf extract are contained in the ligustrazine phosphate and the ginkgo leaf extract, the content of the ligustrazine phosphate is 0.36-0.44 mg in each 1ml of the injection, and the content of the ginkgo leaf extract is such that the total flavonol glycosides and terpene lactones are 36-44 mug and 6.0-13.0 mug in each 1ml of the injection.

Description

Ginkgo leaf extract, injection containing the same and quality control method thereof
Technical Field
The present invention relates to ginkgo leaf extract, injection containing the extract and their quality control method.
Background
In China, the prevalence rate and the death rate of cardiovascular diseases are on the trend of rising year by year. The number of patients suffering from cardiovascular diseases is 2.9 million, wherein 1300 million stroke, 1100 million coronary heart disease and 450 million heart failure are calculated. The cardiovascular disease mortality rate is the top, higher than that of tumors and other diseases, accounting for more than 40% of the resident disease deaths, especially in rural areas, and is continuously higher than the urban level in recent years. Ischemic cardiovascular and cerebrovascular diseases such as blood supply insufficiency, cerebral thrombosis, cerebral embolism, angina, myocardial infarction, ischemic stroke and the like are one of the important lethal disease types.
The practice of the present inventor for several years proves that the Xinxiong sodium chloride injection which is developed and invented before has a significant curative effect for treating ischemic cardiovascular and cerebrovascular diseases. The main component of the injection is folium Ginkgo extract, which is derived from folium Ginkgo.
Although China is a big resource country of ginkgo leaves and the preparation of ginkgo extracts is well known, the components of products obtained by the traditional Chinese medicine extraction method are complex, and the traditional means for performing quality control by taking single components as indexes can not adapt to the requirements of the quality control of the traditional Chinese medicines more and more. In the process of modernization of traditional Chinese medicine, the improvement of the quality standard of traditional Chinese medicine becomes the bottleneck of the development of traditional Chinese medicine, and the modernization of the quality standard is the premise of improving the international competitiveness.
With the improvement of the quality control level of the medicine production in China and the continuous deepening of the attention degree on the problems of the safety, the curative effect, the controllable quality and the like of the injection, the national food and drug administration successively proposes a 'national medicine standard improvement action plan' and 'traditional Chinese medicine injection safety reevaluation work' to comprehensively improve the quality control level of the medicine.
Disclosure of Invention
The invention aims to provide a ginkgo leaf extract with more stable quality and less toxic components, an apricot cnidium sodium chloride injection containing the extract and a quality control method of the ginkgo leaf extract and the apricot cnidium sodium chloride injection.
Specifically, the present invention relates to:
(1) folium Ginkgo extract, wherein the content is calculated according to dry productThe ginkgo biloba extract contains 24.0-37.0% of total flavonol glycoside and 6.0-12.0% of terpene lactone, wherein the content of the total flavonol glycoside is (the content of quercetin, the content of kaempferide and the content of isorhamnetin) multiplied by 2.51, and the content of the terpene lactone is bilobalide (C)15H18O8) Ginkgolide A (C)20H24O9) Ginkgolide B (C)20H24O10) And ginkgolide C (C)20H24O11) To the total amount of (c).
(2) The ginkgo leaf extract according to the above (1), wherein rutin (C)27H30O16) Content of not more than 4.0%, free quercetin (C)15H10O7) The content is not more than 0.4%, preferably not more than 0.2%.
(3) The ginkgo leaf extract according to the above (1) or (2), wherein the heavy metal content is: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg.
(4) The ginkgo biloba leaf extract according to any one of the above (1) to (3), wherein the total ginkgolic acid content is not more than 2mg/kg, preferably not more than 1mg/kg, and the total ginkgolic acid content is ginkgolic acid C13:0 (C)20 H32O3) Ginkgolic acid C15:1 (C)22H34O3) Ginkgolic acid C17:1 (C)24H38O3) To the total amount of (c).
(5) The ginkgo biloba leaf extract of any one of the above (1) to (4), which has a fingerprint having a similarity of not less than 0.90, more preferably not less than 0.93, as compared with the control fingerprint shown in fig. 1, under the same detection conditions as the control fingerprint shown in fig. 1, the similarity being evaluated by the "evaluation software for chromatographic fingerprint similarity of traditional Chinese medicine" version 2.0 ".
(6) A quality control method of folium Ginkgo extract comprises controlling the content of total flavonol glycosides in folium Ginkgo extract at 24.0-37.0% and the content of terpene lactones at 6.0-12.0%.
(7) The method of (6) above, further comprising detecting a fingerprint of the ginkgo biloba extract under the same detection conditions as the control fingerprint shown in fig. 1, and evaluating the similarity using the "evaluation software for similarity of chromatography fingerprints of chinese traditional medicine version 2.0", wherein the similarity of the fingerprint of the extract to the control fingerprint shown in fig. 1 is not less than 0.90, more preferably not less than 0.93.
(8) The method according to any one of (6) or (7), further comprising controlling the content of rutin in the ginkgo biloba extract to be 4.0% or less, and the content of free quercetin to be 0.4% or less, more preferably 0.2% or less.
(9) The method of any one of (6) to (8) above, further comprising controlling the total ginkgolic acid content in the ginkgo biloba extract to be 2mg/kg or less, more preferably 1mg/kg or less.
(10) The method of any one of (6) to (9) above, further comprising controlling the content of heavy metals in the ginkgo biloba extract to be: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg.
(11) The injection contains ligustrazine phosphate and the ginkgo leaf extract in any one of the items (1) to (5), wherein the content of the ligustrazine phosphate is 0.36-0.44 m g in each 1ml of the injection, and the content of the ginkgo leaf extract is 36-44 mu g of total flavonol glycosides and 6.0-13.0 mu g of terpene lactones in each 1ml of the injection.
(12) Under the same detection conditions as the comparison fingerprint shown in the figure 2, the injection of the Xinxiong sodium chloride injection in the (11) has the fingerprint with the similarity not lower than 0.90, more preferably not lower than 0.93 compared with the comparison fingerprint shown in the figure 2, and the similarity is evaluated by the traditional Chinese medicine chromatogram fingerprint similarity evaluation software version 2.0.
(13) A quality control method of the ligustrum lucidum sodium chloride injection comprises the step of using the ginkgo leaf extract in any one of the steps (1) to (5).
(14) The method in (13) above, further comprising detecting fingerprint of the Xinxiong sodium chloride injection under the same detection conditions as the control fingerprint shown in fig. 2, and evaluating the similarity with the traditional Chinese medicine chromatography fingerprint similarity evaluation software version 2.0, wherein the similarity between the fingerprint of the injection and the control fingerprint shown in fig. 2 is not less than 0.90, and more preferably not less than 0.93.
Drawings
FIG. 1 is a fingerprint of Ginkgo biloba extract.
FIG. 2 is a fingerprint of the injection containing the rhizome of apricot and the sodium chloride.
FIG. 3 is a fingerprint of Ginkgo biloba extract having a total flavonol glycoside content outside the scope of the present invention.
FIG. 4 is a fingerprint of Ginkgo biloba extract having a total flavonol glycoside content within the scope of the present invention.
Detailed Description
In the present application, "%" represents a mass percentage content unless otherwise specified.
The ginkgo leaf extract contains 24.0-37.0 percent of total flavonol glycosides and 6.0-12.0 percent of terpene lactones according to the dry product. Wherein the total flavonol glycoside content is (quercetin content + kaempferide content + isorhamnetin content) × 2.51, and the terpene lactone content is bilobalide (C)15H18O8) Ginkgolide A (C)20H24O9) Ginkgolide B (C)20H24O10) And ginkgolide C (C)20H24O11) To the total amount of (c).
In the past, the content of total flavonoids in ginkgo biloba extract is generally evaluated and controlled by measuring the content of quercetin, kaempferide and isorhamnetin after acid hydrolysis, but the method cannot sufficiently reflect the real and accurate content of the flavonoids, so that the quality problem of artificially adding rutin, quercetin and other cheap raw materials to improve the content of the total flavonoids is caused.
The current Chinese pharmacopoeia limits the level of the total flavonol glycoside content in the ginkgo extract to be not less than 24.0 percent, but the inventor of the application finds that the higher the content is, the better the content is, and when reaching a certain level, the similarity of the fingerprint of the extract and the comparison fingerprint is in a descending trend. In other words, the inventors found that there is a correlation between the total flavonol glycoside content and the similarity of fingerprint pattern. Fingerprint spectrum is an effective means for reflecting the internal quality of the extract as a whole. The similarity of the fingerprint spectrum does not reach the specified level, which means that the overall quality of the product is not normal, and artificial adulteration can be involved. The inventor of the application finds that the similarity of the fingerprint spectrum and the comparison fingerprint spectrum of the ginkgo leaf extract reaches a low level of 0.693 and is far lower than the generally accepted similarity requirement of more than 0.90 when the content of the total flavonol glycosides is 41.04 percent, for example. Therefore, the content of total flavonol glycosides in the ginkgo biloba extract should be set below a certain level. Based on the above correlation studies, the present inventors determined that the total flavonol glycoside content in the ginkgo leaf extract should not exceed 37% by considering the general total flavonol glycoside content range in the natural ginkgo leaf extract.
The inventor of the application determines the upper limit of the total flavonol glycoside content by researching the correlation degree between the similarity of the fingerprint and the total flavonol glycoside content, and the method can control the total flavonol glycoside content in natural products in a reasonable range and distinguish the natural products from artificially added products, thereby more effectively controlling and ensuring the quality of the ginkgo leaf extract.
For terpene lactones as another active ingredient in ginkgo leaf extract, the content of terpene lactones in the ginkgo leaf extract is not less than 6.0% as specified in the current Chinese pharmacopoeia. The inventor of the application finds that when the ginkgo biloba extract is used in a medicament injection, if the terpene lactone content in the ginkgo biloba extract is higher than 12.0%, the problems of turbidity and unqualified clarity of the injection can be caused. Therefore, in the invention, the total amount of terpene lactones in the ginkgo biloba extract is controlled to be less than 12.0 percent.
In a preferred embodiment, rutin (C) is contained in the ginkgo biloba extract of the present invention27H30O16) Content of free quercetin (C) below 4.0%15H10O7) The content is 0.4% or less, more preferably 0.2% or less.
The inventor of the application finds that the excessive free quercetin in many ginkgo biloba extracts is serious. This may be due to illegal manufacturers adding cheap quercetin or rutin in order to reach the total flavone content, which is hydrolyzed to produce quercetin. Thus, it was determined that the contents of rutin and free quercetin in the ginkgo biloba extract should be controlled within the above-specified ranges.
In a more preferred embodiment, the content of heavy metals in the ginkgo biloba extract of the present invention is: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg.
With the increasing environmental pollution, the content of heavy metals in the traditional Chinese medicinal materials is increased day by day, and the health of people is seriously harmed. In the present invention, the content of heavy metals in the ginkgo biloba extract is preferably controlled within the above range, thereby further improving the safety of the product.
Further preferably, the total ginkgolic acid content in the ginkgo biloba extract of the invention is not more than 2mg/kg, more preferably not more than 1 mg/kg. The total ginkgolic acid content is ginkgolic acid C13:0 (C)20H32O3) Ginkgolic acid C15:1 (C)22H34O3) Ginkgolic acid C17:1 (C)24H38O3) To the total amount of (c).
Ginkgolic acid is a sensitizing and mutagenic toxic component in ginkgo biloba extract and is related to serious adverse reactions. At present, the limit of total ginkgoic acid in Chinese pharmacopoeia is lower than 10mg/kg, but the international generally accepted standard is lower than 5mg/kg, so that most of ginkgoic preparations in China are exported and limited due to higher content of ginkgoic acid.
In addition, particularly when ginkgo biloba extract is used in pharmaceutical injections, it is desirable to control the amount of total ginkgolic acid to as low a level as possible. The presently widely used dilute alcohol extraction method is difficult to achieve at a lower level, and therefore, water extraction methods have been attempted. However, the water extraction method is inferior to the dilute alcohol extraction method in the kinds and the extraction amount of the extract, and the effect of the product is affected.
In the invention, the total ginkgolic acid content in the ginkgo biloba extract can be controlled below 2mg/kg, more preferably below 1mg/kg by using high-concentration ethanol for extraction and eluting by using macroporous adsorption resin, which is far lower than the current average extraction level limit at home and abroad, so that the safety of the product is further improved, particularly the safety when the product is used for a pharmaceutical injection, and the ginkgo biloba extract and related products in China have more international competitiveness.
The ginkgo leaf extract can be used in foods, health products, medicines, cosmetics and the like, and can be processed with common auxiliary materials in the fields to prepare required forms, such as oral capsules, dripping pills and the like.
The ginkgo biloba extract of the present invention may also be used in combination with other active or active ingredients.
The invention also comprises an ligustrazine phosphate sodium chloride injection, which contains ligustrazine phosphate and the ginkgo leaf extract, wherein the content of the ligustrazine phosphate is 0.36-0.44 m g in each 1ml of the injection, and the content of the ginkgo leaf extract is 36-44 mu g of total flavonol glycosides and 6.0-13.0 mu g of terpene lactones in each 1ml of the injection.
The injection contains total flavonol glycoside (quercetin content + kaempferide content + isorhamnetin content) × 2.51, and terpene lactone (C) as bilobalide (as described in folium Ginkgo extract)15H18O8) Ginkgolide A (C)20H24O9) Ginkgolide B (C)20H24O10) And ginkgolide C (C)20H24O11) To the total amount of (c).
The ligustrum lucidum and sodium chloride injection can be prepared by a known method.
In a preferred embodiment, the ginkgo biloba leaf extract of the present invention has a fingerprint having a similarity of not less than 0.90, more preferably not less than 0.93, as compared to the control fingerprint shown in fig. 1, under the same detection conditions as the control fingerprint shown in fig. 1, and the similarity is evaluated by the "evaluation software for chromatographic fingerprint similarity of traditional Chinese medicine" version 2.0 ".
In another preferred embodiment, the Xinxiong sodium chloride injection disclosed by the invention has a fingerprint with the similarity of not less than 0.90, more preferably not less than 0.93 compared with the control fingerprint shown in figure 2 under the same detection condition as the control fingerprint shown in figure 2, and the similarity is evaluated by using traditional Chinese medicine chromatogram fingerprint similarity evaluation software version 2.0.
In the invention, the fingerprint spectrums of the ginkgo biloba extract and the ligustrum lucidum sodium chloride injection containing the extract can be measured by high performance liquid chromatography according to 0512 of the four ministry of communications in 2015 version of Chinese pharmacopoeia.
In the fingerprint spectrums of the ginkgo biloba extract and the ligustrum japonicum sodium chloride injection containing the extract, characteristic peaks of rutin are obvious, characteristic peaks of other ginkgo flavonoid components exist near rutin chromatographic peaks, and the response of the chromatographic peaks is high, so that rutin is selected as a reference peak to determine the relative peak-appearing positions of the other characteristic components.
Specifically, in the present invention, the control fingerprints shown in fig. 1 and 2 are obtained as follows:
fingerprint spectra of 10 batches of ginkgo biloba extract or 10 batches of the ligustrum lucidum sodium chloride injection containing the extract are respectively detected under the following conditions, the fingerprint spectra are led into the traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation software version 2.0, and 11 Mark peaks with the Mark peak content of more than 2 percent and the Mark peaks accounting for more than 80 percent of all characteristic components in the fingerprint spectra are selected to generate a contrast fingerprint spectra.
Chromatographic conditions are as follows: using octadecylsilane chemically bonded silica as filler (column length 15cm, inner diameter 4.6mm, particle diameter 3.5 μm), acetonitrile as mobile phase A, 5% formic acid solution as mobile phase B, and performing gradient elution according to the specification in Table 1 below; the flow rate was 1.0ml per minute; the column temperature is 20 +/-1 ℃; the detection wavelength was 360 nm. The theoretical plate number is not less than 50000 calculated according to rutin peak.
TABLE 1
Figure RE-GDA0001906217520000061
Adopting LC-Q-TOF to confirm the selected Mark peak, deducing that the structures of the compounds are glycoside conjugates of different forms of quercetin, kaempferide and isorhamnetin, and specifically comprising the following steps: 1. the trisaccharide glycoside of quercetin; 2. trisaccharide glycosides of kaempferol; 3. kaempferol 3-rutinose-7-rhamnoside; 4. rutin; 5. the diglycoside of quercetin; 6. quercetin-3-O- (2 "- β -D-glucose) - α -L-rhamnoside; 7. kaempferol-3-O-rutinoside; 8. isorhamnetin-3-rutinoside; 9. kaempferol-3-O- (2 "- β -D-glucose) - α -L-rhamnoside; 10. quercetin-3-O- (2 ", 6" -alpha-L-dirhamnose) -beta-D-glucoside; 11. kaempferol-3-O-alpha-L-rhamnose-2 '- (6' -p-coumaroyl) -beta-D-glucoside. If glucoside hydrolysis is changed due to factors such as process abnormality, the fingerprint spectrum is likely to be greatly changed, so that the selected Mark peaks have characteristic significance.
The fingerprint of the ginkgo leaf extract is detected as follows:
chromatographic conditions are as follows: as above.
Preparation of reference solutions: taking an appropriate amount of rutin control, precisely weighing, and adding methanol to obtain a solution containing 20 μ g per 1 ml.
Preparation of a test solution: taking about 20mg of the ginkgo biloba extract, precisely weighing, adding 10ml of 75% methanol, carrying out ultrasonic treatment (power 300W and frequency 50KHz) for 10 minutes, centrifuging (4000 rpm) for 5 minutes, and taking supernatant to obtain the ginkgo biloba extract.
The determination method comprises the following steps: precisely sucking 5 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram.
The fingerprint of the test sample is measured under the same condition as the comparison fingerprint, and the similarity of the test sample and the comparison fingerprint is evaluated by using the traditional Chinese medicine chromatogram fingerprint similarity evaluation software 2.0 edition.
The fingerprint of the test sample should show chromatographic peaks consistent with the retention time of the chromatographic peaks of the rutin reference substance, and should show 11 common peaks. Mark peak matching is carried out on the 11 common peaks, and the similarity is not lower than 0.90, and preferably not lower than 0.93.
The fingerprint of the ligustrum lucidum sodium chloride injection containing the ginkgo leaf extract is detected as follows:
chromatographic conditions are as follows: as above.
Preparation of reference solutions: taking appropriate amount of rutin control and ligustrazine phosphate control, accurately weighing, and adding methanol to obtain control solutions each containing 20 μ g per 1 ml.
Preparation of a test solution: and (3) taking a proper amount of the ligustrum lucidum and sodium chloride injection to obtain the injection.
The determination method comprises the following steps: precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram.
After screening the chromatographic peak of ligustrazine phosphate, the fingerprint of the ligustrazine phosphate injection should have chromatographic peaks which are consistent with the retention time of the chromatographic peak of the rutin reference substance, and should have 11 common peaks. Mark peak matching is carried out on the 11 common peaks, and the similarity is not lower than 0.90, and preferably not lower than 0.93.
Examples
The present invention is described below by way of specific examples, but the scope of the present invention is not limited to these examples, and modifications made by those skilled in the art without departing from the spirit and scope of the present invention are within the scope of the present invention.
EXAMPLE 1 preparation and assay of Ginkgo biloba leaf extract
(I) Preparation of
Taking ginkgo leaf decoction pieces, heating and refluxing the ginkgo leaf decoction pieces by using 70-80% ethanol for 2 times, wherein the first time is 2 hours, and the second time is 1 hour. Filtering the extracting solution, concentrating to obtain clear paste with the relative density of 1.05-1.25 (60 +/-2 ℃), precipitating with water for more than 8 hours, filtering, passing the filtrate through a macroporous adsorption resin column with the particle size of 0.12mm, eluting with water and 30-80% ethanol solution in sequence, discarding the washing solution, eluting with 30-80% ethanol solution, passing the eluent through another macroporous adsorption resin column, collecting the effluent, concentrating to a proper amount, refrigerating, filtering, adding the filtrate to a third macroporous adsorption resin column, washing with water, discarding the washing solution, eluting with 30-80% ethanol solution, collecting the eluent, concentrating to a proper amount, refrigerating (0-10 ℃), filtering, passing the filtrate through the macroporous adsorption resin column again, eluting with 30-80% ethanol solution, concentrating the eluent to the relative density of 1.05-1.25 (60 +/-2 ℃), drying, refining, concentrating, drying again, and crushing to obtain light brown to brownish powder.
Multiple batches of ginkgo biloba extract were prepared by the above method.
(II) content determination of total flavonol glycosides, terpene lactones, rutin and free quercetin
The content is determined by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia).
(i) Total flavonol glycosides were determined according to "protocol for high performance liquid chromatography.
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.4% phosphoric acid solution (50: 50) is used as a mobile phase; the detection wavelength was 360 nm. The number of theoretical plates should not be lower than 2500 calculated from the peak of quercetin. The separation degree of the kaempferol peak and the isorhamnetin peak is more than 1.5.
Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 30ug per 1 ml.
Preparation of a test solution: taking about 35mg of the ginkgo leaf extract prepared by the method, precisely weighing, adding 25ml of mixed solution of methanol-25% hydrochloric acid solution (prepared according to the concentration ratio) (4: 1), placing in a water bath, heating and refluxing for 30 minutes, quickly cooling to room temperature, transferring to a 50ml measuring flask, diluting to a scale with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the ginkgo leaf extract.
The determination method comprises the following steps: respectively and precisely sucking 10 μ l of reference solution and test solution, injecting into a liquid chromatograph, measuring, respectively calculating the content of quercetin, kaempferide and isorhamnetin according to the correction factors in the following table 2, and determining the peak positions of quercetin, kaempferide and isorhamnetin according to the relative retention time of the chromatographic peak of the component to be measured and the chromatographic peak of quercetin, wherein the relative retention time is within +/-5% of a specified value (if the relative retention time deviates more than 5%, the corresponding reference is determined to be the standard). The relative retention times and correction factors (F) are shown in table 2 below:
TABLE 2
Figure RE-GDA0001906217520000091
Total flavonol glycoside content (quercetin content + kaempferol content + isorhamnetin content) × 2.51
The multi-batch ginkgo leaf extract prepared by the method contains 27.0 to 34.0 percent of total flavonol glycosides according to dry products.
(ii) Terpene lactones were measured according to "protocol for high performance liquid chromatography.
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the method comprises the following steps of mixing methanol: 0.1% formic acid (29:71) as mobile phase; detection was by evaporative light scattering detector. The number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide.
Preparation of control solutions: respectively taking appropriate amount of bilobalide reference substance, bilobalide A reference substance, bilobalide B reference substance and bilobalide C reference substance, precisely weighing, and adding methanol to obtain mixed solution containing 1.2mg, 0.6mg and 0.3mg per 1 ml; or accurately weighing appropriate amount of folium Ginkgo total lactone control extract, and adding methanol to obtain solution containing 2.5mg per 1 ml.
Preparation of a test solution: taking about 0.15g of the ginkgo biloba extract prepared in the invention, precisely weighing, adding 10ml of water, heating in a water bath to dissolve, adding 2 drops of 2% hydrochloric acid solution, shaking and extracting for 4 times (15ml, 10ml, 10ml) by ethyl acetate, combining the extracting solutions, washing by 20ml of 5% sodium acetate solution, dividing the sodium acetate solution, washing by 10ml of ethyl acetate, combining the ethyl acetate extracting solution and the washing solution, washing by water for 2 times, each time for 20ml, dividing the water solution, washing by 10ml of ethyl acetate, combining the ethyl acetate solutions, recovering the solvent to be dry, dissolving the residue by methanol, transferring to a 5ml measuring flask, adding the methanol to the scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the ginkgo biloba extract.
The determination method comprises the following steps: precisely sucking 3-10 μ l of the reference extract solution and 5-10 μ l of the sample solution, respectively, injecting into a liquid chromatograph, measuring, and calculating the contents of bilobalide, bilobalide A, bilobalide B and bilobalide C by using an external standard two-point method logarithmic equation.
The invention is as followsThe dried product of the above multiple batches of folium Ginkgo extract is calculated by bilobalide (C)15H18O8) Ginkgolide A (C)20H24O9) Ginkgolide B (C)20H24O10) Ginkgolide C (C)20H24O11) The terpene lactone content is 7.0-11.0% of the total amount of the terpene lactone.
(iii) Rutin and free quercetin are measured according to the 'operating procedure for measuring by high performance liquid chromatography'.
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, and 0.4% phosphoric acid solution is taken as a mobile phase B; gradient elution was performed as specified in table 3 below; the detection wavelength was 400 nm. The number of theoretical plates is not less than 8000 according to rutin peak.
TABLE 3
Figure RE-GDA0001906217520000101
Preparation of control solutions: respectively weighing appropriate amount of rutin control and quercetin control, precisely weighing, and adding methanol to obtain mixed solution containing rutin 250 μ g and quercetin 25 μ g per 1 ml.
Preparation of a test solution: taking 0.1g of the ginkgo biloba extract prepared by the method, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of a mixed solution of 70% methanol and 70% ethanol (1: 1), oscillating and extracting for 30 minutes, taking out, centrifuging, and taking supernatant; or filtering to obtain filtrate.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The multi-batch ginkgo leaf extract prepared by the invention contains rutin (C) calculated according to dry products27H30O16) The highest content is about 3.7%, not more than 4.0%, and contains free quercetin (C)15H10O7) Up to 0.18% and not more than 0.20%.
(III) determination of heavy Metal content
Referring to the general rule 2321 of the four parts of the pharmacopoeia 2015 edition, five heavy metals of lead, arsenic, mercury, cadmium, copper and the like in multiple batches of ginkgo biloba extracts prepared by the method are detected by an ICP-MS method, and as a result, cadmium, mercury and lead are not detected in each batch, the arsenic content is less than 0.07mg/kg, and the copper content is less than 0.25 mg/kg.
(IV) determination of Total Ginkgo acid content
According to the general rules 0512 and 0431 of the four departments of the version of 'Chinese pharmacopoeia' 2015, the determination is carried out by a high performance liquid chromatography-mass spectrometry method.
Chromatographic mass spectrum conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking a methanol-1% glacial acetic acid solution (90:10) as a mobile phase, and fully cleaning at least 10 times of column volume by using the methanol-1% glacial acetic acid solution (99:1) after all components to be detected peak; a triple quadrupole mass spectrometer detector was used, multi-reaction monitoring (MRM) in electrospray ionization (ESI) negative ion mode, and the monitored ion pairs are shown in table 4 below.
TABLE 4
Figure RE-GDA0001906217520000111
Preparation of control solutions: respectively taking appropriate amount of ginkgolic acid C13:0 reference substance, ginkgolic acid C15:1 reference substance and ginkgolic acid C17:1 reference substance, precisely weighing, and adding methanol to obtain series of mixed solutions respectively containing 0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml and 50ng/ml as reference substance solutions.
Preparation of a test solution: taking 0.4g of the ginkgo biloba extract prepared by the method, precisely weighing, placing the ginkgo biloba extract in a conical flask with a plug, precisely adding 10ml of methanol, weighing, carrying out ultrasonic treatment (power 180W and frequency 42kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the ginkgo biloba extract.
The determination method comprises the following steps: precisely sucking 1 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatogram-mass spectrometer for measurement.
If a chromatographic peak is detected in the sample solution at the same retention time as that of the control, and the ion abundance ratio of the selected ion abundance ratio and the ion abundance ratio of the control solution at the corresponding concentration satisfy the following table 5, it can be determined that the sample solution has the component.
TABLE 5
Figure RE-GDA0001906217520000121
And (3) taking the peak area of the reference substance quantitative ion pair as a vertical coordinate and the reference substance concentration as a horizontal coordinate, drawing a standard curve, reading the amount of the ginkgolic acid C13:0, the ginkgolic acid C15:1 and the ginkgolic acid C17:1 in the test solution from the standard curve, and calculating to obtain the test solution.
The multi-batch ginkgo extract prepared by the invention is prepared by mixing ginkgolic acid C13:0 (C)20H32O3) Ginkgolic acid C15:1 (C)22H34O3) Ginkgolic acid C17:1 (C)24H38O3) The content of the total ginkgoic acid is 0.7mg/kg at most, and is not more than 1 mg/kg.
(V) determination of fingerprint and evaluation of similarity
The fingerprints of the multiple batches of ginkgo biloba extracts prepared by the method are measured according to the method in the specific embodiment, and similarity evaluation is carried out, and the result shows that the similarity between the fingerprints of the multiple batches of ginkgo biloba extracts and the comparison fingerprint shown in fig. 1 is more than 0.93.
Example 2 determination of Total Flavoneglycoside content Range in Ginkgo biloba leaf extract
The content of total flavonol glycosides in the ginkgo leaves and ginkgo leaf extracts of the multiple batches was determined according to the method described in example 1(II-i), and it was found that the content of total flavonoids in the natural ginkgo leaf extract ranged from 24.5 to 41.4%, with an average value of 33.5%.
In addition, the fingerprint patterns of the ginkgo biloba extract having a total flavonol glycoside content of 41.04% and the ginkgo biloba extract having a total flavonol glycoside content of 30.90% were measured according to the method described in example 1(V), and the results are shown in fig. 3 and 4, respectively. The result of the evaluation of the similarity of the fingerprint shows that the similarity of the fingerprint of the ginkgo biloba extract with the total flavonol glycoside content of 41.04 percent and the comparison fingerprint is reduced to a level less than 0.70 and far lower than the generally required level of more than 0.90, and the ginkgo biloba extract is an abnormal fingerprint and the whole quality of the ginkgo biloba extract cannot be guaranteed. The similarity between the fingerprint of the ginkgo leaf extract with the content of total flavonol glycosides of 30.90 percent and the comparison fingerprint is 0.988, which shows that the ginkgo leaf extract has good consistency with the comparison product.
Therefore, in the invention, on the basis of the correlation degree research, the general total flavonol glycoside content range in the natural ginkgo leaf extract is considered, and the level that the total flavonol glycoside content in the ginkgo leaf extract is not more than 37% is determined, the ginkgo leaf extract in the content range is a natural product and is not added artificially, and the quality control of the ginkgo leaf extract product at the level can better ensure the quality controllability and stability of the product.
Example 3 Erythrochloe and sodium chloride injection
Figure RE-GDA0001906217520000131
According to the method in the specific embodiment, the fingerprint of the prepared multi-batch of the ligustrum sinense sodium chloride injection is measured, and similarity evaluation is carried out, and the result shows that the similarity between the fingerprint of the multi-batch of the ligustrum sinense sodium chloride injection and the contrast fingerprint shown in the figure 2 is more than 0.93.

Claims (7)

1. A folium Ginkgo extract contains total flavonol glycoside 24.0-37.0%, terpene lactone 6.0-12.0%, wherein the total flavonol glycoside content is (quercetin content + kaempferide content + isorhamnetin content) × 2.51, and the terpene lactone content is calculated by the total amount of bilobalide, bilobalide A, bilobalide B and bilobalide C;
in the folium Ginkgo extract, rutin content is not more than 4.0%, and free quercetin content is not more than 0.2%;
in the ginkgo leaf extract, the content of total ginkgolic acid is not more than 0.7mg/kg, and the content of the total ginkgolic acid is calculated by the total amount of C13:0, C15:1 and C17: 1;
the preparation method of the ginkgo leaf extract comprises the following steps:
taking ginkgo leaf decoction pieces, heating and refluxing for 2 times by using 70-80% ethanol, carrying out reflux extraction for the first time for 2 hours and the second time for 1 hour, filtering an extracting solution, concentrating the extracting solution to a clear paste 1 with the relative density of 1.05-1.25 at the temperature of 60 +/-2 ℃, carrying out water precipitation for more than 8 hours, filtering, enabling a filtrate to pass through a macroporous adsorption resin column with the particle size of 0.12mm, eluting by using water and 30-80% ethanol solution in sequence, discarding a water washing solution, eluting by using 30-80% ethanol solution, enabling the eluate to pass through another macroporous adsorption resin column, collecting an effluent, concentrating to a proper amount, refrigerating, filtering, adding the filtrate onto a third macroporous adsorption resin column, washing by using water, discarding a washing solution, eluting by using 30-80% ethanol solution, collecting the eluate, concentrating to a proper amount, refrigerating at the temperature of 0-10 ℃, filtering, enabling the filtrate to pass through the macroporous adsorption resin column again, eluting, concentrating the eluent to obtain clear paste 2 with the relative density of 1.05-1.25 at the temperature of 60 +/-2 ℃, drying, refining, concentrating, drying again, and crushing to obtain light tan to dark brown powder.
2. The ginkgo biloba leaf extract of claim 1, wherein the heavy metal content of the ginkgo biloba leaf extract is: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg.
3. The quality control method of the ginkgo biloba leaf extract according to any one of claims 1 to 2, which is characterized in that the quality control method comprises the steps of controlling the content of total flavonol glycosides in the ginkgo biloba leaf extract to be 24.0-37.0% and controlling the content of terpene lactones to be 6.0-12.0%.
4. The method of claim 3, wherein the quality control method comprises adjusting the content of rutin in the Ginkgo biloba leaf extract to be not more than 4.0% and the content of free quercetin to be not more than 0.2%.
5. The quality control method of ginkgo biloba extract according to claim 3, further comprising controlling the content of heavy metals in the ginkgo biloba extract to be: lead is not more than 5mg/kg, cadmium is not more than 0.3mg/kg, arsenic is not more than 2mg/kg, mercury is not more than 0.2mg/kg, and copper is not more than 20 mg/kg.
6. Use of the ginkgo biloba leaf extract according to any one of claims 1 to 2 for the preparation of a food product, a health product, a pharmaceutical product, or a cosmetic product.
7. An injection containing ligustrazine phosphate and the ginkgo leaf extract as claimed in any one of claims 1-2, wherein the content of ligustrazine phosphate is 0.36-0.44 mg in each 1ml of the injection, and the content of the ginkgo leaf extract is such that the total flavonol glycosides and terpene lactones are 36-44 μ g and 6.0-13.0 μ g in each 1ml of the injection.
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