CN113768957B - Refining method of bee venom, bee venom preparation for injection and preparation method thereof - Google Patents
Refining method of bee venom, bee venom preparation for injection and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of pharmaceutical preparations, and in particular discloses a refining method of bee venom, a bee venom preparation for injection and a preparation method thereof. The refining method of the bee venom crude product comprises the following steps: adding the bee venom crude product into purified water, dispersing uniformly, centrifuging, adjusting the pH of the centrifugate to 3.0-4.0, standing for 1-2 h, and filtering; then adjusting the pH value of the filtrate to 4.5-5.5, and filtering to obtain bee venom filtrate; carrying out constant volume ultrafiltration on the bee venom filtrate by using an ultrafiltration membrane with the molecular weight cutoff of more than 500Da, starting to concentrate when the water adding amount is 8-10 times of the volume of the bee venom filtrate, stopping concentrating when the volume of the residual liquid is 50-60% of the volume of the bee venom filtrate, and collecting the residual liquid to obtain the bee venom ultrafiltrate; filtering and sterilizing the bee venom ultrafiltrate, lyophilizing, and pulverizing to obtain refined bee venom. The prepared refined bee venom is directly used for preparing a freeze-dried preparation for clinical injection, so that the stability of the bee venom preparation can be obviously improved, and the preparation has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a refining method of bee venom, a bee venom preparation for injection and a preparation method thereof.
Background
Bee venom is a yellowish transparent liquid secreted by bee venom glands and accessory glands, and consists of polypeptides, enzymes, biogenic amines and the like. The bee venom has long history of medical care in folks, and modern medical research proves that the bee venom has anti-inflammatory and analgesic effects, and has definite curative effects on treating wind-damp diseases such as rheumatic arthritis, rheumatoid arthritis, ankylosing spondylitis and the like, and peripheral neuritis and myoneuralgia and the like. However, the enzyme substances with large molecular weight and the amine substances with small molecular weight contained in the bee venom easily cause adverse reactions such as allergy, irritation and pain, and the like, so that the application of the bee venom preparation in clinic is greatly limited. The refined bee venom for injection is a novel antirheumatic drug prepared by purifying and refining the whole bee venom and freeze-drying, and has the advantage of reducing adverse reactions such as anaphylaxis, irritation and the like of the whole bee venom for injection.
At present, the conventional bee venom refining method mainly comprises the following steps: dissolving venenum Apis in water, centrifuging, adding acetone into the centrifugate, standing for precipitation, dissolving precipitate with acetic acid solution, separating and purifying with sephadex chromatographic column, adding activated carbon into the eluate, filtering, and lyophilizing the filtrate to obtain refined venenum Apis. The method has the disadvantages of complicated operation and low purity of refined bee venom, and the bee venom preparation prepared from the bee venom is easy to cause unstable clarity and content, short storage period of the product and the like due to unstable bee venom. Therefore, the development of a refining method of bee venom to improve the purity of bee venom and the development of a bee venom preparation with long storage period and stable quality have great significance for expanding the clinical application of bee venom.
Disclosure of Invention
Aiming at the problems of lower purity of bee venom and poor stability of bee venom preparations obtained by the existing refining method, the invention provides a refining method of bee venom, a bee venom preparation for injection and a preparation method thereof.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a refining method of bee venom comprises the following steps:
step a, adding the bee venom crude product into purified water, uniformly dispersing, and centrifuging to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.0-4.0, standing for 1-2 h, and filtering to obtain a first filtrate; then, the pH value of the first filtrate is regulated to be 4.5-5.5, and the second filtrate is obtained by filtering;
c, carrying out constant volume ultrafiltration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of more than 500Da, starting to concentrate when the water adding amount is 8-10 times of the volume of the second filtrate, stopping concentrating when the volume of the residual liquid is 50-60% of the volume of the second filtrate, and collecting the residual liquid to obtain the bee venom ultrafiltrate;
and d, filtering, sterilizing, freeze-drying and crushing the bee venom ultrafiltrate to obtain refined bee venom.
Compared with the prior art, the refining method of the bee venom provided by the invention has the advantages that the water-insoluble impurities are removed by centrifugation after the bee venom crude product is dissolved in purified water, then the pH value of the centrifugate is regulated to 3.0-4.0, part of the impurities in the bee venom crude product can be removed by precipitation under the pH value condition, meanwhile, viruses can be inactivated under the pH value condition, and the pH value is regulated to 4.5-5.5, so that the stability of the bee venom is improved; finally, removing ions, histamine and other small molecular impurities by adopting a constant volume ultrafiltration method to finally obtain the refined bee venom with the content of more than 96 percent.
The invention can separate and remove the sensitization substances and ineffective impurities in the bee venom crude product by simple processes such as centrifugation, pH adjustment, ultrafiltration and the like, thereby realizing the purpose of rapidly producing refined bee venom products in large quantity, wherein the purity of the products can reach more than 96 percent, and the prepared refined bee venom products have higher stability and can be directly used for preparing freeze-dried preparations for clinical injection; in addition, the refining method of bee venom provided by the invention does not need acetone standing precipitation or separation and purification by adopting a sephadex chromatographic column, avoids the use of a large amount of organic solvents, improves the clinical medication safety of refined bee venom, avoids the generation of organic wastewater, is more environment-friendly, has low production cost and has extremely high popularization and application values.
Preferably, in the step a, the mass ratio of the bee venom crude product to the purified water is 1: 8-10.
The preferable ratio of the crude bee venom to purified water can make bee venom fully dissolved, remove water insoluble impurities as much as possible, and improve the purity of bee venom.
Preferably, in step c, the ultrafiltration pressure is between 0.4MPa and 0.8MPa.
The preferable operation pressure can ensure that impurities have higher transmittance and higher membrane flux, and can also avoid membrane pollution caused by overlarge pressure and improve ultrafiltration efficiency.
Preferably, in step d, the freeze-drying specifically includes the following steps:
pre-freezing: cooling the bee venom ultrafiltrate to-40 to-45 ℃, preserving heat for 2-4 hours, vacuumizing to 100-300 mu bar, and entering a drying stage;
and (3) a drying stage: maintaining the vacuum degree at 100-300 mu bar, heating to-20 to-14 ℃, and preserving heat for 10-16 h; heating to-8 to-12 ℃, and preserving heat for 4 to 8 hours; heating to-7 to-3 ℃ and preserving heat for 3 to 5 hours; heating to 0-5 ℃, and preserving heat for 3-6 h; heating to 8-12 ℃, and preserving heat for 3-6 h; heating to 18-22 ℃, and preserving heat for 3-6 h; finally, the temperature is raised to 32-38 ℃, and the temperature is kept for 3-6 hours.
Preferably, in the drying stage, the heating rate is 2-8 ℃/30min.
The pH value of the bee venom filtrate is adjusted to 4.5-5.5, and the stability of the bee venom in the freeze-drying process can be improved by matching with a preferred freeze-drying process, so that the refined bee venom with stable quality is obtained.
Optionally, in the step a, the rotation speed of centrifugation is 4000 r/min-6000 r/min, and the centrifugation time is 15 min-30 min.
The preferred centrifugation speed and centrifugation time remove as much water insoluble impurities as possible.
Optionally, in the step b, 1.0mol/L hydrochloric acid solution is selected to adjust the pH of the bee venom centrifugate to 3.0-4.0.
The preferred acidic conditions can inactivate viruses while precipitating as much as possible of the impurities in the crude bee venom.
Optionally, in the step b, 1.0mol/L sodium hydroxide solution is selected to adjust the pH to 4.5-5.5.
The preferred pH value is favorable for improving the stability of the bee venom in the purification process and ensuring the stable quality of the refined bee venom product.
Alternatively, in step b, the filtration is performed using a 0.45 μm filter.
Alternatively, in step c, ultrafiltration is performed at room temperature.
Optionally, in step d, a filter membrane of 0.22 μm is used for the filter sterilization.
The invention also provides a bee venom preparation for injection, which comprises the refined bee venom prepared by the refining method of the bee venom.
Preferably, the bee venom preparation for injection comprises the following raw material components in parts by weight: 1 part of refined bee venom, 5-15 parts of citric acid and 10-30 parts of monopotassium phosphate.
Further preferably, the bee venom preparation for injection comprises the following raw material components in parts by weight: 1 part of refined bee venom, 10 parts of citric acid and 25 parts of monopotassium phosphate.
The optimized formula can improve the stability of the bee venom in the preparation process, effectively solve the problems of unstable clarity and content of the bee venom preparation in the storage process, and improve the quality stability of the bee venom preparation product.
The invention also provides a preparation method of the bee venom preparation for injection, which comprises the following steps:
weighing the components according to the proportion, dissolving the weighed citric acid and potassium dihydrogen phosphate in water for injection, and regulating the pH value to 5-6 to obtain a citric acid-potassium dihydrogen phosphate solution;
and step two, dissolving the weighed refined bee venom in a citric acid-potassium dihydrogen phosphate solution, uniformly mixing, filtering, sterilizing and freeze-drying to obtain the bee venom preparation for injection.
Preferably, in the first step, the concentration of the citric acid in the citric acid-potassium dihydrogen phosphate solution is 5g/L to 15g/L, and the concentration of the potassium dihydrogen phosphate is 10g/L to 30g/L.
Preferably, in the second step, the specific freeze-drying process includes the following steps:
pre-freezing: cooling the solution after filtration and sterilization to-40 to-45 ℃, preserving heat for 2 to 4 hours, vacuumizing to the vacuum degree of 50 to 150 mu bar, and entering a drying stage;
and (3) a drying stage: heating to-7 to-13 ℃, adjusting the vacuum degree to 200-300 mu bar, and preserving heat for 2-5 h; then heating to-3 ℃, and preserving heat for 3-6 hours; heating to 8-12 ℃, and preserving heat for 2-5 h; finally, heating to 35-40 ℃, adjusting the vacuum degree to 20-80 mu bar, and preserving the heat for 2-5 h.
Preferably, in the drying stage, the heating rate is 5-15 ℃/30min.
The preferred freeze-drying process is favorable for obtaining the bee venom freeze-dried preparation with good physical form, smooth surface, loose and porous interior and water content meeting the requirements, and can lead the product to be quickly and completely rehydrated, has better long-term stability and effectively improves the quality stability of the bee venom preparation product.
Optionally, in the first step, 0.1mol/L sodium hydroxide solution is used for adjusting the pH to 5-6.
Optionally, 5mL size penicillin bottles are selected for filling and then freeze-drying.
Optionally, the specification of the bee venom preparation for injection is 0.5 mg/bottle.
The 0.5mg small-sized bee venom freeze-dried preparation prepared by the invention is convenient for patients to carry, has accurate dosage control, and has the advantages of convenient clinical use, long storage period and stable quality.
The preparation method of the bee venom preparation for injection provided by the invention is matched with a specific prescription, so that the clarity and content stability of the bee venom preparation are effectively improved, the safety of clinical application is improved, and the preparation method is suitable for industrial mass production and has a wide application prospect.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to better illustrate the present invention, the following examples are provided for further illustration.
The protein content in the bee venom crude product used in the embodiment of the invention is 65.7%.
Example 1
The embodiment provides a refining method of bee venom, which comprises the following steps:
step a, adding 25g of bee venom crude product into 200g of purified water, uniformly dispersing, centrifuging at a rotating speed of 4000r/min for 30min, and taking supernatant after centrifugation to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 4.0 by using 0.1mol/L hydrochloric acid solution, standing for 2 hours at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 5.5 by using 0.1mol/L sodium hydroxide solution, and filtering the first filtrate by using a 0.45 mu m filter membrane to obtain a second filtrate;
step c, carrying out constant volume ultrafiltration on the second filtrate by using an ultrafiltration membrane with the molecular weight of 1000Da, wherein the ultrafiltration pressure is 0.4MPa, when the water adding amount is 8 times of the volume of the second filtrate, starting to concentrate, when the volume of the residual liquid is 50% of the volume of the second filtrate, stopping concentrating, and collecting the residual liquid to obtain the bee venom ultrafiltrate;
and d, filtering and sterilizing the bee venom ultrafiltrate by a filter membrane with the diameter of 0.22 mu m, freeze-drying and crushing to obtain 16.8g of refined bee venom.
In the step d, the freeze-drying specifically comprises the following steps:
pre-freezing: cooling the bee venom ultrafiltrate to-40 ℃, preserving heat for 2.5 hours, vacuumizing to the vacuum degree of 250 mu bar, and entering a drying stage;
and (3) a drying stage: maintaining the vacuum degree at 250 μbar, heating to-15deg.C, and maintaining the temperature for 14h; heating to-10deg.C, and maintaining for 5 hr; heating to-5 ℃, and preserving heat for 3.5h; heating to 0 ℃, and preserving heat for 4 hours; heating to 10 ℃, and preserving heat for 4 hours; heating to 20 ℃, and preserving heat for 4 hours; heating to 35 ℃, and preserving heat for 4 hours. The heating rate of the whole drying stage is 3 ℃/30min.
The refined bee venom prepared in this example was purified according to the national pharmaceutical standards (WS) 1 -XG-016-2001) was subjected to quality test, and the test results are shown in Table 1.
TABLE 1 accelerated stability test results 1
| Time | Traits (3) | pH value of | Loss on drying (%) | Activity (%) | Content (%) |
| 0 month | Pale yellow powder | 5.4 | 1.3 | 45.9 | 96.5 |
| Accelerating for 3 months | Pale yellow powder | 5.4 | 1.5 | 44.3 | 94.0 |
| Accelerating for 6 months | Pale yellow powder | 5.5 | 1.6 | 47.6 | 93.9 |
Example 2
The embodiment provides a refining method of bee venom, which comprises the following steps:
step a, adding 25g of bee venom crude product into 225g of purified water, uniformly dispersing, centrifuging at a rotation speed of 5000r/min for 20min, and taking supernatant after centrifugation to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.5 by using 0.1mol/L hydrochloric acid solution, standing for 1.5h at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 5.0 by using 0.1mol/L sodium hydroxide solution, and filtering the first filtrate by using a 0.45 mu m filter membrane to obtain a second filtrate;
step c, carrying out constant volume ultrafiltration on the second filtrate by using an ultrafiltration membrane with the molecular weight of 1000Da, wherein the ultrafiltration pressure is 0.6MPa, when the water adding amount is 9 times of the volume of the second filtrate, starting to concentrate, when the volume of the residual liquid is 55% of the volume of the second filtrate, stopping concentrating, and collecting the residual liquid to obtain the bee venom ultrafiltrate;
and d, filtering and sterilizing the bee venom ultrafiltrate by a filter membrane with the diameter of 0.22 mu m, freeze-drying and crushing to obtain 16.7g of refined bee venom.
In the step d, the freeze-drying specifically comprises the following steps:
pre-freezing: cooling the bee venom ultrafiltrate to-42 ℃, preserving heat for 3 hours, vacuumizing to the vacuum degree of 200 mu bar, and entering a drying stage;
and (3) a drying stage: maintaining the vacuum degree at 100 μbar, heating to-20deg.C, and maintaining the temperature for 15h; heating to-8deg.C, and maintaining for 6 hr; heating to-3 ℃, and preserving heat for 4 hours; heating to 3 ℃, and preserving heat for 3 hours; heating to 10 ℃, and preserving heat for 4 hours; heating to 22 ℃, and preserving heat for 3 hours; heating to 32 ℃, and preserving heat for 5 hours. The heating rate of the whole drying stage is 5 ℃/30min.
The refined bee venom prepared in this example was purified according to the national pharmaceutical standards (WS) 1 -XG-016-2001) was subjected to quality test, and the test results are shown in Table 2.
TABLE 2 accelerated stability test results 2
| Time | Traits (3) | pH value of | Loss on drying (%) | Activity (%) | Content (%) |
| 0 month | Pale yellow powder | 5.1 | 1.5 | 46.7 | 97.7 |
| Accelerating for 3 months | Pale yellow powder | 5.0 | 1.9 | 49.1 | 94.1 |
| Accelerating for 6 months | Pale yellow powder | 5.1 | 1.9 | 45.9 | 94.7 |
Example 3
The embodiment provides a refining method of bee venom, which comprises the following steps:
step a, adding 25g of bee venom crude product into 250g of purified water, uniformly dispersing, centrifuging at 6000r/min for 15min, and taking supernatant after centrifugation to obtain bee venom centrifugate;
step b, regulating the pH of the bee venom centrifugate to 3.0 by using 0.1mol/L hydrochloric acid solution, standing for 1h at room temperature, and filtering to obtain a first filtrate; then adjusting the pH value of the first filtrate to 4.6 by using 0.1mol/L sodium hydroxide solution, and filtering the first filtrate by using a 0.45 mu m filter membrane to obtain a second filtrate;
step c, carrying out constant volume ultrafiltration on the second filtrate by using an ultrafiltration membrane with the molecular weight of 500Da, wherein the ultrafiltration pressure is 0.8MPa, when the water adding amount is 10 times of the volume of the second filtrate, starting to concentrate, when the volume of the residual liquid is 60% of the volume of the second filtrate, stopping concentrating, and collecting the residual liquid to obtain the bee venom ultrafiltrate;
and d, filtering and sterilizing the bee venom ultrafiltrate by a filter membrane with the diameter of 0.22 mu m, freeze-drying and crushing to obtain 16.1g of refined bee venom.
In the step d, the freeze-drying specifically comprises the following steps:
pre-freezing: cooling the bee venom ultrafiltrate to-45 ℃, preserving heat for 4 hours, vacuumizing to a vacuum degree of 100 mu bar, and entering a drying stage;
and (3) a drying stage: maintaining the vacuum degree at 100 μbar, heating to-17 ℃, and preserving heat for 10h; heating to-12 ℃, and preserving heat for 7h; heating to-5 ℃, and preserving heat for 5 hours; heating to 0 ℃, and preserving heat for 4 hours; heating to 12 ℃, and preserving heat for 3 hours; heating to 18 ℃, and preserving heat for 5 hours; heating to 38 ℃, and preserving heat for 3 hours. The heating rate of the whole drying stage is 8 ℃/30min.
The refined bee venom prepared in this example was purified according to the national pharmaceutical standards (WS) 1 -XG-016-2001) was subjected to quality test, and the test results are shown in Table 3.
TABLE 3 accelerated stability test results 3
| Time | Traits (3) | pH value of | Loss on drying (%) | Activity (%) | Content of(％) |
| 0 month | Pale yellow powder | 4.6 | 1.8 | 47.9 | 98.5 |
| Accelerating for 3 months | Pale yellow powder | 4.6 | 2.1 | 50.6 | 96.4 |
| Accelerating for 6 months | Pale yellow powder | 4.7 | 2.2 | 50.1 | 95.9 |
Example 4
The embodiment provides a bee venom preparation for injection, the specification is 0.5 mg/bottle, and the prescription is as follows:
1g of refined bee venom prepared in example 1, 9g of citric acid, 18g of monopotassium phosphate and 1000mL of water for injection.
The preparation method of the bee venom preparation for injection comprises the following steps:
firstly, weighing the components according to the proportion, dissolving the weighed citric acid and potassium dihydrogen phosphate in 1000mL of water for injection, and regulating the pH value to 6.0 by using 0.1mol/L NaOH solution to obtain a citric acid-potassium dihydrogen phosphate solution;
dissolving the weighed refined bee venom in a citric acid-potassium dihydrogen phosphate solution, uniformly mixing, filtering, sterilizing, sampling, detecting an intermediate, and press-filtering the qualified feed liquid into a liquid storage tank;
and thirdly, filling the bee venom preparation into 5mL penicillin bottles, filling 0.5mL of each penicillin bottle, and freeze-drying to obtain the bee venom preparation for injection.
In the third step, the specific freeze-drying process comprises the following steps:
pre-freezing: cooling the solution after filtration and sterilization to-45 ℃, preserving heat for 2 hours, vacuumizing to the vacuum degree of 100 mu bar, and entering a drying stage;
and (3) a drying stage: heating to-10deg.C, adjusting vacuum degree to 250 μbar, and maintaining for 3 hr; then heating to-1 ℃, and preserving heat for 4 hours; heating to 10 ℃, and preserving heat for 2 hours; heating to 40deg.C, maintaining the temperature for 2h, adjusting vacuum degree to 25 μbar, and maintaining for 2h. The temperature rising rate is 8 ℃/30min in the whole drying stage.
The bee venom preparation for injection prepared in this example was subjected to quality detection according to the national pharmaceutical standard (WS-10001- (HD-1463) -2003), and the detection results are shown in Table 4.
TABLE 4 accelerated stability test results 4
| Time | Traits (3) | pH value of | Loss on drying (%) | Clarity of the product | Indicated amount (%) |
| 0 month | White freeze-dried block | 5.9 | 1.4 | ＜0.5 | 99.8 |
| Accelerating for 3 months | White freeze-dried block | 5.9 | 1.6 | ＜0.5 | 98.9 |
| Accelerating for 6 months | White freeze-dried block | 5.9 | 1.7 | ＜0.5 | 101.2 |
Example 5
The embodiment provides a bee venom preparation for injection, the specification is 0.5 mg/bottle, and the prescription is as follows:
1g of refined bee venom prepared in example 1, 5g of citric acid, 30g of monopotassium phosphate and 1000mL of water for injection.
The preparation method of the bee venom preparation for injection comprises the following steps:
firstly, weighing the components according to the proportion, dissolving the weighed citric acid and potassium dihydrogen phosphate in 1000mL of water for injection, and regulating the pH value to 5.5 by using 0.1mol/LNaOH solution to obtain a citric acid-potassium dihydrogen phosphate solution;
dissolving the weighed refined bee venom in a citric acid-potassium dihydrogen phosphate solution, uniformly mixing, filtering, sterilizing, sampling, detecting an intermediate, and press-filtering the qualified feed liquid into a liquid storage tank;
and thirdly, filling the bee venom preparation into 5mL penicillin bottles, filling 0.5mL of each penicillin bottle, and freeze-drying to obtain the bee venom preparation for injection.
In the third step, the specific freeze-drying process comprises the following steps:
pre-freezing: cooling the solution after filtration and sterilization to-42 ℃, preserving heat for 3 hours, vacuumizing to the vacuum degree of 80 mu bar, and entering a drying stage;
and (3) a drying stage: heating to-7deg.C, adjusting vacuum degree to 200 μbar, and maintaining for 2 hr; then heating to 0 ℃, and preserving heat for 5 hours; heating to 8 ℃, and preserving heat for 3 hours; heating to 35 ℃, preserving heat for 3 hours, adjusting the vacuum degree to 50 mu bar, and maintaining for 3 hours. The temperature rising rate is 10 ℃/30min in the whole drying stage.
The bee venom preparation for injection prepared in this example was subjected to quality detection according to the national pharmaceutical standard (WS-10001- (HD-1463) -2003), and the detection results are shown in Table 5.
TABLE 5 accelerated stability test results 5
| Time | Traits (3) | pH value of | Loss on drying (%) | Clarity of the product | Indicated amount (%) |
| 0 month | White freeze-dried block | 5.5 | 1.7 | ＜0.5 | 100.2 |
| Accelerating for 3 months | White freeze-dried block | 5.6 | 2.1 | ＜0.5 | 100.6 |
| Accelerating for 6 months | White freeze-dried block | 5.5 | 2.2 | ＜0.5 | 102.4 |
Example 6
The embodiment provides a bee venom preparation for injection, the specification is 0.5 mg/bottle, and the prescription is as follows:
1g of refined bee venom prepared in example 1, 15g of citric acid, 11g of monopotassium phosphate and 1000mL of water for injection.
The preparation method of the bee venom preparation for injection comprises the following steps:
firstly, weighing the components according to the proportion, dissolving the weighed citric acid and potassium dihydrogen phosphate in 1000mL of water for injection, and regulating the pH value to 5.0 by using 0.1mol/LNaOH solution to obtain a citric acid-potassium dihydrogen phosphate solution;
dissolving the weighed refined bee venom in a citric acid-potassium dihydrogen phosphate solution, uniformly mixing, filtering, sterilizing, sampling, detecting an intermediate, and press-filtering the qualified feed liquid into a liquid storage tank;
and thirdly, filling the bee venom preparation into 5mL penicillin bottles, filling 0.5mL of each penicillin bottle, and freeze-drying to obtain the bee venom preparation for injection.
In the third step, the specific freeze-drying process comprises the following steps:
pre-freezing: cooling the solution after filtration and sterilization to-40 ℃, preserving heat for 4 hours, vacuumizing to the vacuum degree of 150 mu bar, and entering a drying stage;
and (3) a drying stage: heating to-12deg.C, adjusting vacuum degree to 300 μbar, and maintaining for 4 hr; then heating to 3 ℃, and preserving heat for 4 hours; heating to 12 ℃, and preserving heat for 2 hours; heating to 38deg.C, maintaining the temperature for 2h, adjusting vacuum degree to 40 μbar, and maintaining for 2h. The temperature rising rate is 12 ℃/30min in the whole drying stage.
The bee venom preparation for injection prepared in this example was subjected to quality detection according to the national pharmaceutical standard (WS-10001- (HD-1463) -2003), and the detection results are shown in Table 6.
TABLE 6 accelerated stability test results 6
| Time | Traits (3) | pH value of | Loss on drying (%) | Clarity of the product | Indicated amount (%) |
| 0 month | White freeze-dried block | 5.0 | 1.8 | ＜0.5 | 101.5 |
| Accelerating for 3 months | White freeze-dried block | 4.9 | 1.9 | ＜0.5 | 99.8 |
| Accelerating for 6 months | White freeze-dried block | 5.0 | 2.0 | ＜0.5 | 102.9 |
Comparative example 1
The comparative example provides a method for refining bee venom, wherein the refining step is identical to that of the embodiment 3, except that in the step b, the pH of the bee venom centrifugate is not adjusted, the pH of the original solution of the bee venom centrifugate is 4.95, and the bee venom centrifugate is directly subjected to ultrafiltration in the step c.
The stability of the purified bee venom prepared in comparative example 1 was examined according to the national pharmaceutical standards (WS 1 -XG-016-2001) was measured, and the measurement results are shown in Table 7.
TABLE 7 accelerated stability test results 7
| Time | Traits (3) | pH value of | Weight loss on drying% | Activity (%) | Content (%) |
| 0 month | Pale yellow powder | 4.6 | 2.1 | 45.2 | 93.5 |
| Accelerating for 3 months | Pale yellow powder | 4.6 | 2.2 | 46.4 | 91.4 |
| Accelerating for 6 months | Pale yellow powder | 4.7 | 2.4 | 50.3 | 90.9 |
Comparative example 2
The preparation method of the bee venom preparation for injection is completely the same as that of the example 5, except that citric acid and potassium dihydrogen phosphate are replaced by mannitol in the same amount.
The bee venom preparation for injection prepared in comparative example 2 was subjected to quality detection according to the national pharmaceutical standard (WS-10001- (HD-1463) -2003), and the detection results are shown in Table 8.
TABLE 8 accelerated stability test results 8
| Time | Traits (3) | pH value of | Loss on drying (%) | Clarity of the product | Indicated amount (%) |
| 0 month | White freeze-dried block | 5.5 | 1.9 | ＜0.5 | 99.5 |
| Accelerating for 3 months | White freeze-dried block | 5.6 | 2.2 | 0.5 to 1 No. | 105.2 |
| Accelerating for 6 months | White freeze-dried block | 5.6 | 2.4 | No. 1-No. 2 | 108.1 |
Examples 4 to 6 the purified bee venom prepared in examples 2 and 3 according to the present invention was used to obtain effects substantially equivalent to those of examples 4 to 6.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (9)
1. A refining method of bee venom is characterized by comprising the following steps:
step a, adding the bee venom crude product into purified water, uniformly dispersing, and centrifuging to obtain bee venom centrifugate; the mass ratio of the bee venom crude product to the purified water is 1 (8-10);
step b, adjusting the pH of the bee venom centrifugate to 3.0-4.0, standing for 1-2 h, and filtering to obtain a first filtrate; then, the pH value of the first filtrate is regulated to be 4.5-5.5, and the second filtrate is obtained through filtration;
c, carrying out constant volume ultrafiltration on the second filtrate by using an ultrafiltration membrane with the molecular weight cutoff of more than 500Da, starting to concentrate when the water addition amount is 8-10 times of the volume of the second filtrate, stopping concentrating when the volume of the residual liquid is 50-60% of the volume of the second filtrate, and collecting the residual liquid to obtain the bee venom ultrafiltrate;
and d, filtering, sterilizing, freeze-drying and crushing the bee venom ultrafiltrate to obtain refined bee venom.
2. The method for purifying bee venom according to claim 1, wherein in the step c, the ultrafiltration pressure is 0.4 to 0.8mpa.
3. The method of refining bee venom according to claim 1, wherein in the step d, the freeze-drying comprises the steps of:
pre-freezing: cooling the bee venom ultrafiltrate to-40 to-45 ℃, preserving heat for 2-4 hours, vacuumizing to 100-300 mu bar, and entering a drying stage;
and (3) a drying stage: maintaining the vacuum degree at 100-300 mu bar, heating to-20 to-14 ℃, and preserving heat for 10-16 h; heating to-8 to-12 ℃, and preserving heat for 4-8 hours; heating to-7 to-3 ℃, and preserving heat for 3-5 hours; heating to 0-5 ℃, and preserving heat for 3-6 hours; heating to 8-12 ℃, and preserving heat for 3-6 hours; heating to 18-22 ℃, and preserving heat for 3-6 hours; and finally, heating to 32-38 ℃, and preserving heat for 3-6 hours.
4. The method for purifying bee venom according to claim 3, wherein the heating rate in the drying stage is 2-8 ℃/30min.
5. A bee venom preparation for injection, which is characterized by comprising the refined bee venom prepared by the refining method of bee venom as defined in any one of claims 1-4.
6. The bee venom preparation for injection according to claim 5, which comprises the following raw materials in parts by mass: 1 part of refined bee venom, 5-15 parts of citric acid and 10-30 parts of monopotassium phosphate.
7. The method for preparing bee venom preparation for injection as defined in claim 6, comprising the steps of:
firstly, weighing the components according to the proportion, dissolving the weighed citric acid and potassium dihydrogen phosphate in water for injection, and regulating the pH value to 5-6 to obtain a citric acid-potassium dihydrogen phosphate solution;
and step two, dissolving the weighed refined bee venom in a citric acid-potassium dihydrogen phosphate solution, uniformly mixing, filtering, sterilizing and freeze-drying to obtain the bee venom preparation for injection.
8. The method for preparing bee venom preparation for injection according to claim 7, wherein in the second step, the specific freeze-drying process comprises the following steps:
pre-freezing: cooling the solution after filtration and sterilization to-40 to-45 ℃, preserving heat for 2-4 hours, vacuumizing to a vacuum degree of 50-150 mu bar, and entering a drying stage;
and (3) a drying stage: heating to-7 to-13 ℃, adjusting the vacuum degree to 200-300 mu bar, and preserving heat for 2-5 h; then heating to-3 ℃, and preserving heat for 3-6 hours; heating to 8-12 ℃, and preserving heat for 2-5 hours; finally, heating to 35-40 ℃, adjusting the vacuum degree to 20-80 mu bar, and preserving heat for 2-5 h.
9. The method for preparing a bee venom preparation for injection according to claim 8, wherein the heating rate in the drying stage is 5-15 ℃/30min.
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| WO2014196674A1 (en) * | 2013-06-07 | 2014-12-11 | 주식회사 청진바이오텍 | Preparation method for isolated, purified bee venom having allergic components isolated |
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