CN102338782B - Fresh animal medicinal composition and method for measuring content of Chinese medicines of fresh animal medicinal composition - Google Patents

Fresh animal medicinal composition and method for measuring content of Chinese medicines of fresh animal medicinal composition Download PDF

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CN102338782B
CN102338782B CN 201110163974 CN201110163974A CN102338782B CN 102338782 B CN102338782 B CN 102338782B CN 201110163974 CN201110163974 CN 201110163974 CN 201110163974 A CN201110163974 A CN 201110163974A CN 102338782 B CN102338782 B CN 102338782B
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amino acid
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volume
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CN102338782A (en
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李建生
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BEIJING JIANSHENG PHARMACEUTICAL CO LTD
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BEIJING JIANSHENG PHARMACEUTICAL CO LTD
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Abstract

The invention discloses a fresh animal medicinal composition and a method for measuring the content of Chinese medicines of the fresh animal medicinal composition. The method comprises the following steps of: putting the fresh animal medicinal composition and the Chinese medicines into a measuring flask, dissolving by adding water, performing ultrasonic extraction and centrifugation, taking supernate, and sieving the supernate with a microporous filtering film to obtain a free amino acid test sample solution; and weighing the fresh animal medicinal composition and the Chinese medicines, fully dissolving by adding water, then performing ultrasonic extraction and centrifugation, taking the supernate, putting the supernate into an ampoule bottle, adding hydrochloric acid, performing vacuum nitrogenization and hydrolyzation, taking the mixture out, cooling the mixture to room temperature, adding a NaOH solution, uniformly mixing, transferring to the measuring flask, diluting by adding water, and fixing the volume to a scale, uniformly mixing, centrifuging, taking the supernate, and sieving the supernate with the microporous filtering film to obtain the total amino acid test sample solution. A chromatographic column is used for detecting primary amino acid and secondary amino acid. A mobile phase A comprising acetonitrile, methanol and water and a mobile phase B comprising Na2HPO4 of which the pH is 7.8 are used for performing gradient elution. The method is simple in operation and high in measurement resolution and sensitivity, and by the method, 12 to 26 types of amino acids in the fresh animal medicinal composition and the Chinese medicines thereof can be separated in a short time, so the method is a reliable method for measuring the amino acid content.

Description

The content assaying method of a kind of bright animal drugs pharmaceutical composition and bulk drug thereof
Technical field
The present invention relates to a kind of assay method, particularly relate to a kind of bright animal drugs pharmaceutical composition of primary carcinoma of liver blood stasis pent-up card and content assaying method of bulk drug thereof for the treatment of.
Background technology
Jinlong capsule of the present invention (the accurate word Z10980041 of traditional Chinese medicines) is cancer treatment drugs, with the compound preparation of medicinal animal (bright house lizard, bright long-nosed pit viper, bright Bungarus Parvus) prescription, at the anti-tumor aspect determined curative effect.
(WS3-158 (Z-036)-2001Z) is to the control of its product quality for the drug standards that Jinlong capsule has been promulgated, comprise: proterties, differentiate, 4 of inspection and assays, from the requirement of present SFDA to the Chinese medicine quality control standard, and the bright house lizard of Jinlong capsule bulk drug, bright Bungarus Parvus, chemistry and the pharmacological research present situation of bright long-nosed pit viper, the assay project has adopted visible spectrophotometry at present, the Folin-phenol reagent is measured total protein, the specificity of this method is relatively poor, in addition for ubiquitous amino acid in the animal drugs, free monosaccharide, some little molecule life base substances of polysaccharide and endogenous all do not carry out quality controling research.
According to Jinlong capsule quality control standard present situation, the present invention rises to starting point with quality control standard, amino acid in the Jinlong capsule is measured, determined the chromatographic condition that Jinlong capsule amino acid detects, this chromatographic condition adopts pre-column derivatization method, and precision, stability and reappearance are good in the methodological study.And contain a large amount of amino acid in the Jinlong capsule, use determined amino acid and can better control drug quality.
Summary of the invention
The object of the present invention is to provide a kind of content assaying method of bright animal drugs pharmaceutical composition.Another object of the present invention is to provide a kind of content assaying method of bright animal drugs pharmaceutical composition bulk drug.
The present invention is achieved through the following technical solutions:
The content assaying method of pharmaceutical composition of the present invention comprises the steps:
1, the preparation of standard solution
A, 0.1mol/L hydrochloric acid solution: get concentrated hydrochloric acid 0.83 parts by volume of 36%-38% in 100 parts by volume measuring bottles, thin up also is settled to scale;
B, 17 seed amino acid hybrid standard product solution: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 0.5-1.5 parts by volume volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.2-0.8 weight portion in the brown measuring bottle of 30-70 parts by volume, add concentrated hydrochloric acid 15-35 parts by volume, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: get NaOH 10-30 weight portion, place 30-70 parts by volume measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: get NaH 2PO 4The 3-8 weight portion, being dissolved in water also, constant volume with 10mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 500-1500 parts by volume measuring bottle;
2. the preparation of need testing solution
The preparation of a, free amino acid need testing solution: take by weighing pharmaceutical composition 0.05-0.15 weight portion of the present invention, place 25-75 parts by volume measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 15-45 minute, centrifugal 5-15 minute, get supernatant, cross 0.3-0.6 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: take by weighing pharmaceutical composition 0.05-0.15 weight portion of the present invention, place 10-20 parts by volume plastic centrifuge tube, after adding water 5-15 parts by volume and fully dissolving, ultrasonic extraction 15-45 minute centrifugal 5-15 minute, is got supernatant, get 0.05-1.5 parts by volume supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 2-6 parts by volume that contains 1% phenol, vacuum nitrogen N 215-45 second, rapidly sealing placed in the 80-150 ℃ of thermostatic drying chamber hydrolysis 12-32 hour, take out, let cool to room temperature, add 1.2-3.6 parts by volume 10mol/L NaOH solution, mixing, be transferred in the 5-15 parts by volume measuring bottle, thin up also is settled to scale, mixing, centrifugal 5-15 minute, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
3, chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=25-65: 25-65: 5-15 is as mobile phase A, and pH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, the gradient elution program is as follows:
0-2.5min mobile phase A is 0%, Mobile phase B is 100%;
2.5-16min mobile phase A is 30%, Mobile phase B is 70%;
The 16-24min mobile phase A is 57%, and Mobile phase B is 43%;
24-24.8min mobile phase A is 100%, Mobile phase B is 0%;
24.8-29.7min mobile phase A is 100%, Mobile phase B is 0%;
29.7-30.9min mobile phase A is 0%, Mobile phase B is 100%;
30.9-34.7min mobile phase A is 0%, Mobile phase B is 100%;
4, measurement result
Detect 14 seed amino acids in the pharmaceutical composition of the present invention, wherein the content of gross protein is 1.9%-2.85%, and the content of total free amino acid is 12.52%-18.78%, and the content of total amino acid is 14.41%-21.62%;
The content assaying method of pharmaceutical composition of the present invention is preferably as follows step:
1, the preparation of standard solution
A, 0.1mol/L hydrochloric acid solution: get 37.5% concentrated hydrochloric acid, 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
The preparation of b, 17 seed amino acid hybrid standard product solution: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 1 parts by volume measuring bottle, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.5 weight portion in the brown measuring bottle of 50 parts by volume, add concentrated hydrochloric acid 25 parts by volume, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: get NaOH 20.0 weight portions, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: get NaH 2PO 45.5 weight portion, being dissolved in water also, constant volume with 10mol/LNaOH solution adjusting pH value to 7.8, mixing, and get final product in 1000 parts by volume measuring bottles;
2. the preparation of need testing solution
The preparation of a, free amino acid need testing solution: take by weighing pharmaceutical composition 0.1 weight portion of the present invention, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: take by weighing pharmaceutical composition 0.1 weight portion of the present invention, place 15 parts by volume plastic centrifuge tubes, after adding water 10 parts by volume and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get 1 parts by volume supernatant, place peace to cut open bottle, add 6mol/L hydrochloric acid 4 parts by volume that contain 1% phenol, vacuum nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4 parts by volume 10mol/L NaOH solution, mixing, be transferred in the 10 parts by volume measuring bottles, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
3, chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=PH was 7.8 40mmol/L Na as mobile phase A in 45: 45: 10 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
4, measurement result
Detect 14 seed amino acids in the pharmaceutical composition of the present invention, wherein the content of gross protein is 1.9%, and the content of total free amino acid is 12.52%, and the content of total amino acid is 14.41%;
14 seed amino acids are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline in the pharmaceutical composition of the present invention.
The content Mid-Heaven Gate winter histidine content of total free amino acid is 0.55%-0.83% in the pharmaceutical composition of the present invention, content of glutamic acid is 1.79%-2.79%, serine content is 0.54%-0.81%, histidine content is 0.52%-0.78%, glycocoll content is 1.15%-1.73%, threonine content is 0.62%-0.93%, alanine content is 2.01%-3.02%, valine content is 0.86%-1.29%, methionine content is 0.36%-0.54%, phenylalanine content is 0.5%-0.75%, isoleucine content is 0.53%-0.80%, leucine content is 0.96%-1.44%, lysine content is 1.30%-1.95%, proline content is 0.82%-1.23%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.80%-1.20%, content of glutamic acid is 2.29%-3.44%, serine content is 0.72%-1.08%, histidine content is 0.73%-1.10%, glycocoll content is 1.53%-2.30%, threonine content is 0.73%-1.10%, alanine content is 2.08%-3.12%, valine content is 0.94%-1.41%, methionine content is 0.42%-0.63%, phenylalanine content is 0.72%-1.08%, isoleucine content is 0.72%-1.08%, leucine content is that content is 1.15%-2.27%, lysine content is 0.82%-1.23%, proline content is 0.76%-1.14%.
The content Mid-Heaven Gate winter histidine content of total free amino acid is 0.55% in the pharmaceutical composition of the present invention, content of glutamic acid is 1.79%, serine content is 0.54%, histidine content is 0.52%, glycocoll content is 1.15%, threonine content is 0.62%, alanine content is 2.01%, valine content is 0.86%, methionine content is 0.36%, phenylalanine content is 0.5%, isoleucine content is 0.53%, leucine content is 0.96%, lysine content is 1.30%, proline content is 0.82%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.80%, content of glutamic acid is 2.29%, serine content is 0.72%, histidine content is 0.73%, glycocoll content is 1.53%, threonine content is 0.73%, alanine content is 2.08%, valine content is 0.94%, methionine content is 0.42%, phenylalanine content is 0.72%, isoleucine content is 0.72%, leucine content is that 1.15 content are %, lysine content is 0.82%, proline content is 0.76%;
The bulk drug of pharmaceutical composition of the present invention consists of:
The bright Bungarus Parvus 400-1100 of bright house lizard 700-2300 weight portion weight portion
Bright long-nosed pit viper 400-1100 weight portion.
The bulk drug composition of pharmaceutical composition of the present invention is preferably:
Bright house lizard 1500 weight portions, bright Bungarus Parvus 750 weight portions, bright long-nosed pit viper 750 weight portions;
Or bright house lizard 800 weight portions, bright Bungarus Parvus 1000 weight portions, bright long-nosed pit viper 500 weight portions;
Or bright house lizard 2000 weight portions, bright Bungarus Parvus 500 weight portions, bright long-nosed pit viper 100 weight portions.
The preparation method of pharmaceutical composition of the present invention is:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, the water gaging homogenate such as add after, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draw supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying, be ground into fine grained, tablet, capsule, granule or pill are made in low temperature drying.
The preparation method of pharmaceutical composition of the present invention can also for:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, add the homogenate of 3-5 times of volume water after, place-18 ℃ to-22 ℃ freezing 24 hours, take out, place 15 ℃ of-30 ℃ of thawings, filter, filtrate is centrifugal with 12000-16000 rev/min, draws supernatant, ultrafiltration is got filtrate and is added the freeze drying of beta-schardinger dextrin-20-60 weight portion inclusion final vacuum, is ground into fine grained, add starch 66-198 weight portion, mixing, tablet, capsule, granule or pill are made in low temperature drying.
The preparation method of pharmaceutical composition of the present invention can also be preferably:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, add the homogenate of 4 times of volume water after, place-20 ℃ freezing 24 hours, take out, place 25 ℃ of thawings, filter, filtrate is centrifugal with 14000 rev/mins, draws supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-40 weight portion inclusion final vacuum freeze dryings, is ground into fine grained, add starch 132 weight portions, mixing, tablet, capsule, granule or pill are made in low temperature drying.
The content assaying method of pharmaceutical composition bulk drug of the present invention comprises the steps:
1, the preparation of standard solution
A, 0.1mol/L hydrochloric acid solution: get 36%-38% concentrated hydrochloric acid 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
B, the accurate solution of 17 seed amino acid mixing mark product: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 0.5-1.5 parts by volume volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.2-0.8 weight portion in the brown measuring bottle of 30-70 parts by volume, add concentrated hydrochloric acid 15-35 parts by volume, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: get NaOH 10-30 weight portion, place 30-70 parts by volume measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: get NaH 2PO 4The 3-38 weight portion, being dissolved in water also, constant volume with 10mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 500-1500 parts by volume measuring bottle;
2. the preparation of need testing solution
The preparation of a, free amino acid need testing solution: take by weighing each 0.05-0.15 weight portion of bright Bungarus Parvus, bright long-nosed pit viper and bright house lizard, place 25-75 parts by volume measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 15-45 minute, centrifugal 5-15 minute, get supernatant, cross 0.3-0.6 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: take by weighing each 0.05-0.15 weight portion of bright Bungarus Parvus, bright long-nosed pit viper and bright house lizard, place 10-20 parts by volume plastic centrifuge tube, after adding water 5-15 parts by volume and fully dissolving, ultrasonic extraction 15-45 minute centrifugal 5-15 minute, is got supernatant, get 0.05-1.5 parts by volume supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 2-6 parts by volume that contains 1% phenol, vacuum nitrogen N 215-45 second, rapidly sealing placed in the 80-150 ℃ of thermostatic drying chamber hydrolysis 12-32 hour, take out, let cool to room temperature, add respectively 1.2-3.6 parts by volume 10mol/L NaOH solution, mixing, be transferred in the 5-15 parts by volume measuring bottle, thin up also is settled to scale, mixing, centrifugal 5-15 minute, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
3, chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place.Take acetonitrile: methyl alcohol: water=25-65: 25-65: 5-15 is as mobile phase A, and pH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, the gradient elution program is as follows:
0-2.5min mobile phase A is 0%, Mobile phase B is 100%;
2.5-16min mobile phase A is 30%, Mobile phase B is 70%;
The 16-24min mobile phase A is 57%, and Mobile phase B is 43%;
24-24.8min mobile phase A is 100%, Mobile phase B is 0%;
24.8-29.7min mobile phase A is 100%, Mobile phase B is 0%;
29.7-30.9min mobile phase A is 0%, Mobile phase B is 100%;
30.9-34.7min mobile phase A is 0%, Mobile phase B is 100%;
4, measurement result
Detect 14 seed amino acids in the bright house lizard of bulk drug of the present invention, wherein the content of gross protein is 1.77%-2.66%, and the content of total free amino acid is 22.50%-33.75%, and the content of total amino acid is 24.26%-36.39%;
Or detect 12 seed amino acids in the bright Bungarus Parvus, and wherein the content of gross protein is 0.13%-0.20%, and the content of total free amino acid is 6.02%-9.03%, and the content of total amino acid is 6.15%-9.23%;
Or detect 15 seed amino acids in the bright long-nosed pit viper, and wherein the content of gross protein is 18.03%-27.05%, and the content of total free amino acid is 5.09%-7.64%, and the content of total amino acid is 23.12%-34.68%;
The content assaying method of pharmaceutical composition of the present invention is preferably as follows step:
1, the preparation of standard solution
A, 0.1mol/L hydrochloric acid solution: get 37.5% concentrated hydrochloric acid, 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
The preparation of b, 17 seed amino acid hybrid standard product solution: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 1 parts by volume volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.5 weight portion in the brown measuring bottle of 50 parts by volume, add concentrated hydrochloric acid 25 parts by volume, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: get NaOH 20.0 weight portions, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: get NaH 2PO 45.5 weight portion, being dissolved in water also, constant volume with 10mol/LNaOH solution adjusting pH value to 7.8, mixing, and get final product in 1000 parts by volume measuring bottles;
2. the preparation of need testing solution
The preparation of a, free amino acid need testing solution: take by weighing each 0.1 weight portion of bright Bungarus Parvus, bright long-nosed pit viper and bright house lizard, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: take by weighing each 0.1 weight portion of bright Bungarus Parvus, bright long-nosed pit viper product and bright house lizard, place 15 parts by volume plastic centrifuge tubes, after adding water 10 parts by volume and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get 1 parts by volume supernatant, place peace to cut open bottle, add 6mol/L hydrochloric acid 4 parts by volume that contain 1% phenol, vacuum nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4 parts by volume 10mol/L NaOH solution, mixing, be transferred in the 10 parts by volume measuring bottles, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
3, chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=PH was 7.8 40mmol/L Na as mobile phase A in 45: 45: 10 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
4, measurement result
Detect 14 seed amino acids in the bright house lizard of bulk drug of the present invention, wherein the content of gross protein is 1.77%, and the content of total free amino acid is 22.50%, and the content of total amino acid is 24.26%;
Or detect 12 seed amino acids in the bright Bungarus Parvus, and wherein the content of gross protein is 0.13%, and the content of total free amino acid is 6.02%, and the content of total amino acid is 6.15%;
Or detect 15 seed amino acids in the bright long-nosed pit viper, and wherein the content of gross protein is 18.03%, and the content of total free amino acid is 5.09%, and the content of total amino acid is 23.12%.
14 seed amino acids in the bright house lizard of bulk drug of the present invention are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline;
Or 12 seed amino acids in the bright Bungarus Parvus are respectively asparatate, glutamic acid, serine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine and proline;
Or 15 seed amino acids in the bright long-nosed pit viper are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
The content Mid-Heaven Gate winter histidine content of total free amino acid is 1.33%-2.00% in the bright house lizard of bulk drug of the present invention, content of glutamic acid is 3.29%-4.94%, serine content is 0.95%-1.43%, histidine content is 0.72%-1.08%, glycocoll content is 1.64%-2.46%, threonine content is 0.98%-1.47%, alanine content is 2.48%-3.72%, valine content is 1.36%-2.04%, methionine content is 0.68%-1.02%, phenylalanine content is 0.93%-1.40%, isoleucine content is 1.00%-1.50%, leucine content is 1.67%-2.51%, lysine content is 4.13%-6.20%, proline content is 1.36%-2.04%; The content Mid-Heaven Gate winter histidine content of total amino acid is 1.92%-2.88%, content of glutamic acid is 4.39%-6.59%, serine content is 1.19%-1.79%, histidine content is 0.95%-1.43%, glycocoll content is 2.16%-3.24%, threonine content is 1.22%-1.83%, alanine content is 2.62%-3.93%, valine content is that content is 1.60%-2.40%, methionine content is 0.71%-1.07%, phenylalanine content is 1.20%-1.80%, isoleucine content is 1.20%-1.80%, leucine content is 1.89%-2.84%, lysine content is 2.06%-3.09%, proline content is 1.17%-1.76%;
Or the content Mid-Heaven Gate winter histidine content of total free amino acid is 0.25%-0.38% in the bright Bungarus Parvus, content of glutamic acid is 0.57%-0.86%, serine content is 0.15%-0.23%, glycocoll content is 0.68%-1.02%, threonine content is 0.39%-0.59%, alanine content is 1.48%-2.22%, valine content is 0.62%-0.93%, methionine content is 0.24%-0.36%, phenylalanine content is 0.51%-0.77%, isoleucine content is 0.40%-0.60%, leucine content is 0.60%-0.90%, proline content is 0.15%-0.23%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.39%-0.59%, content of glutamic acid is 0.70%-1.05%, serine content is 0.30%-0.45%, glycocoll content is 0.74%-1.11%, threonine content is 0.39%-0.59%, alanine content is 1.20%-1.80%, valine content is 0.53%-0.80%, methionine content is 0.28%-0.42%, phenylalanine content is 0.39%-0.59%, isoleucine content is 0.40%-0.60%, leucine content is 0.59%-0.89%, proline content is 0.23%-0.35%;
Or the content Glutamic Acid content of total free amino acid is 0.17%-0.26% in the bright long-nosed pit viper, serine content is 0.12%-0.18%, histidine content is 0.18%-0.27%, glycocoll content is 0.51%-0.77%, threonine content is 0.38%-0.57%, alanine content is 1.13%-1.70%, valine content is 0.40%-0.60, isoleucine content is 0.22%-0.33%, leucine content is 0.34%-0.51%, lysine content is 1.64%-2.72%; The content Mid-Heaven Gate winter histidine content of total amino acid is 2.28%-3.42%, content of glutamic acid is 3.17%-4.76%, serine content is 1.18%-1.77%, histidine content is 1.09%-1.64%, glycocoll content is 1.52%-2.28%, threonine content is 1.39%-2.39%, alanine content is 2.16%-3.24%, tyrosine content is 0.91%-1.37, valine content is 1.60%-2.40%, methionine content is 0.51%-0.77%, phenylalanine content is 1.11%-1.67%, isoleucine content is 1.22%-1.83%, leucine content is 2.17%-3.26%, lysine content is 2.11%-3.17%, the content of proline is 0.74%-1.11%.
The content Mid-Heaven Gate winter histidine content of total free amino acid is 1.33% in the bright house lizard of bulk drug of the present invention, content of glutamic acid is 3.29%, serine content is 0.95%, histidine content is 0.72%, glycocoll content is 1.64%, threonine content is 0.98%, alanine content is 2.48%, valine content is 1.36%, methionine content is 0.68%, phenylalanine content is 0.93%, isoleucine content is 1.00%, leucine content is 1.67%, lysine content is 4.13%, proline content is 1.36%; The content Mid-Heaven Gate winter histidine content of total amino acid is 1.92%, content of glutamic acid is 4.39%, serine content is 1.19%, histidine content is 0.95%, glycocoll content is 2.16%, threonine content is 1.22%, alanine content is 2.62%, valine content is that content is 1.60%, methionine content is 0.71%, phenylalanine content is 1.20%, isoleucine content is 1.20%, leucine content is 1.89%, lysine content is 2.06%, proline content is 1.17%;
Or the content Mid-Heaven Gate winter histidine content of total free amino acid is 0.25%, content of glutamic acid is 0.57% in the bright Bungarus Parvus, serine content is 0.15%, glycocoll content is 0.68%, threonine content is 0.39%, alanine content is 1.48%, valine content is 0.62%, methionine content is 0.24%, phenylalanine content is 0.51%, isoleucine content is 0.40%, leucine content is 0.60%, proline content is 0.15%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.39%, content of glutamic acid is 0.70%, serine content is 0.30%, glycocoll content is 0.74%, threonine content is 0.39%, alanine content is 1.20%, valine content is 0.53%, methionine content is 0.28%, phenylalanine content is 0.39%, isoleucine content is 0.40%, leucine content is 0.59%, proline content is 0.23%;
Or the content Glutamic Acid content of total free amino acid is 0.17%, serine content is 0.12% in the bright long-nosed pit viper, histidine content is 0.18%, glycocoll content is 0.51%, threonine content is 0.38%, alanine content is 1.13%, valine content is 0.40%, isoleucine content is 0.22%, leucine content is 0.34%, lysine content is 1.64%; The content Mid-Heaven Gate winter histidine content of total amino acid is 2.28%, content of glutamic acid is 3.17%, serine content is 1.18%, histidine content is 1.09%, glycocoll content is 1.52%, threonine content is 1.39%, alanine content is 2.16%, tyrosine content is 0.91%, valine content is 1.60%, methionine content is 0.51%, phenylalanine content is 1.11%, isoleucine content is 1.22%, leucine content is 2.17%, lysine content is 2.11%, the content of proline is 0.74%.
The preparation method of the bright house lizard of pharmaceutical composition Raw medicine of the present invention is: get bright house lizard 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
Or the preparation method of bright Bungarus Parvus is: get bright Bungarus Parvus 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
Or the preparation method of bright long-nosed pit viper is: get bright long-nosed pit viper 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
The pass of weight portion of the present invention and parts by volume is g/ml or kg/l.
The preparation method of pharmaceutical composition of the present invention changes into derivant with amino acid first, carries out chromatographic resolution again, operate relatively simple, high resolving power, high sensitivity, and can separate at 12～40min a kind of reliable method of 12～26 seed amino acids in the short time.
Description of drawings
Fig. 1 is 17 seed amino acid standard items HPLC figure;
Fig. 2 is mixed material HPLC figure;
Fig. 3 is house lizard HPLC figure
Fig. 4 is long-noded pit viper HPLC figure
Fig. 5 is long-nosed pit viper HPLC figure.
Wherein: 1, asparatate, 2, glutamic acid, 3, serine, 4, histidine, 5, glycocoll, 6, threonine, 7, arginine, 8, alanine, 9, tyrosine, 10, cystine, 11, valine, 12, methionine, 13, phenylalanine, 14, isoleucine, 15, leucine, 16, lysine, 17, proline.
Following experimental example is used for further specifying the present invention, but is not limited to the present invention.
Experimental example: amino acid whose quantitative test in pharmaceutical composition Jinlong capsule of the present invention and each bulk drug
1. experiment material and instrument and equipment
1.1 experiment material: 17 seed amino acids (asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline) hybrid standard product solution, borate buffer, OPA and FMOC derivatization reagent all are purchased from Agilent company; Methyl alcohol and acetonitrile are by B﹠amp; J company provides, chromatographically pure; It is pure that sodium dihydrogen phosphate, hydrochloric acid and NaOH are analysis, is purchased from the Beijing Chemical Plant; Pharmaceutical composition Jinlong capsule of the present invention (according to the method preparation of embodiment 1) and the bright Bungarus Parvus of pharmaceutical composition bulk drug of the present invention (according to the method preparation of embodiment 2) thereof, bright long-nosed pit viper (according to the method preparation of embodiment 3), bright house lizard (according to the method preparation of embodiment 4) are provided by the Beijing JianSheng pharmacy Co., Ltd.
1.2 instrument and equipment: high performance liquid chromatograph is Agilent 1100 HPLC, is equipped with UV-detector, automatic sampler, column oven and quaternary gradient elution pump.
2. solution preparation
2.10.1mol/L hydrochloric acid solution: get 37.5% concentrated hydrochloric acid 0.83mL in the 100mL measuring bottle, thin up also is settled to scale.
2.2 the preparation of series concentration 17 seed amino acid hybrid standard product solution: get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in the 1mL volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration.
The 6mol/L hydrochloric acid solution of 2.31% phenol: get phenol approximately 0.5g in the brown measuring bottle of 50mL, add concentrated hydrochloric acid 25mL, be dissolved in water and be settled to scale.
2.410mol/LNaOH solution: get approximately 20.0g of NaOH, place the 50mL measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use.
2.540mmol/L Na 2HPO 4Damping fluid (pH7.8): get NaH 2PO 4About 5.5g, be dissolved in water and constant volume in the 1L measuring bottle, regulate pH value to 7.8 with 10mol/LNaOH solution, mixing, and get final product.
3. the preparation of need testing solution
3.1 the preparation of free amino acid need testing solution: get pharmaceutical composition of the present invention (according to the preparation of embodiment 1 method) and the bright Bungarus Parvus powder of pharmaceutical composition bulk drug of the present invention (according to the method preparation of embodiment 2), bright long-nosed pit viper sample powder (according to the method preparation of embodiment 3), bright house lizard sample powder (according to the method preparation of embodiment 4), get respectively 0.1g, place successively 50mL, 25mL, 25mL, in the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30min, centrifugal 10min, get supernatant, cross 0.45 μ m miillpore filter, get respectively the free amino acid need testing solution.
3.2 the preparation of total amino acid need testing solution: get pharmaceutical composition of the present invention (according to the preparation of embodiment 1 method) and the bright Bungarus Parvus powder of pharmaceutical composition bulk drug of the present invention (according to the method preparation of embodiment 2), bright long-nosed pit viper powder (according to the method preparation of embodiment 3), bright house lizard powder (according to the method preparation of embodiment 4), get respectively 0.1g, place respectively the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30min, centrifugal 10min, get supernatant, draw respectively supernatant 1mL, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is filled N 230s, rapidly sealing places hydrolysis 22hr in 110 ℃ of thermostatic drying chambers, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10min, get supernatant, cross 0.45 μ m miillpore filter, get respectively the total amino acid need testing solution.
4. chromatographic condition
Chromatographic column: Zorbax Eclipse-AAA post (4.6 * 150mm, 3.5 μ m), flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place.With (acetonitrile: methyl alcohol: water=45: 45: 10) (A) and 40mmol/L Na 2HPO 4(pH 7.8) (B) for mobile phase carries out gradient elution, the gradient elution program is as follows:
Time A(％) B(％)
0 0 100
2.5 0 100
16 30 70
24 57 43
24.8 100 0
29.7 100 0
30.9 0 100
34.7 0 100
5, derivative reaction
Get respectively standard solution or each 0.5 μ L of need testing solution of series concentration, add borate buffer 2.5 μ L, then add successively OPA derivatization reagent 0.5 μ L and FMOC derivatization reagent 0.5 μ L, reacted 1-2 minute, inject high performance liquid chromatograph and carry out stratographic analysis.
6. methodology checking
6.1 method specificity: with 17 seed amino acid hybrid standard product solution and each bulk drug of Jinlong capsule, carry out HPLC behind the derivatization and analyze, chromatogram is seen Fig. 1-Fig. 5, and each amino acid is better separated.
6.2 linear: each the 0.5 μ L of standard solution that gets respectively series concentration, add borate buffer 2.5 μ L, then add successively OPA derivatization reagent 0.5 μ L and FMOC derivatization reagent 0.5 μ L, reacted 1-2 minute, inject high performance liquid chromatograph and carry out stratographic analysis, record amino acid whose peak area response, with concentration peak area is made typical curve, carry out linear regression, obtain equation of linear regression and see Table 1.
6.3 the precision of standard solution is investigated
In testing process, only detect 15 seed amino acids by spectrum, wherein arginine and cystine do not detect, get 100,200, the 15 seed amino acid hybrid standard product solution except arginine and cystine of 400pmol/ μ L, carry out respectively above-mentioned derivative reaction, then inject high performance liquid chromatograph analysis, repeat 6 times, record the peak area response of 15 seed amino acids, calculate the RSD of peak area, the results are shown in Table 2 to table 4, the RSD of the peak area precision of 15 seed amino acid hybrid standard product solution of three kinds of concentration can satisfy the requirement of analysis all less than 5%.
The amino acid whose linear equation of table 1 and related coefficient
Figure BDA0000069169050000101
Figure BDA0000069169050000111
The precision of table 2 standard solution is investigated (100pmol/ μ L)
Figure BDA0000069169050000112
The precision of table 3 standard solution is investigated (200pmol/ μ L)
Figure BDA0000069169050000113
Figure BDA0000069169050000121
The precision of table 4 standard solution is investigated (400pmol/ μ L)
Figure BDA0000069169050000122
6.4 the study on the stability of standard solution
Get respectively 100,200, the 15 seed amino acid hybrid standard product solution 0.5 μ L of 400pmol/ μ L except arginine and cystine, carry out above-mentioned derivative reaction respectively at 0h, 4h, 8h, 12h, 16h, 24h, then inject high performance liquid chromatograph analysis, record the peak area response of 15 seed amino acids, calculate the RSD of peak area, get in a few days stability result of standard items, see Table 5 to table 7, the RSD of 15 seed amino acid peak areas is less than 5%, show 15 seed amino acid hybrid standard product solution 24h with interior be stable; Respectively at preparing first day, second day, carrying out above-mentioned derivative reaction in the 3rd day, the 4th day, the 5th day, injecting high performance liquid chromatograph analyzes, record the peak area response of 15 seed amino acids, calculate the RSD of peak area, get in the daytime stability result of standard items, see Table 8 to table 10, the RSD of 15 seed amino acid peak areas less than 5% show 15 seed amino acid hybrid standard product solution five days with interior be stable.
The in a few days study on the stability of table 5 standard solution (100pmol/ μ L)
Figure BDA0000069169050000131
The in a few days study on the stability of table 6 standard solution (200pmol/ μ L)
Figure BDA0000069169050000141
The in a few days study on the stability of table 7 standard solution (400pmol/ μ L)
Figure BDA0000069169050000142
In the daytime the study on the stability of table 8 standard solution (100pmol/ μ L)
Figure BDA0000069169050000143
Figure BDA0000069169050000151
In the daytime the study on the stability of table 9 standard solution (200pmol/ μ L)
Figure BDA0000069169050000152
In the daytime the study on the stability of table 10 standard solution (400pmol/ μ L)
Figure BDA0000069169050000153
Figure BDA0000069169050000161
6.5 the test sample Free Amino Acids is measured the investigation of repeatability
Repeat to prepare the free amino acid need testing solution of 6 parts of pharmaceutical compositions of the present invention (according to the method preparation of embodiment 1) sample, get every part of need testing solution 0.5 μ L, carry out above-mentioned derivative reaction, inject high performance liquid chromatograph and analyze, detect 14 seed amino acids, record the peak area response of 14 seed amino acids, calculate the RSD of peak area, the results are shown in Table the RSD of peak area response of 11,14 seed amino acids less than 5%, can satisfy the analysis requirement.
Table 11 pharmaceutical composition test sample of the present invention Free Amino Acids is measured the investigation of repeatability
Figure BDA0000069169050000162
6.6 the stability of need testing solution Free Amino Acids
Get pharmaceutical composition of the present invention (according to the method preparation of embodiment 1) and the bright house lizard powder of bulk drug of the present invention (according to the method preparation of implementing 2), bright Bungarus Parvus powder (according to the method preparation of implementing 3), the free amino acid need testing solution 0.5 μ L of bright long-nosed pit viper powder (according to the method preparation of implementing 4) bulk drug, respectively at 0h after the preparation, 2h, 4h, 8h, 12h, 24h carries out above-mentioned derivative reaction, then inject high performance liquid chromatograph analysis, record amino acid whose peak area response, calculate the RSD of peak area, the results are shown in Table 12 to table 15, the RSD of amino acid whose peak area response is less than 5% in the bright Bungarus Parvus need testing solution of pharmaceutical composition need testing solution of the present invention and bulk drug, illustrate the amino acid of surveying in 24 hours, be stable.In the bright long-nosed pit viper need testing solution of bulk drug amino acid whose peak area response 8 hours with interior RSD less than 5%, illustrate that institute's amino acid of surveying stablized (particularly lysine) in 8 hours.In the bright house lizard need testing solution of bulk drug amino acid whose peak area response 12 hours with interior RSD less than 5%, illustrate that institute's amino acid of surveying stablized (particularly lysine) in 12 hours.In view of study on the stability result in a few days, respectively at first day after the preparation, second day, the 3rd day, the bright Bungarus Parvus need testing solution of pharmaceutical composition test sample of the present invention and bulk drug carried out above-mentioned derivative reaction in the 4th day, injecting high performance liquid chromatograph analyzes, record amino acid whose peak area response, calculate the RSD of peak area, the results are shown in Table 16-table 17, amino acid was stable (particularly lysine and proline) in one day only in the pharmaceutical composition of the present invention, the RSD of amino acid whose peak area response in four days be less than 5% in the bright Bungarus Parvus need testing solution of bulk drug, illustrate that institute's amino acid of surveying stablized in four days.
The in a few days study on the stability of table 12 pharmaceutical composition need testing solution of the present invention Free Amino Acids
Figure BDA0000069169050000171
The in a few days study on the stability of the bright Bungarus Parvus need testing solution of table 13 bulk drug Free Amino Acids
Figure BDA0000069169050000172
The in a few days study on the stability of the bright long-nosed pit viper need testing solution of table 14 bulk drug Free Amino Acids
Figure BDA0000069169050000181
The in a few days study on the stability of the bright house lizard need testing solution of table 15 bulk drug Free Amino Acids
Figure BDA0000069169050000182
In the daytime the study on the stability of table 16 pharmaceutical composition need testing solution of the present invention Free Amino Acids
Figure BDA0000069169050000183
Figure BDA0000069169050000191
In the daytime the study on the stability of the bright Bungarus Parvus need testing solution of table 17 bulk drug Free Amino Acids
Figure BDA0000069169050000192
6.7 the study on the stability of total amino acid in the test sample
Get pharmaceutical composition of the present invention (according to the method preparation of embodiment 1) and the bright Bungarus Parvus powder of bulk drug of the present invention (according to the method preparation of embodiment 2), bright long-nosed pit viper powder (according to the method preparation of embodiment 3), bright house lizard powder (according to the method preparation of embodiment 4) total amino acid need testing solution 0.5 μ L, respectively at 0h after the preparation, 2h, 4h, 8h, 12h, 24h carries out above-mentioned derivative reaction, injecting high performance liquid chromatograph analyzes, record amino acid whose peak area response, calculate the RSD of peak area, get the in a few days stability result of total amino acid in the test sample, the results are shown in Table 18-table 21, pharmaceutical composition need testing solution of the present invention, the mensuration of total amino acid all needs to carry out immediately lysine after opening ampulla in the bright long-nosed pit viper need testing solution of bulk drug of the present invention and the bright house lizard need testing solution, proline, the less stable of phenylalanine and isoleucine; The RSD of the peak area response of total amino acid less than 5%, illustrated in 12 hours it is stable in the bright Bungarus Parvus need testing solution of bulk drug of the present invention in 12 hours.
The in a few days study on the stability of total amino acid in the table 18 pharmaceutical composition need testing solution of the present invention
Figure BDA0000069169050000201
The in a few days study on the stability of total amino acid in the bright house lizard need testing solution of table 19
Figure BDA0000069169050000202
The in a few days study on the stability of total amino acid in the bright long-nosed pit viper need testing solution of table 20
Figure BDA0000069169050000203
Figure BDA0000069169050000211
The in a few days study on the stability of total amino acid in the bright Bungarus Parvus need testing solution of table 21
Figure BDA0000069169050000212
6.8 the precision of need testing solution free amino acid is investigated
Get the need testing solution 0.5 μ L of pharmaceutical composition free amino acid of the present invention, carry out above-mentioned derivative reaction, injecting high performance liquid chromatograph analyzes, repeat sample introduction 6 times, record amino acid whose peak area response, calculate the RSD of peak area, the results are shown in Table 22, the amino acid whose peak area RSD that surveys less than 5%, can satisfy the requirement of amino acid analysis.
The precision of table 22 need testing solution free amino acid is investigated
Figure BDA0000069169050000213
Figure BDA0000069169050000221
6.9 the recovery is investigated
Get the free amino acid need testing solution 0.5mL of drug regimen matter sample of the present invention in the 1mL measuring bottle, add 17 seed amino acid standard items mixing storing solutions, 200 μ L, with water dilution and constant volume, mixing repeats to prepare 6 parts, carry out above-mentioned derivative reaction, inject high performance liquid chromatograph and carry out stratographic analysis, record amino acid whose peak area response, the substitution typical curve calculates amino acid whose content in the test sample, calculate recovery rate the results are shown in Table 23.
Table 23 determination of recovery rates result
Figure BDA0000069169050000222
7, the mensuration of test sample
Get respectively pharmaceutical composition of the present invention (according to preparation method's preparation of embodiment 1) and the bright Bungarus Parvus powder of pharmaceutical composition bulk drug of the present invention (according to preparation method's preparation of embodiment 2), bright long-nosed pit viper powder (according to preparation method's preparation of embodiment 3), the need testing solution 0.5 μ L of bright house lizard powder (according to preparation method's preparation of embodiment 4) free amino acid and total amino acid, carry out above-mentioned derivative reaction, injecting high performance liquid chromatograph analyzes, record corresponding amino acid whose peak area response, the substitution typical curve, calculate amino acid whose content, the results are shown in Table 24 to table 27.
The assay of table 24 pharmaceutical composition test sample of the present invention
Figure BDA0000069169050000231
The assay of the bright house lizard test sample of table 25 bulk drug
Figure BDA0000069169050000232
Figure BDA0000069169050000241
The assay of the bright Bungarus Parvus test sample of table 26 bulk drug
Figure BDA0000069169050000242
The assay of the bright long-nosed pit viper test sample of table 27 bulk drug
Figure BDA0000069169050000243
Figure BDA0000069169050000251
Six, conclusion
Contain a large amount of amino acid in the bright medicine Jinlong capsule of the present invention, uses the quality that content assaying method mensuration amino acid of the present invention can better be controlled fresh medicine preparation, and the chromatographic condition of content assaying method of the present invention can be effectively separates amino acid in 17.Precision, stability and reappearance are good in the methodological study of content assaying method of the present invention simultaneously.Amino acid content is large in the especially bright animal drugs of bright medicine, and the present invention has established the method for bright medicine Contents of Amino Acids, lays a good foundation for bright medicine quality standard improves.
Following embodiment all can realize the effect of above-mentioned experimental example.
The content assaying method of embodiment 1 medicament composition capsule agent of the present invention
1, the preparation of capsule
Bright house lizard 1500kg, bright Bungarus Parvus 750kg, bright long-nosed pit viper 750kg cut bright house lizard, bright Bungarus Parvus altogether with bright long-nosed pit viper and to cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172kg inclusion, the filtrate vacuum freeze drying is ground into fine grained, capsule is made in low temperature drying;
2, assay
(1) preparation of standard solution:
A, 0.1mol/L hydrochloric acid solution: get 37.5% concentrated hydrochloric acid 0.83mL in the 100mL measuring bottle, thin up also is settled to scale;
The preparation of b, 17 seed amino acid hybrid standard product solution: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in the 1mL volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.5g in the brown measuring bottle of 50mL, add concentrated hydrochloric acid 25mL, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: get NaOH 20.0g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: get NaH 2PO 45.5g being dissolved in water also, constant volume with 10mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in the 1L measuring bottle;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get capsule 0.1g of the present invention, place the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get capsule 0.1g of the present invention, place the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=PH was 7.8 40mmol/L Na as mobile phase A in 45: 45: 10 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
(4) measurement result
Detect 14 seed amino acids in the medicament composition capsule of the present invention, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; Wherein the content of gross protein is 1.9%, the content of total free amino acid is 12.52%, and wherein aspartic acid content is 0.55%, content of glutamic acid is 1.79%, serine content is 0.54%, histidine content is 0.52%, glycocoll content is 1.15%, threonine content is 0.62%, alanine content is 2.01%, valine content is 0.86%, methionine content is 0.36%, phenylalanine content is 0.5%, isoleucine content is 0.53%, leucine content is 0.96%, lysine content is 1.30%, proline content is 0.82%; The content of total amino acid is 14.41%, and wherein aspartic acid content is 0.80%, content of glutamic acid is 2.29%, serine content is 0.72%, histidine content is 0.73%, glycocoll content is 1.53%, threonine content is 0.73%, alanine content is 2.08%, valine content is 0.94%, methionine content is 0.42%, phenylalanine content is 0.72%, isoleucine content is 0.72%, leucine content is 1.15%, lysine content is 0.82%, proline content is 0.76%.
The content assaying method of the bright Bungarus Parvus of embodiment 2 pharmaceutical composition bulk drugs of the present invention
1, the preparation of bright Bungarus Parvus powder
Get bright Bungarus Parvus 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172g inclusion, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
2, assay
(1) preparation of standard solution: (with embodiment 1)
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get the bright Bungarus Parvus sample powder of bulk drug of the present invention 0.1g, place the 25mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get the bright Bungarus Parvus sample powder of bulk drug of the present invention 0.1g, place the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 1)
(4) measurement result
Detect 12 seed amino acids in the bright Bungarus Parvus of bulk drug of the present invention, be respectively asparatate, glutamic acid, serine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine and proline; Wherein the content of gross protein is 0.13%, the content of total free amino acid is 6.02%, and wherein aspartic acid content is 0.25%, content of glutamic acid is 0.57%, serine content is 0.15%, glycocoll content is 0.68%, threonine content is 0.39%, alanine content is 1.48%, valine content is 0.62%, methionine content is 0.24%, phenylalanine content is 0.51%, isoleucine content is 0.40%, leucine content is 0.60%, proline content is 0.15%; The content of total amino acid is 6.15%, and wherein aspartic acid content is 0.39%, content of glutamic acid is 0.70%, serine content is 0.30%, glycocoll content is 0.74%, threonine content is 0.39%, alanine content is 1.20%, valine content is 0.53%, methionine content is 0.28%, phenylalanine content is 0.39%, isoleucine content is 0.40%, leucine content is 0.59%, proline content is 0.23%.
The content assaying method of the bright long-nosed pit viper of embodiment 3 pharmaceutical composition bulk drugs of the present invention
1, the preparation of bright long-nosed pit viper powder
Get bright long-nosed pit viper 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172g inclusion, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
2, assay
(1) preparation of standard solution: (with embodiment 1)
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get bright long-nosed pit viper sample powder 0.1g, place the 25mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get bright long-nosed pit viper sample powder 0.1g, place respectively the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 1)
(4) measurement result
Detect 15 seed amino acids in the bright long-nosed pit viper of bulk drug of the present invention, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline; Wherein the content of gross protein is 18.03%, the content of total free amino acid is 5.09%, and wherein content of glutamic acid is 0.17%, serine content is 0.12%, histidine content is 0.18%, glycocoll content is 0.51%, threonine content is 0.38%, alanine content is 1.13%, valine content is 0.40%, isoleucine content is 0.22%, leucine content is 0.34%, lysine content is 1.64%; The content of total amino acid is 23.12%, and wherein aspartic acid content is 2.28%, content of glutamic acid is 3.17%, serine content is 1.18%, histidine content is 1.09%, glycocoll content is 1.52%, threonine content is 1.39%, alanine content is 2.16%, tyrosine content is 0.91%, valine content is 1.60%, methionine content is 0.51%, phenylalanine content is 1.11%, isoleucine content is 1.22%, leucine content is 2.17%, lysine content is 2.11%, the content of proline is 0.74%.
The content assaying method of the bright house lizard of embodiment 4 pharmaceutical composition bulk drugs of the present invention
1, the preparation of bright house lizard powder
Get bright house lizard 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172g inclusion, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
2, assay
(1) preparation of standard solution: (with embodiment 1)
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get bright house lizard powder 0.1g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get bright house lizard powder 0.1g, place the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 1)
(4) measurement result
Detect 14 seed amino acids in the bright house lizard of pharmaceutical composition bulk drug of the present invention, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; Wherein the content of gross protein is 1.77%; The content of total free amino acid is 22.50%, and wherein aspartic acid content is 1.33%, content of glutamic acid is 3.29%, serine content is 0.95%, histidine content is 0.72%, glycocoll content is 1.64%, threonine content is 0.98%, alanine content is 2.48%, valine content is 1.36%, methionine content is 0.68%, phenylalanine content is 0.93%, isoleucine content is 1.00%, leucine content is 1.67%, lysine content is 4.13%, proline content is 1.36%; The content of total amino acid is 24.26%, and wherein aspartic acid content is 1.92%, content of glutamic acid is 4.39%, serine content is 1.19%, histidine content is 0.95%, glycocoll content is 2.16%, threonine content is 1.22%, alanine content is 2.62%, valine content is that content is 1.60%, methionine content is 0.71%, phenylalanine content is 1.20%, isoleucine content is 1.20%, leucine content is 1.89%, lysine content is 2.06%, proline content is 1.17%.
The assay of embodiment 5 medicinal composition tablets of the present invention
1, the preparation of medicinal composition tablets of the present invention
Bright house lizard 800kg, bright Bungarus Parvus 1000kg, bright long-nosed pit viper 500kg cut bright house lizard, bright Bungarus Parvus altogether with bright long-nosed pit viper and to cut into meat gruel, grind to form pasty state, after adding the homogenate of 4 times of volume water, place-20 ℃ freezing 24 hours, take out, place 25 ℃ of thawings, filter, filtrate is centrifugal with 14000 rev/mins, draw supernatant, ultrafiltration is got filtrate and is added the freeze drying of beta-schardinger dextrin-40kg inclusion final vacuum, be ground into fine grained, add starch 132kg, mixing, tablet is made in low temperature drying.
2, assay
(1) preparation of standard solution:
A, 0.1mol/L hydrochloric acid solution: get 36.5% concentrated hydrochloric acid 0.83mL in the 100mL measuring bottle, thin up also is settled to scale;
The preparation of b, series concentration 17 seed amino acid hybrid standard product solution: 17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution (being purchased from Agilent company), 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in the 1mL volumetric flask, with 0.1mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 251000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol: get phenol 0.5g in the brown measuring bottle of 50mL, add concentrated hydrochloric acid 25mL, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution: take by weighing NaOH 20.0g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40mol/L Na 2HPO 4Damping fluid: take by weighing NaH 2PO 45.5g being dissolved in water also, constant volume with 10mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in the 1L measuring bottle;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get tablet 0.1g of the present invention, place successively the 25mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get tablet 0.1g of the present invention, place respectively the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=PH was 7.8 40mmol/L Na as mobile phase A in 45: 45: 10 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
(4), measurement result
Detect 14 seed amino acids in the medicinal composition tablets of the present invention, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; Wherein the content of gross protein is 2.8%, the content of total free amino acid is 15.73%, and wherein aspartic acid content is 0.75%, content of glutamic acid is 1.99%, serine content is 0.74%, histidine content is 0.62%, glycocoll content is 1.30%, threonine content is 0.84%, alanine content is 2.61%, valine content is 1.06%, methionine content is 0.40%, phenylalanine content is 0.6%, isoleucine content is 0.73%, leucine content is 1.26%, lysine content is 1.71%, proline content is 1.12%; The content of total amino acid is 17.05%, and wherein aspartic acid content is 1.10%, content of glutamic acid is 2.39%, serine content is 1.02%, histidine content is 1.03%, glycocoll content is 1.63%, threonine content is 0.75%, alanine content is 3.08%, valine content is 1.04%, methionine content is 0.62%, phenylalanine content is 0.92%, isoleucine content is 0.94%, leucine content is that content is 1.65%, lysine content is 0.92%, proline content is 0.96%.The assay of the bright Bungarus Parvus powder of embodiment 6 pharmaceutical composition Raw medicines of the present invention
1, the preparation of the bright Bungarus Parvus of bulk drug
Get bright Bungarus Parvus 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
2, assay
(1) preparation of standard solution: (with embodiment 5)
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get bright Bungarus Parvus powder 0.1g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get bright Bungarus Parvus powder 0.1g, place respectively the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/L NaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 5)
(4) measurement result
Detect 12 seed amino acids in the bright Bungarus Parvus of above-mentioned raw materials medicine, be respectively asparatate, glutamic acid, serine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine and proline; Wherein the content of gross protein is 0.18%, the content of total free amino acid is 7.79%, and wherein aspartic acid content is 0.43%, content of glutamic acid is 0.78%, serine content is 0.20%, glycocoll content is 0.98%, threonine content is 0.19%, alanine content is 1.93%, valine content is 0.62%, methionine content is 0.33%, phenylalanine content is 0.71%, isoleucine content is 0.55%, leucine content is 0.87%, proline content is 0.20%; The content of total amino acid is 8.19%, and wherein aspartic acid content is 0.55%, content of glutamic acid is 0.98%, serine content is 0.44%, glycocoll content is 1.02%, threonine content is 0.51%, alanine content is 1.68%, valine content is 0.73%, methionine content is 0.40%, phenylalanine content is 0.41%, isoleucine content is 0.35%, leucine content is 0.79%, proline content is 0.33%.
The assay of the bright long-nosed pit viper powder of embodiment 7 pharmaceutical composition Raw medicines of the present invention
1, the preparation of the bright long-nosed pit viper powder of bulk drug
Get bright long-nosed pit viper 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172g inclusion, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
2, assay
(1) preparation of standard solution: (with embodiment 5)
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution: get the bright long-nosed pit viper powder of bulk drug 0.1g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get the bright long-nosed pit viper powder of bulk drug 0.1g, place the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add 2.4mL10mol/LNaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 5)
(4) measurement result
Detect 15 seed amino acids in the bright long-nosed pit viper of above-mentioned raw materials medicine, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline; Wherein the content of gross protein is 25.13%, the content of total free amino acid is 6.9%, and wherein content of glutamic acid is 0.21%, serine content is 0.17%, histidine content is 0.25%, glycocoll content is 0.68%, threonine content is 0.42%, alanine content is 1.53%, valine content is 0.58%, isoleucine content is 0.30%, leucine content is 0.42%, lysine content is 2.34%; The content of total amino acid is 32.88%, and wherein aspartic acid content is 3.21%, content of glutamic acid is 4.37%, serine content is 1.56%, histidine content is 1.43%, glycocoll content is 2.23%, threonine content is 1.89%, alanine content is 3.06%, tyrosine content is 1.31%, valine content is 2.35%, methionine content is 0.70%, phenylalanine content is 1.88%, isoleucine content is 1.68%, leucine content is 3.07%, lysine content is 3.02%, the content of proline is 1.12%.
The assay of the bright house lizard powder of embodiment 8 pharmaceutical composition Raw medicines of the present invention
1, the preparation of the bright house lizard powder of bulk drug
Get bright house lizard 3000g, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172g inclusion, the filtrate vacuum freeze drying is ground into fine grained, low temperature drying adds conventional auxiliary material according to common process and makes tablet.
2, assay
(1) preparation of standard solution: (with embodiment 5)
(2) preparation of need testing solution:
The preparation of a, free amino acid need testing solution: get the bright house lizard powder of bulk drug of the present invention 0.1g, place the 50mL measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution: get the bright house lizard powder of bulk drug of the present invention 0.1g, place the 15mL plastic centrifuge tube, after adding water 10mL and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get the 1mL supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 4mL that contains 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4mL10mol/LNaOH solution, mixing, be transferred in the 10mL measuring bottle, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition (with embodiment 5)
(4) measurement result
Detect 14 seed amino acids in the bright house lizard of bulk drug of the present invention, be respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline; Wherein the content of gross protein is 2.42%; The content of total free amino acid is 29.94%, and wherein aspartic acid content is 1.95%, content of glutamic acid is 4.19%, serine content is 1.22%, histidine content is 0.82%, glycocoll content is 1.98%, threonine content is 1.35%, alanine content is 3.28%, valine content is 1.86%, methionine content is 0.83%, phenylalanine content is 1.25%, isoleucine content is 1.32%, leucine content is 1.98%, lysine content is 6.03%, proline content is 1.88%; The content of total amino acid is 32.33%, and wherein aspartic acid content is 1.98%, content of glutamic acid is 6.29%, serine content is 1.59%, histidine content is 0.99%, glycocoll content is 3.12%, threonine content is 1.42%, alanine content is 3.72%, valine content is that content is 1.95%, methionine content is 0.81%, phenylalanine content is 1.76%, isoleucine content is 1.65%, leucine content is 2.32%, lysine content is 3.05%, proline content is 1.68%.

Claims (19)

1. the content assaying method of a pharmaceutical composition, its feature in the method for comprising the steps:
(1) preparation of standard solution
A, 0.1 mol/L hydrochloric acid solution
Get concentrated hydrochloric acid 0.83 parts by volume of 36%-38% in 100 parts by volume measuring bottles, thin up also is settled to scale;
B, series concentration 17 seed amino acid hybrid standard product solution
17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution, 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 0.5-1.5 parts by volume volumetric flask, with 0.1 mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol
Get phenol 0.2-0.8 weight portion in the brown measuring bottle of 30-70 parts by volume, add concentrated hydrochloric acid 15-35 parts by volume, be dissolved in water and be settled to scale;
D, 10 mol/L NaOH solution
Get NaOH 10-30 weight portion, place 30-70 parts by volume measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40 mol/L Na 2HPO 4Damping fluid
Get NaH 2PO 4The 3-8 weight portion, being dissolved in water also, constant volume with 10 mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 500-1500 parts by volume measuring bottle;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution
Take by weighing pharmaceutical composition 0.05-0.15 weight portion, place 25-75 parts by volume measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 15-45 minute, centrifugal 5-15 minute, get supernatant, cross 0.3-0.6 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution
Take by weighing pharmaceutical composition 0.05-0.15 weight portion, place 10-20 parts by volume plastic centrifuge tube, after adding water 5-15 parts by volume and fully dissolving, ultrasonic extraction 15-45 minute centrifugal 5-15 minute, is got supernatant, get 0.05-1.5 parts by volume supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 2-6 parts by volume that contains 1% phenol, vacuum nitrogen N 215-45 second, rapidly sealing placed in the 80-150 ℃ of thermostatic drying chamber hydrolysis 12-32 hour, take out, let cool to room temperature, add 1.2-3.6 parts by volume 10 mol/L NaOH solution, mixing, be transferred in the 5-15 parts by volume measuring bottle, thin up also is settled to scale, mixing, centrifugal 5-15 minute, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=25-65:25-65:5-15 is as mobile phase A, and pH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, the gradient elution program is as follows:
0-2.5min mobile phase A is 0%, Mobile phase B is 100%;
2.5-16min mobile phase A is 30%, Mobile phase B is 70%;
The 16-24min mobile phase A is 57%, and Mobile phase B is 43%;
24-24.8min mobile phase A is 100%, Mobile phase B is 0%;
24.8-29.7min mobile phase A is 100%, Mobile phase B is 0%;
29.7-30.9min mobile phase A is 0%, Mobile phase B is 100%;
30.9-34.7min mobile phase A is 0%, Mobile phase B is 100%;
(4) measurement result
Detect 14 seed amino acids in the described pharmaceutical composition, wherein the content of gross protein is 1.9%-2.85%, and the content of total free amino acid is 12.52%-18.78%, and the content of total amino acid is 14.41%-21.62%;
The bulk drug of described pharmaceutical composition consists of:
The bright Bungarus Parvus 400-1100 of bright house lizard 700-2300 weight portion weight portion
Bright long-nosed pit viper 400-1100 weight portion.
2. the content assaying method of pharmaceutical composition as claimed in claim 1 is characterized in that the method comprises the steps:
(1) preparation of standard solution
A, 0.1 mol/L hydrochloric acid solution
Get 37.5% concentrated hydrochloric acid, 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
The preparation of b, 17 seed amino acid hybrid standard product solution
17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution, 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 1 parts by volume measuring bottle, with 0.1 mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol
Get phenol 0.5 weight portion in the brown measuring bottle of 50 parts by volume, add concentrated hydrochloric acid 25 parts by volume, be dissolved in water and be settled to scale;
D, 10 mol/L NaOH solution
Get NaOH 20.0 weight portions, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40 mol/L Na 2HPO 4Damping fluid
Get NaH 2PO 45.5 weight portion, being dissolved in water also, constant volume with 10 mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 1000 parts by volume measuring bottles;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution
Take by weighing pharmaceutical composition 0.1 weight portion, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution
Take by weighing pharmaceutical composition 0.1 weight portion, place 15 parts by volume plastic centrifuge tubes, after adding water 10 parts by volume and fully dissolving, ultrasonic extraction 30 minutes, centrifugal 10 minutes, get supernatant, get 1 parts by volume supernatant, place peace to cut open bottle, add 6mol/L hydrochloric acid 4 parts by volume that contain 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4 parts by volume, 10 mol/L NaOH solution, mixing, be transferred in the 10 parts by volume measuring bottles, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=45:45:10 is as mobile phase A, and PH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
(4) measurement result
Detect 14 seed amino acids in the aforementioned pharmaceutical compositions, wherein the content of gross protein is 1.9%, and the content of total free amino acid is 12.52%, and the content of total amino acid is 14.41%.
3. the content assaying method of pharmaceutical composition as claimed in claim 1 or 2 is characterized in that 14 seed amino acids are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline in the described pharmaceutical composition.
4. the content assaying method of pharmaceutical composition as claimed in claim 3 is characterized in that the content Mid-Heaven Gate winter histidine content of described total free amino acid is 0.55%-0.83%, content of glutamic acid is 1.79%-2.79%, serine content is 0.54%-0.81%, histidine content is 0.52%-0.78%, glycocoll content is 1.15%-1.73%, threonine content is 0.62%-0.93%, alanine content is 2.01%-3.02%, valine content is 0.86%-1.29%, methionine content is 0.36%-0.54%, phenylalanine content is 0.5%-0.75%, isoleucine content is 0.53%-0.80%, leucine content is 0.96%-1.44%, lysine content is 1.30%-1.95%, proline content is 0.82%-1.23%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.80%-1.20%, content of glutamic acid is 2.29%-3.44%, serine content is 0.72%-1.08%, histidine content is 0.73%-1.10%, glycocoll content is 1.53%-2.30%, threonine content is 0.73%-1.10%, alanine content is 2.08%-3.12%, valine content is 0.94%-1.41%, methionine content is 0.42%-0.63%, phenylalanine content is 0.72%-1.08%, isoleucine content is 0.72%-1.08%, leucine content is 1.15%-2.27%, lysine content is 0.82%-1.23%, proline content is 0.76%-1.14%.
5. the content assaying method of pharmaceutical composition as claimed in claim 4 is characterized in that the content Mid-Heaven Gate winter histidine content of described total free amino acid is 0.55%, content of glutamic acid is 1.79%, serine content is 0.54%, histidine content is 0.52%, glycocoll content is 1.15%, threonine content is 0.62%, alanine content is 2.01%, valine content is 0.86%, methionine content is 0.36%, phenylalanine content is 0.5%, isoleucine content is 0.53%, leucine content is 0.96%, lysine content is 1.30%, proline content is 0.82 %; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.80%, content of glutamic acid is 2.29%, serine content is 0.72%, histidine content is 0.73%, glycocoll content is 1.53%, threonine content is 0.73%, alanine content is 2.08%, valine content is 0.94%, methionine content is 0.42%, phenylalanine content is 0.72%, isoleucine content is 0.72%, leucine content is that 1.15 content are that %, lysine content are 0.82%, proline content is 0.76%.
6. such as the content assaying method of claim 1,2,4 or 5 described pharmaceutical compositions, it is characterized in that the bulk drug of described pharmaceutical composition consists of:
Bright house lizard 1500 weight portions, bright Bungarus Parvus 750 weight portions, bright long-nosed pit viper 750 weight portions;
Or bright house lizard 800 weight portions, bright Bungarus Parvus 1000 weight portions, bright long-nosed pit viper 500 weight portions;
Or bright house lizard 2000 weight portions, bright Bungarus Parvus 500 weight portions, bright long-nosed pit viper 100 weight portions.
7. the content assaying method of pharmaceutical composition as claimed in claim 3 is characterized in that the bulk drug of described pharmaceutical composition consists of:
Bright house lizard 1500 weight portions, bright Bungarus Parvus 750 weight portions, bright long-nosed pit viper 750 weight portions;
Or bright house lizard 800 weight portions, bright Bungarus Parvus 1000 weight portions, bright long-nosed pit viper 500 weight portions;
Or bright house lizard 2000 weight portions, bright Bungarus Parvus 500 weight portions, bright long-nosed pit viper 100 weight portions.
8. such as the content assaying method of claim 1,2,4,5 or 7 described pharmaceutical compositions, it is characterized in that described pharmaceutical composition makes by the following method:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, the water gaging homogenate such as add after, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draw supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying, be ground into fine grained, tablet, capsule, granule or pill are made in low temperature drying;
Or bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, after adding the homogenate of 3-5 times of volume water, place-18 ℃ to-22 ℃ freezing 24 hours, take out, place 15 ℃ of-30 ℃ of thawings, filter, filtrate is centrifugal with 12000-16000 rev/min, draw supernatant, ultrafiltration is got filtrate and is added the freeze drying of beta-schardinger dextrin-20-60 weight portion inclusion final vacuum, is ground into fine grained, add starch 66-198 weight portion, mixing, tablet, capsule, granule or pill are made in low temperature drying.
9. the content assaying method of pharmaceutical composition as claimed in claim 3 is characterized in that described pharmaceutical composition makes by the following method:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, the water gaging homogenate such as add after, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draw supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying, be ground into fine grained, tablet, capsule, granule or pill are made in low temperature drying;
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, add the homogenate of 3-5 times of volume water after, place-18 ℃ to-22 ℃ freezing 24 hours, take out, place 15 ℃ of-30 ℃ of thawings, filter, filtrate is centrifugal with 12000-16000 rev/min, draws supernatant, ultrafiltration is got filtrate and is added the freeze drying of beta-schardinger dextrin-20-60 weight portion inclusion final vacuum, is ground into fine grained, add starch 66-198 weight portion, mixing, tablet, capsule, granule or pill are made in low temperature drying.
10. the content assaying method of pharmaceutical composition as claimed in claim 6 is characterized in that described pharmaceutical composition makes by the following method:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, the water gaging homogenate such as add after, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draw supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying, be ground into fine grained, tablet, capsule, granule or pill are made in low temperature drying;
Or bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, after adding the homogenate of 3-5 times of volume water, place-18 ℃ to-22 ℃ freezing 24 hours, take out, place 15 ℃ of-30 ℃ of thawings, filter, filtrate is centrifugal with 12000-16000 rev/min, draw supernatant, ultrafiltration is got filtrate and is added the freeze drying of beta-schardinger dextrin-20-60 weight portion inclusion final vacuum, is ground into fine grained, add starch 66-198 weight portion, mixing, tablet, capsule, granule or pill are made in low temperature drying.
11. the content assaying method of pharmaceutical composition as claimed in claim 8 is characterized in that described pharmaceutical composition makes by the following method:
Bright house lizard, bright Bungarus Parvus cut altogether with bright long-nosed pit viper cut into meat gruel, grind to form pasty state, add the homogenate of 4 times of volume water after, place-20 ℃ freezing 24 hours, take out, place 25 ℃ of thawings, filter, filtrate is centrifugal with 14000 rev/mins, draws supernatant, ultrafiltration is got filtrate and is added beta-schardinger dextrin-40 weight portion inclusion final vacuum freeze dryings, is ground into fine grained, add starch 132 weight portions, mixing, tablet, capsule, granule or pill are made in low temperature drying.
12. the content assaying method such as claim 9 or 10 described pharmaceutical compositions, the preparation method who it is characterized in that described pharmaceutical composition is: with bright house lizard, bright Bungarus Parvus is cut altogether with bright long-nosed pit viper and cuts into meat gruel, grind to form pasty state, after adding the homogenate of 4 times of volume water, place-20 ℃ freezing 24 hours, take out, place 25 ℃ of thawings, filter, filtrate is centrifugal with 14000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-40 weight portion inclusion final vacuum freeze dryings, be ground into fine grained, add starch 132 weight portions, mixing, tablet is made in low temperature drying, capsule, granule or pill.
13. the content assaying method of a pharmaceutical composition bulk drug is characterized in that the method comprises the steps:
(1) preparation of standard solution
A, 0.1 mol/L hydrochloric acid solution
Get 36%-38% concentrated hydrochloric acid 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
B, the accurate solution of 17 seed amino acid mixing mark product
17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution, 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 0.5-1.5 parts by volume volumetric flask, with 0.1 mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol
Get phenol 0.2-0.8 weight portion in the brown measuring bottle of 30-70 parts by volume, add concentrated hydrochloric acid 15-35 parts by volume, be dissolved in water and be settled to scale;
D, 10 mol/L NaOH solution
Get NaOH 10-30 weight portion, place 30-70 parts by volume measuring bottle, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40 mol/L Na 2HPO 4Damping fluid
Get NaH 2PO 4The 3-8 weight portion, being dissolved in water also, constant volume with 10 mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 500-1500 parts by volume measuring bottle;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution
Take by weighing each 0.05-0.15 weight portion of bright Bungarus Parvus, bright long-nosed pit viper and bright house lizard, place 25-75 parts by volume measuring bottle, be dissolved in water and be settled to scale, ultrasonic extraction 15-45 minute centrifugal 5-15 minute, is got supernatant, cross 0.3-0.6 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution
Take by weighing each 0.05-0.15 weight portion of bright long-noded pit viper, bright long-nosed pit viper and bright house lizard, place 10-20 parts by volume plastic centrifuge tube, after adding water 5-15 parts by volume and fully dissolving, ultrasonic extraction 15-45 minute centrifugal 5-15 minute, is got supernatant, get 0.05-1.5 parts by volume supernatant, place peace to cut open bottle, add the 6mol/L hydrochloric acid 2-6 parts by volume that contains 1% phenol, vacuum is rushed nitrogen N 215-45 second, rapidly sealing placed in the 80-150 ℃ of thermostatic drying chamber hydrolysis 12-32 hour, take out, let cool to room temperature, add respectively 1.2-3.6 parts by volume 10 mol/L NaOH solution, mixing, be transferred in the 5-15 parts by volume measuring bottle, thin up also is settled to scale, mixing, centrifugal 5-15 minute, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=25-65:25-65:5-15 is as mobile phase A, and pH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, the gradient elution program is as follows:
0-2.5min mobile phase A is 0%, Mobile phase B is 100%;
2.5-16min mobile phase A is 30%, Mobile phase B is 70%;
The 16-24min mobile phase A is 57%, and Mobile phase B is 43%;
24-24.8min mobile phase A is 100%, Mobile phase B is 0%;
24.8-29.7min mobile phase A is 100%, Mobile phase B is 0%;
29.7-30.9min mobile phase A is 0%, Mobile phase B is 100%;
30.9-34.7min mobile phase A is 0%, Mobile phase B is 100%;
(4) measurement result
Detect 14 seed amino acids in the bright house lizard of bulk drug, wherein the content of gross protein is 1.77%-2.66%, and the content of total free amino acid is 22.50%-33.75%, and the content of total amino acid is 24.26%-36.39%;
Detect 12 seed amino acids in the bright Bungarus Parvus, wherein the content of gross protein is 0.13%-0.20%, and the content of total free amino acid is 6.02%-9.03%, and the content of total amino acid is 6.15%-9.23%;
Detect 15 seed amino acids in the bright long-nosed pit viper, wherein the content of gross protein is 18.03%-27.05%, and the content of total free amino acid is 5.09%-7.64%, and the content of total amino acid is 23.12%-34.68%.
14. the content assaying method of pharmaceutical composition bulk drug as claimed in claim 13 is characterized in that the method comprises the steps:
(1) preparation of standard solution
A, 0.1 mol/L hydrochloric acid solution
Get 37.5% concentrated hydrochloric acid, 0.83 parts by volume in 100 parts by volume measuring bottles, thin up also is settled to scale;
The preparation of b, 17 seed amino acid hybrid standard product solution
17 seed amino acids difference: asparatate, glutamic acid, serine, histidine, glycocoll, threonine, arginine, alanine, tyrosine, cystine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline;
Get 17 seed amino acid hybrid standard product solution, 25 μ L, 50 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L in 1 parts by volume volumetric flask, with 0.1 mol/L aqueous hydrochloric acid solution dilution and be settled to scale, get the amino-acid mixed standardization product solution of 25-1000pmol/ μ L series concentration;
The 6mol/L hydrochloric acid solution of c, 1% phenol
Get phenol 0.5 weight portion in the brown measuring bottle of 50 parts by volume, add concentrated hydrochloric acid 25 parts by volume, be dissolved in water and be settled to scale;
D, 10mol/L NaOH solution
Get NaOH 20.0 weight portions, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, mixing is transferred in the plastics storage bottle for subsequent use;
E, 40 mol/L Na 2HPO 4Damping fluid
Get NaH 2PO 45.5 weight portion, being dissolved in water also, constant volume with 10 mol/L NaOH solution adjusting pH value to 7.8, mixing, and get final product in 1000 parts by volume measuring bottles;
(2) preparation of need testing solution
The preparation of a, free amino acid need testing solution
Take by weighing each 0.1 weight portion of bright Bungarus Parvus, bright long-nosed pit viper and bright house lizard, place 50 parts by volume measuring bottles, be dissolved in water and be settled to scale, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, cross 0.45 μ m miillpore filter, get the free amino acid need testing solution;
The preparation of b, total amino acid need testing solution
Take by weighing each 0.1 weight portion of bright Bungarus Parvus, bright long-nosed pit viper product and bright house lizard, place 15 parts by volume plastic centrifuge tubes, after adding water 10 parts by volume and fully dissolving, ultrasonic extraction 30 minutes centrifugal 10 minutes, is got supernatant, get 1 parts by volume supernatant, place peace to cut open bottle, add 6mol/L hydrochloric acid 4 parts by volume that contain 1% phenol, vacuum is rushed nitrogen N 230 seconds, rapidly sealing placed in 110 ℃ of thermostatic drying chambers and is hydrolyzed 22 hours, take out, let cool to room temperature, add respectively 2.4 parts by volume, 10 mol/L NaOH solution, mixing, be transferred in the 10 parts by volume measuring bottles, thin up also is settled to scale, mixing, centrifugal 10 minutes, get supernatant, cross 0.45 μ m miillpore filter, get the total amino acid need testing solution;
(3) chromatographic condition
Chromatographic column: 4.6 * 150mm, 3.5 μ m Zorbax Eclipse-AAA posts, flow velocity: 1.5ml/min, column temperature are 35 ℃, auto injection program, UV-detector detect one-level amino acid in the 338nm place, detect secondary amino acid in 262nm wavelength place; Take acetonitrile: methyl alcohol: water=45:45:10 is as mobile phase A, and PH is 7.8 40mmol/L Na 2HPO 4Be Mobile phase B, carry out gradient elution, elution program is as follows:
0-2.5min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
2.5-16min mobile phase A concentration is 30%, Mobile phase B concentration is 70%;
16-24min mobile phase A concentration is 57%, and Mobile phase B concentration is 43%;
24-24.8min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
24.8-29.7min mobile phase A concentration is 100%, Mobile phase B concentration is 0%;
29.7-30.9min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
30.9-34.7min mobile phase A concentration is 0%, Mobile phase B concentration is 100%;
(4) measurement result
Detect 14 seed amino acids in the bright house lizard of described bulk drug, wherein the content of gross protein is 1.77%, and the content of total free amino acid is 22.50%, and the content of total amino acid is 24.26%;
Detect 12 seed amino acids in the described bright Bungarus Parvus, wherein the content of gross protein is 0.13%, and the content of total free amino acid is 6.02%, and the content of total amino acid is 6.15%;
Detect 15 seed amino acids in the described bright long-nosed pit viper, wherein the content of gross protein is 18.03%, and the content of total free amino acid is 5.09%, and the content of total amino acid is 23.12%.
15. such as the content assaying method of claim 13 or 14 described pharmaceutical composition bulk drugs, it is characterized in that 14 seed amino acids in the bright house lizard of described bulk drug are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine, lysine and proline;
12 seed amino acids in the bright Bungarus Parvus of described bulk drug are respectively asparatate, glutamic acid, serine, glycocoll, threonine, alanine, valine, methionine, phenylalanine, isoleucine, leucine and proline;
15 seed amino acids in the bright long-nosed pit viper of described bulk drug are respectively asparatate, glutamic acid, serine, histidine, glycocoll, threonine, alanine, tyrosine, valine, methionine, phenylalanine, isoleucine, leucine, lysine, proline.
16. the content assaying method of pharmaceutical composition bulk drug as claimed in claim 15 is characterized in that the content Mid-Heaven Gate winter histidine content of total free amino acid in the bright house lizard of described bulk drug is 1.33%-2.00%, content of glutamic acid is 3.29%-4.94%, serine content is 0.95%-1.43%, histidine content is 0.72%-1.08%, glycocoll content is 1.64%-2.46%, threonine content is 0.98%-1.47%, alanine content is 2.48%-3.72%, valine content is 1.36%-2.04%, methionine content is 0.68%-1.02%, phenylalanine content is 0.93%-1.40%, isoleucine content is 1.00%-1.50%, leucine content is 1.67%-2.51%, lysine content is 4.13%-6.20%, proline content is 1.36%-2.04%; The content Mid-Heaven Gate winter histidine content of total amino acid is 1.92%-2.88%, content of glutamic acid is 4.39%-6.59%, serine content is 1.19%-1.79%, histidine content is 0.95%-1.43%, glycocoll content is 2.16%-3.24%, threonine content is 1.22%-1.83%, alanine content is 2.62%-3.93%, valine content is that content is 1.60%-2.40%, methionine content is 0.71%-1.07%, phenylalanine content is 1.20%-1.80%, isoleucine content is 1.20%-1.80%, leucine content is 1.89%-2.84%, lysine content is 2.06%-3.09%, proline content is 1.17%-1.76%;
The content Mid-Heaven Gate winter histidine content of total free amino acid is 0.25%-0.38% in the bright Bungarus Parvus of described bulk drug, content of glutamic acid is 0.57%-0.86%, serine content is 0.15%-0.23%, glycocoll content is 0.68%-1.02%, threonine content is 0.39%-0.59%, alanine content is 1.48%-2.22%, valine content is 0.62%-0.93%, methionine content is 0.24%-0.36%, phenylalanine content is 0.51%-0.77%, isoleucine content is 0.40%-0.60%, leucine content is 0.60%-0.90%, proline content is 0.15%-0.23%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.39%-0.59%, content of glutamic acid is 0.70%-1.05%, serine content is 0.30%-0.45%, glycocoll content is 0.74%-1.11%, threonine content is 0.39%-0.59%, alanine content is 1.20%-1.80%, valine content is 0.53%-0.80%, methionine content is 0.28%-0.42%, phenylalanine content is 0.39%-0.59%, isoleucine content is 0.40%-0.60%, leucine content is 0.59%-0.89%, proline content is 0.23%-0.35%;
The content Glutamic Acid content of total free amino acid is that 0.17%-0.26%, serine content are that 0.12%-0.18%, histidine content are that 0.18%-0.27%, glycocoll content are that 0.51%-0.77%, threonine content are that 0.38%-0.57%, alanine content are that 1.13%-1.70%, valine content are that 0.40%-0.60, isoleucine content are that 0.22%-0.33%, leucine content are that 0.34%-0.51%, lysine content are 1.64%-2.72% in the bright long-nosed pit viper of described bulk drug; The content Mid-Heaven Gate winter histidine content of total amino acid is 2.28%-3.42%, content of glutamic acid is 3.17%-4.76%, serine content is 1.18%-1.77%, histidine content is 1.09%-1.64%, glycocoll content is 1.52%-2.28%, threonine content is 1.39%-2.39%, alanine content is 2.16%-3.24%, tyrosine content is 0.91%-1.37, valine content is 1.60%-2.40%, methionine content is 0.51%-0.77%, phenylalanine content is 1.11%-1.67%, isoleucine content is 1.22%-1.83%, leucine content is 2.17%-3.26%, lysine content is 2.11%-3.17%, the content of proline is 0.74%-1.11%.
17. the content assaying method of pharmaceutical composition bulk drug as claimed in claim 16 is characterized in that the content Mid-Heaven Gate winter histidine content of total free amino acid in the bright house lizard of described bulk drug is 1.33%, content of glutamic acid is 3.29%, serine content is 0.95%, histidine content is 0.72%, glycocoll content is 1.64%, threonine content is 0.98%, alanine content is 2.48%, valine content is 1.36%, methionine content is 0.68%, phenylalanine content is 0.93%, isoleucine content is 1.00%, leucine content is 1.67%, lysine content is 4.13%, proline content is 1.36%; The content Mid-Heaven Gate winter histidine content of total amino acid is 1.92%, content of glutamic acid is 4.39%, serine content is 1.19%, histidine content is 0.95%, glycocoll content is 2.16%, threonine content is 1.22%, alanine content is 2.62%, valine content is that content is 1.60%, methionine content is 0.71%, phenylalanine content is 1.20%, isoleucine content is 1.20%, leucine content is 1.89%, lysine content is 2.06%, proline content is 1.17%;
The content Mid-Heaven Gate winter histidine content of total free amino acid is 0.25% in the bright Bungarus Parvus of described bulk drug, content of glutamic acid is 0.57%, serine content is 0.15%, glycocoll content is 0.68%, threonine content is 0.39%, alanine content is 1.48%, valine content is 0.62%, methionine content is 0.24%, phenylalanine content is 0.51%, isoleucine content is 0.40%, leucine content is 0.60%, proline content is 0.15%; The content Mid-Heaven Gate winter histidine content of total amino acid is 0.39%, content of glutamic acid is 0.70%, serine content is 0.30%, glycocoll content is 0.74%, threonine content is 0.39%, alanine content is 1.20%, valine content is 0.53%, methionine content is 0.28%, phenylalanine content is 0.39%, isoleucine content is 0.40%, leucine content is 0.59%, proline content is 0.23%;
The content Glutamic Acid content of total free amino acid is 0.17% in the bright long-nosed pit viper of described bulk drug, serine content is 0.12%, histidine content is 0.18%, glycocoll content is 0.51%, threonine content is 0.38%, alanine content is 1.13%, valine content is 0.40%, isoleucine content is 0.22%, leucine content is 0.34%, lysine content is 1.64%; The content Mid-Heaven Gate winter histidine content of total amino acid is 2.28%, content of glutamic acid is 3.17%, serine content is 1.18%, histidine content is 1.09%, glycocoll content is 1.52%, threonine content is 1.39%, alanine content is 2.16%, tyrosine content is 0.91%, valine content is 1.60%, methionine content is 0.51%, phenylalanine content is 1.11%, isoleucine content is 1.22%, leucine content is 2.17%, lysine content is 2.11%, the content of proline is 0.74%.
18. such as the content assaying method of claim 13,14,16 or 17 described pharmaceutical composition bulk drugs, it is characterized in that described bulk drug makes by the following method:
Get bright house lizard 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying;
Get bright Bungarus Parvus 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying;
Get bright long-nosed pit viper 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
19. the content assaying method of pharmaceutical composition bulk drug as claimed in claim 15 is characterized in that described bulk drug makes by the following method:
Get bright house lizard 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying;
Get bright Bungarus Parvus 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying;
Get bright long-nosed pit viper 3000 weight portions, cut and cut into meat gruel, grind to form pasty state, after the water gaging homogenate such as adding, place-25 ℃ freezing 24 hours, take out, place 37 ℃ of water-baths to melt, three times repeatedly, filter, filtrate is centrifugal with 8000 rev/mins, draws supernatant, ultrafiltration, get filtrate and add beta-schardinger dextrin-172 weight portion inclusions, the filtrate vacuum freeze drying is ground into fine grained, powder is made in low temperature drying.
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