CN108186698B - Preparation method of stauntonvine injection - Google Patents
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Abstract
The invention discloses a preparation method and a detection method of stauntonvine injection. The invention relates to a preparation method and the current standard (WS) of stauntonvine injection3Compared with the preparation method in-B-4003-98), the preparation method has the difference that the first alcohol precipitation concentrated solution is heated and then refrigerated after being subjected to acid adjustment. The prepared stauntonvine injection has small content change of total flavone, but the content of total saponin is obviously reduced, and the safety of the product is improved. The invention also provides a method for measuring the content of the total saponins in the stauntonvine injection.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to a preparation method and a detection method of stauntonvine injection.
Background
The stauntonvine injection is collected in the twentieth volume of the pharmaceutical standard Chinese medicinal prescription preparation of the Ministry of health, is a sterilized aqueous solution prepared by extracting stauntonvine, has the effects of dispelling wind, relieving pain, relaxing tendons and activating collaterals, and is used for treating pathogenic wind blocking collateral type trigeminal neuralgia and sciatica.
The preparation method loaded in the stauntonvine injection standard (WS3-B-4003-98) comprises the following steps: collecting 2500g wild fructus Chaenomelis, decocting in water for three times, each for 2 hr, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 nl; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, refrigerating, standing for precipitation, filtering, adjusting pH of the filtrate to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain soft extract, adding 8 times of ethanol, stirring, refrigerating, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding 500ml of water for injection, adjusting the pH value to 7.0-7.5 by using 8% sodium hydroxide solution, adding 10g of polysorbate 80 and 10ml of benzyl alcohol, adding 1000ml of water for injection, adding 2g of activated carbon, stirring, filtering, filling and sealing, and sterilizing to obtain the injection.
The stauntonvine is rich in active substances such as triterpenoid saponin, polysaccharide, flavone, SOD and the like, the total saponin content in per gram of dry pawpaw powder is 0.01765g by adopting a spectrophotometry method and taking oleanolic acid as a standard substance, and the water solution of the saponin can damage erythrocytes greatly to generate a hemolysis phenomenon. The hemolysis test under the examination item in the stauntonvine injection standard stipulates that no hemolysis phenomenon can be observed for half an hour, but the hemolysis test method in appendix XIIIH hemolysis and coagulation examination method of the edition of Chinese pharmacopoeia 2015 requires observation for 3 hours, and the applicant finds that the hemolysis occurs after 3 hours when the hemolysis test is carried out on the stauntonvine injection, so that the preparation method of the stauntonvine injection needs to be improved to improve the safety.
Because the hemolytic effect of the stauntonvine injection is mainly caused by saponin, the establishment of the method for measuring the content of the total saponin in the stauntonvine injection has important significance for controlling the safety of the stauntonvine injection.
Disclosure of Invention
The invention aims to provide a preparation method of a stauntonvine injection, the content change of total flavonoids of the stauntonvine injection prepared by the method is small, but the content of total saponins is obviously reduced, and the safety of the product is improved.
The invention also aims to provide a method for measuring the content of the total saponins in the stauntonvine injection, which is beneficial to controlling the safety of the stauntonvine injection.
The stauntonvine injection is a sterilized aqueous solution prepared by extracting stauntonvine.
The preparation method of the stauntonvine injection comprises the steps of regulating acid of the first alcohol precipitation concentrated solution, heating at 60-100 ℃ and refrigerating.
The preparation method of the stauntonvine injection provided by the invention is preferably as follows: extracting a stauntonvine medicinal material with water, combining extracting solutions, filtering, concentrating a filtrate, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate until each milliliter of the solution is equal to not less than 5.0g of a crude drug, adding water until each milliliter of the solution is equal to not less than 5.0g of the crude drug, adjusting the pH value to 1-3, heating at 60-100 ℃, refrigerating, filtering, adjusting the pH value of the filtrate to be neutral, concentrating, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate, recovering the ethanol, adding water, adjusting the pH value to 6.5-8.0, adding Tween 80 and benzyl alcohol, adding water until each milliliter of the solution is equal to 2.5g of the crude drug, adding active carbon, stirring, filtering, encapsulating and sterilizing to obtain the stauntonvine.
The preparation method of the stauntonvine injection provided by the invention is more preferably as follows: extracting a stauntonvine medicinal material with water for 2-3 times, combining extracting solutions, filtering, concentrating a filtrate until each milliliter of the solution is equivalent to 2.0-3.0 g of a crude medicinal material, adding ethanol until the alcohol content reaches 75-85%, refrigerating for 12-48 hours, filtering, concentrating a filtrate until each milliliter of the solution is equivalent to not less than 10.0g of the crude medicinal material, adding water until each milliliter of the solution is equivalent to not less than 5.0g of the crude medicinal material, adjusting the pH value to 1-3, heating at 60-100 ℃, refrigerating for 12-72 hours, filtering, adjusting the pH value of the filtrate to be neutral, concentrating into a thick paste, adding ethanol until the alcohol content reaches 85-95%, refrigerating for 12-48 hours, filtering, concentrating the filtrate to recover ethanol, adding water until each milliliter of the solution is equivalent to 2.6-3.5 g of the crude medicinal material, adjusting the pH value to 6.5-8.0, adding 2-10 g of tween 80 and 2-10 ml of benzyl alcohol, adding water until each milliliter of the solution is equivalent to 2.5g of the crude medicinal material, adding active carbon, stirring, filtering, bottling, and sterilizing.
The method for measuring the content of the total saponins in the stauntonvine injection comprises the following steps: measuring absorbance at 545nm by using 5% vanillin-glacial acetic acid solution and perchloric acid as developer, and calculating total saponin content in the Stauntonia chinensis injection.
The saponin components in the stauntonvine are mainly triterpenoid saponin taking oleanolic acid, ursolic acid and betulinic acid as sapogenins, so the total saponin content determination method provided by the invention can select the above sapogenins as a reference substance, preferably the oleanolic acid as the reference substance, and the reference substance is dissolved by methanol or absolute ethanol and is subjected to constant volume to a specified concentration, thus obtaining a reference substance solution.
The preparation method of the test solution comprises the following steps: extracting the stauntonvine injection by adding n-butyl alcohol, combining n-butyl alcohol extract, concentrating to be dry, dissolving residues by using 0-30% ethanol solution, adsorbing by using pretreated macroporous adsorption resin, eluting by using pure water and 50-100% ethanol solution in sequence, collecting alcohol eluent, concentrating to be dry, dissolving residues by using methanol or absolute ethanol, and fixing the volume to the specified volume to obtain the stauntonvine injection.
In the preparation method of the test solution, after the stauntonvine injection is adsorbed by macroporous adsorption resin, not only pure water can be used for eluting to remove impurities, but also 0-30% ethanol solution can be used for eluting to remove impurities, and then 50-100% ethanol solution is used for eluting.
The colorimetric determination method is preferably: placing the control solution or the sample solution in a test tube with a plug, volatilizing the solvent, adding 0.2ml of 5% vanillin-glacial acetic acid solution, adding 0.8ml perchloric acid solution, heating in 70 deg.C water bath for 15 min, taking out, cooling with cold water or ice water, adding 5ml glacial acetic acid, mixing, and measuring absorbance at 545 nm.
In the colorimetric measurement method, the amount of the color-developing agent, the temperature for heating in a water bath, and the amount of glacial acetic acid may be appropriately adjusted according to the measurement requirements. For example, the temperature of the water bath can be adjusted to 60 ℃ according to the measurement requirements.
The innovation of the invention is that: on the premise of not influencing the content of total flavonoids in the stauntonvine injection, the content of total saponins is effectively reduced, and the safety of the product is improved; according to the method for measuring the content of the total saponins, the maximum absorption wavelength of the sample solution is consistent with that of the standard solution after color development, and the detection method is more exclusive and sensitive.
The present invention will be further illustrated by the following experimental examples.
Experimental example 1 method for measuring the content of stauntonvine total saponins
The spectrophotometry is a common method for measuring the content of the total saponin at present, and is simple, convenient and sensitive, and has wide application range.
The content of total saponins in Stauntonia chinensis is measured by Palmaria azalea et al (see book 27, 11 th stage of 2006) by spectrophotometry, and the content of total saponins in per gram of dry pawpaw powder is 0.01765g, so that the method has good stability and reproducibility.
In the measuring method adopted by the jonquil and the like, the maximum absorption wavelength is inconsistent after the sample solution and the standard solution are developed, and the invention improves the processing method of the sample.
The method comprises the following steps: extracting herba Stauntoniae injection (intermediate) with n-butanol, mixing n-butanol extractive solutions, concentrating under reduced pressure to dry, and dissolving with anhydrous ethanol.
The method 2 comprises the following steps: adding n-butanol into the wild papaya injection (intermediate) for extraction, mixing n-butanol extract, concentrating under reduced pressure to dryness, adding water for dissolution, adsorbing with pretreated D101 macroporous adsorbent resin, sequentially eluting with pure water, 10%, 30%, 50%, 70% and 95% ethanol solutions, respectively collecting eluates, concentrating under reduced pressure to dryness, and dissolving residue with anhydrous ethanol.
Precisely absorbing 0.2ml of the sample solution and 0.2ml of the oleanolic acid control solution, respectively placing the sample solution and the oleanolic acid control solution in colorimetric tubes with plugs, after the solvent is volatilized by water bath, precisely adding 0.4ml of 5% vanillin-glacial acetic acid solution and 0.6ml of perchloric acid, placing the mixture in water bath at 60 ℃ for heating for 15 minutes, then rapidly placing the mixture in the ice water bath for cooling for 2 minutes, further precisely adding 5ml of glacial acetic acid, uniformly mixing, and scanning by using an ultraviolet-visible spectrophotometer in the wavelength range of 400-800nm, so that the oleanolic acid control solution and the sample solutions prepared by eluting the ethanol solutions of 30%, 50% and 70% in the method 2 have maximum absorption at 545nm, the sample solution prepared by the method 1 has maximum absorption at 533nm, and the sample solutions prepared by the methods 2 by eluting the ethanol solutions of 10%, 30%, 50%, 70% and 95% in the method 2 respectively have absorption values measured at 545nm of 0.08, 0, 0.11, 1.36, 0.09 and 0.09.
Experimental example 2 preparation method of Stauntonia chinensis injection
The stauntonvine is rich in saponin components, and the water solution of the saponin can destroy red blood cells to cause hemolysis. The observation time of the hemolysis test in the current standard of the stauntonvine injection is 30 minutes, if the hemolysis is detected according to the method of appendix XIIIH in the section 2015 of Chinese pharmacopoeia, the hemolysis occurs, so that in order to further improve the safety of the stauntonvine injection, the concentration of total saponins in the stauntonvine injection needs to be properly reduced.
Sample 1: collecting 2500g of stauntonvine, adding water, decocting for three times, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, adding water to 500ml, adjusting the pH value to 2.0 with 6mol/L hydrochloric acid solution, refrigerating, standing for precipitation, filtering, adjusting the pH value of the filtrate to neutrality with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure; adding 500ml of water for injection, adjusting the pH value to 7.3 by using 8% sodium hydroxide solution, adding 10g of polysorbate 80 and 10ml of benzyl alcohol, adding 1000ml of water for injection, adding 2g of activated carbon, stirring, filtering, encapsulating and sterilizing to obtain the measurement result: the content of total flavone is 1.79 mg/ml; the content of total saponin is 1.69mg/ml.
Sample 2: collecting 2500g of stauntonvine, adding water, decocting for three times, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, adding water to 500ml, adjusting the pH value to 2.0 with 6mol/L hydrochloric acid solution, repeatedly refrigerating and filtering until no precipitation is separated out at the bottom, adjusting the pH value of the filtrate to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.3 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the content of total flavone is 0.93 mg/ml; the content of total saponin is 0.91mg/ml.
Samples 3-11: collecting 2500g of stauntonvine, adding water, decocting for three times, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, adding water to 500ml, adjusting the pH value to 2.0 with 6mol/L hydrochloric acid solution, respectively heating according to the conditions in Table 1, refrigerating, standing for precipitation, filtering, adjusting the pH value of the filtrate with 20% sodium hydroxide to be neutral, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.3 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing. The results of the content determination of total flavonoids and total saponins are shown in table 1.
TABLE 1 content of Total Flavonoids and Total Saponins in samples prepared under different heating conditions
As is clear from Table 1, the total flavone content in samples 3 to 11 was comparable to that in sample 1 of Experimental example 1, and the total saponin content was much reduced.
EXAMPLE 3 hemolysis test
In experimental example 2, sample 1 and sample 3 were respectively operated according to the hemolysis and coagulation test method in appendix XIIIH of the first edition of Chinese pharmacopoeia 2015, and after 0.5H, 1.0H, 2.0H and 3.0H, whether hemolysis occurred or not was observed, and the test results are shown in Table 2.
TABLE 2 results of hemolysis test
| Sample name | Observe for 0.5h | Observe for 1.0h | Observe for 2.0h | Observe for 3.0h |
| Sample 1 | Without hemolysis | Without hemolysis | Has hemolysis | Has hemolysis |
| Sample 3 | Without hemolysis | Without hemolysis | Without hemolysis | Has hemolysis |
As can be seen from Table 2, the safety of sample 3 was relatively high after the time delay of hemolysis.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 80 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
[ CONTENT DETERMINATION ] Total Flavonoids
Preparation of reference solution rutin reference 100mg dried under reduced pressure at 120 deg.C to constant weight is precisely weighed, added with 60% ethanol 70ml in 100ml measuring flask, put on water bath for slightly heating to dissolve, and then is anti-cold, diluted with 60% ethanol to scale, shaken up, precisely measured 10ml, put in 50ml measuring flask, added with water to scale, shaken up, and the final product is obtained.
Preparation of standard curve reference solutions 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0ml were precisely measured, placed in 25ml measuring flasks respectively, adding water to 6ml, adding 5% sodium nitrite solution 1ml, mixing, standing for 6 min, adding 10% aluminum nitrate solution 1ml, shaking, standing for 6 min, adding sodium hydroxide solution 10ml, adding water to scale, shaking, standing for 15 min, irradiating spectrophotometry (appendix V B), measuring absorbance at 500nm wavelength, drawing standard curve with absorbance as ordinate and concentration as abscissa, precisely measuring 5ml of the product, placing in 25ml measuring flask, the absorbance was measured according to the method under the standard curve preparation from "each water was added to 6 ml", adding water 5ml into the product to 25ml as blank, measuring absorbance by the method, reading out rutin content in the product from the standard curve, and calculating to obtain the final product.
[ CONTENT DETERMINATION ] Total Saponin
Preparing a reference solution, namely precisely absorbing 0ml, 0.1ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml of the reference solution by absolute ethyl alcohol to prepare a solution containing 300ug per 1ml, preparing a standard curve, respectively placing the reference solution in colorimetric tubes with plugs, volatilizing the solvent, adding 0.2ml of 5% vanillin-glacial acetic acid solution, adding 0.8ml of perchloric acid solution, heating in a 60 ℃ water bath for 15 minutes, rapidly placing in an ice water bath for cooling for 2 minutes, adding 5ml glacial acetic acid, uniformly mixing, measuring the absorbance value at 545nm, taking the absorbance value A as a vertical coordinate and the concentration C as a horizontal coordinate, drawing the standard curve, precisely absorbing 5ml of the residue of the product by a measuring method, adding n-butanol, repeatedly extracting until the n-butanol is colorless, combining n-butanol layers, concentrating to be dry in vacuum, dissolving the residue in 10ml of water to obtain a macroporous adsorbent resin D101 which is pretreated, eluting with pure water, discarding water washing solution, eluting with 75% ethanol solution, collecting ethanol washing solution, concentrating under reduced pressure to dryness, dissolving residue with anhydrous ethanol to constant volume of 25ml, precisely sucking 0.2ml, placing in colorimetric tube with plug, measuring absorbance by following method from "volatilizing solvent", reading out oleanolic acid content from standard curve, and calculating.
And (3) measuring results: the content of total flavone is 1.78 mg/ml; the content of total saponin is 1.30 mg/ml.
Example 2 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 60 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.79 mg/ml; the content of total saponin is 1.52 mg/ml.
Example 3 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 70 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.79 mg/ml; the content of total saponin is 1.27 mg/ml.
Example 4 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 90 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.78 mg/ml; the content of total saponin is 1.28 mg/ml.
Example 5 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 100 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.76 mg/ml; the content of total saponin is 1.17 mg/ml.
Example 6 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 100 deg.C water bath for 4 hr, refrigerating for 72 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.48 mg/ml; the content of total saponin is 1.04 mg/ml.
Example 7 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 100 deg.C water bath for 4 hr, refrigerating for 72 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 4ml 8g, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.48 mg/ml; the content of total saponin is 1.04 mg/ml.
Example 8 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 100 deg.C water bath for 4 hr, refrigerating for 72 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml 4g, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.48 mg/ml; the content of total saponin is 1.04 mg/ml.
Example 9 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 400 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 80 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 10ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the content of total flavone is 1.43 mg/ml; the content of total saponin is 1.01 mg/ml.
Example 10 Stauntonia chinensis injection
2500g of stauntonvine are taken and decocted with water for three times. Mixing decoctions for 2 hr each time, filtering, concentrating the filtrate under reduced pressure to 1000ml, adding 5000ml ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, recovering ethanol from the filtrate under reduced pressure, and adding water to 500 ml; adjusting pH to 2.0 with 6mol/L hydrochloric acid solution, heating in 70 deg.C water bath for 4 hr, refrigerating for 24 hr, standing for precipitation, filtering, adjusting pH to neutral with 20% sodium hydroxide, concentrating under reduced pressure to obtain thick paste, adding 8 times of ethanol, stirring, refrigerating for 24 hr, standing for precipitation, filtering, and recovering ethanol from the filtrate under reduced pressure; adding water for injection 500ml, adjusting pH to 7.5 with 8% sodium hydroxide solution, adding polysorbate 80 and benzyl alcohol 8ml, adding water for injection 1000ml, adding activated carbon 2g, stirring, filtering, bottling, and sterilizing.
And (3) measuring results: the determination method is the same as that of example 1, and the content of the total flavone is 1.79 mg/ml; the content of total saponin is 1.27 mg/ml.
Claims (3)
1. A preparation method of stauntonvine injection is characterized by comprising the following steps: extracting a stauntonvine medicinal material with water, combining extracting solutions, filtering, concentrating a filtrate, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate until each milliliter of the solution is equal to not less than 6.0g of a crude drug, adding water until each milliliter of the solution is equal to not less than 5.0g of the crude drug, adjusting the pH value to 1-3, heating at the temperature of 80-100 ℃, refrigerating, filtering, adjusting the pH value of the filtrate to be neutral, concentrating, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate, recovering the ethanol, adding the water, adjusting the pH value to 6.5-8.0, adding Tween 80 and benzyl alcohol, adding the water until each milliliter of the solution is equal to 2.5g of the crude drug, adding active carbon, stirring, filtering, encapsulating and sterilizing to obtain the stauntonvine.
2. The method of claim 1, wherein the method comprises: extracting a stauntonvine medicinal material with water, combining extracting solutions, filtering, concentrating a filtrate, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate until each milliliter of the solution is equal to not less than 10.0g of a crude drug, adding water until each milliliter of the solution is equal to not less than 5.0g of the crude drug, adjusting the pH value to 1-3, heating at the temperature of 80-100 ℃, refrigerating, filtering, adjusting the pH value of the filtrate to be neutral, concentrating, carrying out alcohol precipitation, refrigerating, filtering, concentrating the filtrate, recovering the ethanol, adding the water, adjusting the pH value to 6.5-8.0, adding Tween 80 and benzyl alcohol, adding the water until each milliliter of the solution is equal to 2.5g of the crude drug, adding active carbon, stirring, filtering, encapsulating and sterilizing to obtain the stauntonvine.
3. The method of claim 1 or 2, wherein: extracting a stauntonvine medicinal material with water for 2-3 times, combining extracting solutions, filtering, concentrating a filtrate until each milliliter of the solution is equivalent to 2.0-3.0 g of a crude medicinal material, adding ethanol until the alcohol content reaches 75-85%, refrigerating for 12-48 hours, filtering, concentrating a filtrate until each milliliter of the solution is equivalent to not less than 10.0g of the crude medicinal material, adding water until each milliliter of the solution is equivalent to not less than 5.0g of the crude medicinal material, adjusting the pH value to 1-3, heating at 80-100 ℃, refrigerating for 12-72 hours, filtering, adjusting the pH value of the filtrate to be neutral, concentrating into a thick paste, adding ethanol until the alcohol content reaches 85-95%, refrigerating for 12-48 hours, filtering, concentrating the filtrate to recover ethanol, adding water until each milliliter of the solution is equivalent to 2.6-3.5 g of the crude medicinal material, adjusting the pH value to 6.5-8.0, adding 2-10 g of tween 80 and 2-10 ml of benzyl alcohol, adding water until each milliliter of the solution is equivalent to 2.5g of the crude medicinal material, adding active carbon, stirring, filtering, bottling, and sterilizing.
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