CN100382842C - Orally administered cervus and cucumis polypeptide composition and preparation method thereof - Google Patents
Orally administered cervus and cucumis polypeptide composition and preparation method thereof Download PDFInfo
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Abstract
The present invention provides an oral administration cervus and cucumis polypeptide composition and a preparation method thereof, which relates to an oral administration medicament and a preparation method thereof. The present invention solve the problem that more than 10KD of protein and polypeptide are discarded in the existing preparation process of cervus and cucumis polypeptide composition, and the existing cervus and cucumis polypeptide composition is unsuitable for oral administration and has poor curative effect of oral administration. The oral administration cervus and cucumis polypeptide composition comprises 5 to 95 portions of polypeptide and 5 to 95 portions of excipient according to weight proportions, wherein the polypeptide is composed of deer bone polypeptide and muskmelon seed polypeptide, the weight ratio of the deer bone polypeptide to the muskmelon seed polypeptide is 4: 1 to 1: 4, and the molecular weight of the deer bone polypeptide and the muskmelon seed polypeptide is no greater than 6KD. The preparation method has the steps that (A) preparation of the extracting solution of the deer bone polypeptide; (B) preparation of the extracting solution of the muskmelon seed polypeptide; (C) preparation of the oral administration polypeptide composition. The present invention uses a biologic enzymolysis technology to carry out enzymatic hydrolysis on protein and polypeptide which have high molecular weight in deer bones and muskmelon seeds, so that the deer bone polypeptide and the muskmelon seed polypeptide which have molecular weight no greater than 6 KD, centralized molecular weight distribution and favorable absorption effect on oral administration are obtained.
Description
Technical field
The present invention relates to a kind of oral drugs and preparation method thereof.
Background technology
Protein runs off too much in the cervus and cucumis polypeptide composition preparation process at present, as patent CN1579542A (application number is 200410013602.7) and CN1742778A (application number is 200510108157.7), the above protein and the polypeptide overwhelming majority of molecular weight 10KD is dropped in its preparation process; And existing cervus and cucumis polypeptide composition mostly is the injection injection, all exist be not suitable for oral, the problem of oral curative effect difference.
Summary of the invention
The objective of the invention is to be dropped, to waste serious and existing cervus and cucumis polypeptide composition and be not suitable for problem oral, the oral curative effect difference in order to solve in the existing cervus and cucumis polypeptide composition preparation process protein more than the 10KD and the polypeptide overwhelming majority, and a kind of Orally administered cervus and cucumis polypeptide composition that provides and preparation method thereof.The preparation method of Orally administered cervus and cucumis polypeptide composition is carried out according to the following steps: (A) preparation Os Cervi polypeptide extracting solution: 1. clean fresh Os Cervi, and with deer bone powder essence, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained the Os Cervi crude extract; 4. measure the content of protein and polypeptide in the Os Cervi crude extract, regulating Os Cervi crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Os Cervi crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains the Os Cervi polypeptide extracting solution; (B) preparation Fructus Melo seed polypeptide extracting solution: 1. clean fresh Fructus Melo seed, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained Fructus Melo seed crude extract; 4. measure the content of protein and polypeptide in the Fructus Melo seed crude extract, regulating Fructus Melo seed crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Fructus Melo seed crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains Fructus Melo seed polypeptide extracting solution; (C) prepare oral peptide composition: 1. the polypeptide in the Os Cervi polypeptide extracting solution mixes by 4: 1～1: 4 weight ratio with polypeptide in the Fructus Melo seed polypeptide extracting solution; 2. add the ratio of weight and number adding excipient of 5～95 parts of excipient by 5～95 parts of polypeptide, promptly obtain Orally administered cervus and cucumis polypeptide composition.Orally administered cervus and cucumis polypeptide composition comprises 5～95 parts of polypeptide and 5～95 parts of excipient by ratio of weight and the number of copies, wherein polypeptide is described Os Cervi polypeptide of said method A step and the described Fructus Melo seed of said method B step polypeptide, the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 4: 1～1: 4, Os Cervi polypeptide and Fructus Melo seed polypeptide molecular weight≤6KD.The present invention is a unit of account with the weight of Os Cervi polypeptide and Fructus Melo seed polypeptide, the different of effective ingredient in each batch medicine that the difference because of the place of production, collection phase, raw material individuality and extraction process causes have been avoided, effective ingredient difference is little in each batch medicine, efficacy stability.The present invention adopts comparatively demulcent reaction condition, high temperature, strong acid, highly basic have been reduced to proteinic destruction, also non-active ingredient in the extract be can fully remove simultaneously, thereby the effective ingredient in Os Cervi and the Fructus Melo seed and the precursor component of effective ingredient utilized fully.The present invention adopts biological enzymolysis technology, orderly to the high-molecular weight protein in Os Cervi and the Fructus Melo seed and polypeptide (〉=10KD) carry out enzyme hydrolysis, thereby obtain molecular weight≤6KD, and molecular weight distribution is concentrated, is beneficial to the Os Cervi polypeptide and the Fructus Melo seed polypeptide of oral absorption.
The specific embodiment
The specific embodiment one: the present embodiment Orally administered cervus and cucumis polypeptide composition is made by 5～95 parts of polypeptide and 5～95 parts of excipient by ratio of weight and the number of copies, wherein polypeptide is Os Cervi polypeptide and Fructus Melo seed polypeptide, the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 4: 1～1: 4, Os Cervi polypeptide and Fructus Melo seed polypeptide molecular weight≤6KD.
The specific embodiment two: the difference of the present embodiment and the specific embodiment one is: excipient is made up of in Radix Glycyrrhizae, sucrose, simple syrup, saccharin sodium, sodium carboxymethyl cellulose, dextrin, tween 80, magnesium stearate, carboxymethyl starch sodium, hyprolose, calcium hydrogen phosphate, the starch one or more.Other is identical with embodiment one.
The specific embodiment three: present embodiment and the specific embodiment one or twos' difference is: excipient is made up of in Radix Glycyrrhizae, sucrose, the simple syrup one or more.Other is identical with embodiment one or two.
Present embodiment is made into the cervus and cucumis polypeptide oral liquid.
The specific embodiment four: present embodiment and the specific embodiment one or twos' difference is: excipient is made up of in saccharin sodium, sodium carboxymethyl cellulose, dextrin, tween 80, magnesium stearate, carboxymethyl starch sodium, hyprolose, calcium hydrogen phosphate, the starch one or more.Other is identical with embodiment one or two.
Present embodiment is made into cervus and cucumis polypeptide tablet, capsule, dispersible tablet or suspensoid.
The specific embodiment five: the difference of the present embodiment and the specific embodiment one is: the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 3: 1～1: 3.Other is identical with embodiment one.
The specific embodiment six: the difference of the present embodiment and the specific embodiment one is: the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 2: 1～1: 2.Other is identical with embodiment one.
The specific embodiment seven: the difference of the present embodiment and the specific embodiment one is: Orally administered cervus and cucumis polypeptide composition is made by 10～90 parts of polypeptide and 10～90 parts of excipient by ratio of weight and the number of copies.Other is identical with embodiment one.
The specific embodiment eight: the difference of the present embodiment and the specific embodiment one is: Orally administered cervus and cucumis polypeptide composition is made by 20～80 parts of polypeptide and 20～80 parts of excipient by ratio of weight and the number of copies.Other is identical with embodiment one.
The specific embodiment nine: the preparation method of present embodiment Orally administered cervus and cucumis polypeptide composition is carried out according to the following steps:
(A) preparation Os Cervi polypeptide extracting solution: 1. clean fresh Os Cervi, and with deer bone powder essence, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained the Os Cervi crude extract; 4. measure the content of protein and polypeptide in the Os Cervi crude extract, regulating Os Cervi crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Os Cervi crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains the Os Cervi polypeptide extracting solution;
(B) preparation Fructus Melo seed polypeptide extracting solution: 1. clean fresh Fructus Melo seed, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained Fructus Melo seed crude extract; 4. measure the content of protein and polypeptide in the Fructus Melo seed crude extract, regulating Fructus Melo seed crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Fructus Melo seed crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains Fructus Melo seed polypeptide extracting solution;
(C) prepare oral peptide composition: 1. the polypeptide in the Os Cervi polypeptide extracting solution mixes by 4: 1～1: 4 weight ratio with polypeptide in the Fructus Melo seed polypeptide extracting solution; 2. add the ratio of weight and number adding excipient of 5～95 parts of excipient by 5～95 parts of polypeptide, promptly obtain Orally administered cervus and cucumis polypeptide composition.
Present embodiment is regulated pH value with hydrochloric acid and/or sodium hydroxide.Polypeptide molecular weight concentrates on 4KD～6KD, accounts for 65%～80% of polypeptide thing gross mass.The enzymatic hydrolysis condition that present embodiment is selected is the protein and the polypeptide of enzymolysis macromolecule fully, polypeptide molecular weight is concentrated be distributed in 4KD～6KD, and too small the or enzymolysis of protein and polypeptide enzymolysis can not become aminoacid, and the cervus and cucumis polypeptide composition of molecular weight≤6KD has good oral medication effect again.
According to " the enzyme process polypeptide opinion " of world-renowned polypeptide scientist Zou Yuandong monograph, the proteinic principal mode of absorption of human body is not an aminoacid as can be known, but absorb with the form of polypeptide; The absorption of polypeptide has following characteristics: 1. need not digest, directly absorb.The synthetic polypeptide of human body self is that human body carries out the enzymatic hydrolysis gained with protein and (decomposed by short enzyme, digestive enzyme, pancreatin, pepsin, gastric acid, digestive tract alkaline matter, what have is broken down into little peptide, what have is broken down into amino acid residue, what have is broken down into free amino acid, little peptide finally absorbs by small intestinal, then through human body cell, tissue, organ and blood systemic circulation).And there is layer protecting film on external artificial synthetic little peptide surface; can not be subjected to the various enzymes and the hydrolysis of acid-base material secondary of human body after being taken by human body, its same small intestinal that directly enters with the synthetic peptide of human body is absorbed by small intestinal; enter the human recycle system, the performance biological function.
2. absorb fast.The outer synthetic polypeptide of prosthesis, the oral human body that enters, it is than the speed of aminoacid fast 70%, pass people's oral cavity, stomach apace, directly enter small intestinal, by little intestinal absorption, finally enter blood of human body blood circulation, organ and cell tissue, bring into play its physiological action and biological function rapidly.
3. absorb with complete form.Polypeptide is to be absorbed by the body and to utilize with complete form.
4. polypeptide has 100% characteristics that are absorbed by the body.After the absorption, do not have any Excreta, and be all to be absorbed by the body and to utilize.
5. polypeptide has the advantages that initiatively be absorbed by the body.For because of digestive system defective, obstacle, damage, and can not absorb nutrition person, polypeptide has the advantages that initiatively allow absorption of human body or force absorption of human body.This is poor for those digestion powers, malnutrition, physical weakness person, has great significance.
6. polypeptide has the advantages that preferentially be absorbed by the body.The usual nutrient substance of eating of people, and the amino acid residue and the aminoacid of human body degraded, in the absorption competition of polypeptide, polypeptide has the characteristics of preferential absorption.This and its be undivided by the characteristics of active absorption.
7. human body does not have and need expend body energy the absorption of polypeptide, need not increase digestive tract, particularly the characteristics of gastrointestinal function burden.Polypeptide self has extremely strong activity and energy, its active absorption, forces absorption, be exactly self activity and energy in action.Therefore, it is not that the energy that human body expends self goes to absorb it when being absorbed by the body, but polypeptide with self energy by absorption of human body.Its this distinguishing feature is to the infant of digestive system undeveloped mature, the old people that digestive system is begun to degenerate and be badly in need of nitrogenous source because of hyperkinesia, and athlete, the physical labourer that can not increase the gastrointestinal function burden have significance.
8. polypeptide shows carrier function in human body, can be with the nutrient substance that the mediocrity ate, and particularly calcium etc. adsorbs, pastes, is loaded on the body human body beneficial's trace element.
9. polypeptide can play the transportation instrument in human body.Be transported to each cell of human body, organ, tissue after can being adsorbed on various nutrient substance on the body, be absorbed by the body together and utilize, bring into play different separately functions with body.Here it is, and present people in the world its objective is the reason of polypeptide raw material midbody as medicine and food formula will strengthen drug effect and enriched nutritive, strengthen human body to its absorbance.
10. after polypeptide is absorbed by the body, can in human body, play courier's effect.The courier that it transmits information as neurotransmitter, commander is neural, brings into play self-acting, safeguards the team spirit and the group effect of human nerve, makes human body become flexible more, sensitive, wise.
Illustrate that oral way picked-up polypeptide and peptide composition have the incomparable advantage of other picked-up approach.
The specific embodiment ten: the difference of the present embodiment and the specific embodiment nine is: fresh deer bone powder is broken into the osseous granules of particle diameter less than 1cm.Other is identical with embodiment nine.
The specific embodiment 11: the difference of the present embodiment and the specific embodiment nine is: protease is made up of in papain, bromelain, ficin, cysteine proteinase, the soybean protein enzyme one or more, and enzyme work is 8 * 10
5～11 * 10
5U/g.Other is identical with embodiment nine.
The specific embodiment 12: the difference of the present embodiment and the specific embodiment nine is: the weight of the water for injection of adding is 1.5～3 times of Os Cervi weight.Other is identical with embodiment nine.
The specific embodiment 13: the difference of the present embodiment and the specific embodiment nine is: the weight of the water for injection of adding is 1.5～3 times of Fructus Melo seed weight.Other is identical with embodiment nine.
The specific embodiment 14: the difference of the present embodiment and the specific embodiment nine is: the temperature of stand at low temperature is 0～20 ℃.Other is identical with embodiment nine.
The specific embodiment 15: the difference of the present embodiment and the specific embodiment nine is: adopt micro-Kjeldahl, biuret method, forint phenol reagent process or Coomassie brilliant blue method to measure the content of protein and polypeptide in Os Cervi polypeptide crude extract and the Fructus Melo seed polypeptide crude extract.Other is identical with embodiment nine.
The specific embodiment 16: the difference of the present embodiment and the specific embodiment nine is: protease is papain, and enzyme work is 9 * 10
5～10 * 10
5U/g.Other is identical with embodiment nine.
The specific embodiment 17: the difference of the present embodiment and the specific embodiment nine is: the Orally administered cervus and cucumis polypeptide composition of preparation is made oral liquid, tablet, capsule, dispersible tablet or suspensoid.Other is identical with embodiment nine.
The specific embodiment 18: the difference of the present embodiment and the specific embodiment nine is: be to be excipient with Radix Glycyrrhizae and sucrose, the distilled water that adds 2～10 times of himself quality again in the Orally administered cervus and cucumis polypeptide composition of making is made the cervus and cucumis polypeptide oral liquid.Other is identical with embodiment nine.
The specific embodiment 19: the difference of the present embodiment and the specific embodiment nine is: be to be excipient with saccharin sodium, dextrin and calcium hydrogen phosphate, with the Orally administered cervus and cucumis polypeptide composition made lyophilization 10～30h under＜10Pa ,-40～20 ℃ condition.Other is identical with embodiment nine.
Present embodiment can be used for preparing cervus and cucumis polypeptide tablet, capsule, dispersible tablet or suspensoid.
The specific embodiment 20: the Orally administered cervus and cucumis polypeptide composition with embodiment nine preparations carries out pharmacodynamic experiment:
(1) the pharmacodynamics contrast experiment of the Orally administered cervus and cucumis polypeptide composition of different molecular weight treatment adjuvant-induced arthritis:
Selecting the adjuvant-induced arthritis rat is animal model, and this experiment is carried out by minutes 5 groups, 12 of every group of rat: the 1st group of normal rat; The 2nd group of adjuvant-induced arthritis rat, not administration; The 3rd group of adjuvant-induced arthritis rat, oral cervus and cucumis polypeptide composition by the 10KD filter membrane; The 4th group of adjuvant-induced arthritis rat, oral cervus and cucumis polypeptide composition by the 6KD filter membrane; The 5th group of adjuvant-induced arthritis rat, oral cervus and cucumis polypeptide composition by the 3KD filter membrane.Experimental result is as shown in table 1.
Table 1
The effect that shows the 4th group (oral cervus and cucumis polypeptide composition by the 6KD filter membrane) by contrast experiment () is ideal, compares with the 2nd group to have utmost point significant difference P＜0.01.There are not state of an illness bounce-back after the drug withdrawal, invalid situation.
(2) the swollen joint expansibility of different dosage form or Drug therapy adjuvant-induced arthritis experiment:
Selecting the adjuvant-induced arthritis rat is animal model, and this experiment is carried out by minutes 9 groups, 12 of every group of rat: the 1st group of normal rat; The 2nd group of adjuvant-induced arthritis rat, not administration; The 3rd group of adjuvant-induced arthritis rat is by the dosage injection cervus and cucumis polypeptide composition (now disclosed cervus and cucumis polypeptide goods) of every 1kg rat injection 2.0mL; The 4th group of adjuvant-induced arthritis rat, per injection adds the 5mg prednisone on the basis of the 3rd group of dosage; The 5th group of adjuvant-induced arthritis rat is by the oral 2.5mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times); The 6th group of adjuvant-induced arthritis rat by the oral 10mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times), and adds the 5mg prednisone at every turn when taking medicine; The 7th group of adjuvant-induced arthritis rat, each medication 5mg prednisone; The 8th group of adjuvant-induced arthritis rat, each medication 200mg aspirin; The 9th group of adjuvant-induced arthritis rat, each medication 10mg prednisone.Experimental result is as shown in table 2.
Table 2
Show the satisfactory for result of the 5th group (by the oral 2.5mL cervus and cucumis polypeptide of every 1kg rat oral liquid) by contrast experiment (two), compare with the 2nd group and have utmost point significant difference P＜0.01.There are not state of an illness bounce-back after the drug withdrawal, invalid situation.
(3) the interleukin-2 contrast experiment of different dosage form or Drug therapy adjuvant-induced arthritis:
Selecting the adjuvant-induced arthritis rat is animal model, and this experiment is carried out by minutes 9 groups, 12 of every group of rat: the 1st group of normal rat; The 2nd group of adjuvant-induced arthritis rat, not administration; The 3rd group of adjuvant-induced arthritis rat is by the dosage injection cervus and cucumis polypeptide composition (now disclosed cervus and cucumis polypeptide goods) of every 1kg rat injection 2.0mL; The 4th group of adjuvant-induced arthritis rat, per injection adds the 5mg prednisone on the basis of the 3rd group of dosage; The 5th group of adjuvant-induced arthritis rat is by the oral 2.5mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times); The 6th group of adjuvant-induced arthritis rat by the oral 10mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times), and adds the 5mg prednisone at every turn when taking medicine; The 7th group of adjuvant-induced arthritis rat, each medication 5mg prednisone; The 8th group of adjuvant-induced arthritis rat, each medication 200mg aspirin; The 9th group of adjuvant-induced arthritis rat, each medication 10mg prednisone.Experimental result is as shown in table 3.
Table 3
| Group | The scorching back of system adjuvant-induced arthritis interleukin-2 value |
| 1 | 8.36 |
| 2 | 8.32 |
| 3 | 8.09 |
| 4 | 8.86 |
| 5 | 76.43 |
| 6 | 69.92 |
| 7 | 65.73 |
| 8 | 40.26 |
| 9 | 33.31 |
Illustrate that by contrast experiment (three) numerical value of taking cervus and cucumis polypeptide oral liquid (Orally administered cervus and cucumis polypeptide composition) interleukin-2 significantly raises, compare with the 2nd group and have significant difference P＜0.05.
(4) tumor necrosis factor of different dosage form or Drug therapy adjuvant-induced arthritis experiment:
Selecting the adjuvant-induced arthritis rat is animal model, and this experiment is carried out by minutes 9 groups, 12 of every group of rat: the 1st group of normal rat; The 2nd group of adjuvant-induced arthritis rat, not administration; The 3rd group of adjuvant-induced arthritis rat is by the dosage injection cervus and cucumis polypeptide composition (now disclosed cervus and cucumis polypeptide goods) of every 1kg rat injection 2.0mL; The 4th group of adjuvant-induced arthritis rat, per injection adds the 5mg prednisone on the basis of the 3rd group of dosage; The 5th group of adjuvant-induced arthritis rat is by the oral 2.5mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times); The 6th group of adjuvant-induced arthritis rat by the oral 10mL cervus and cucumis polypeptide of every 1kg rat oral liquid (dilution of Orally administered cervus and cucumis polypeptide adding distil water is made for 3 times), and adds the 5mg prednisone at every turn when taking medicine; The 7th group of adjuvant-induced arthritis rat, each medication 5mg prednisone; The 8th group of adjuvant-induced arthritis rat, each medication 200mg aspirin; The 9th group of adjuvant-induced arthritis rat, each medication 10mg prednisone.Experimental result is as shown in table 4.
Table 4
| Group | The scorching back of system adjuvant-induced arthritis anti-tumor necrosis factor value |
| 1 | 0.99 |
| 2 | 0.25 |
| 3 | 0.19 |
| 4 | 0.15 |
| 5 | 0.08 |
| 6 | 0 |
| 7 | 0.31 |
| 8 | 0.24 |
| 9 | 0.14 |
Illustrate that by contrast experiment (four) cervus and cucumis polypeptide oral liquid (Orally administered cervus and cucumis polypeptide composition) obviously has the effect of anti-tumor necrosis factor, compare with the 2nd group and have significant difference P＜0.05.
The effect of the orthopaedic diseases such as Orally administered cervus and cucumis polypeptide composition treatment of arthritis of experimental result explanation embodiment nine preparations obviously is better than existing cervus and cucumis polypeptide medicine.
Claims (10)
1. the preparation method of Orally administered cervus and cucumis polypeptide composition is characterized in that the preparation method of Orally administered cervus and cucumis polypeptide composition is carried out according to the following steps:
(A) preparation Os Cervi polypeptide extracting solution: 1. clean fresh Os Cervi, and with deer bone powder essence, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained the Os Cervi crude extract; 4. measure the content of protein and polypeptide in the Os Cervi crude extract, regulating Os Cervi crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Os Cervi crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains the Os Cervi polypeptide extracting solution;
(B) preparation Fructus Melo seed polypeptide extracting solution: 1. clean fresh Fructus Melo seed, add behind the water for injection under 90～105 ℃, the condition of 0.05～0.5MPa and extract 3～5 times; 2. regulating pH value behind the merge extractive liquid, is 4～5, leaves standstill 15～30h again under cryogenic conditions, then filtration, centrifugal; 3. regulating pH value behind the collection supernatant is 8～9, leaves standstill 15～30h again under cryogenic conditions, and filtration, centrifugal is then collected supernatant and obtained Fructus Melo seed crude extract; 4. measure the content of protein and polypeptide in the Fructus Melo seed crude extract, regulating Fructus Melo seed crude extract pH value is 6～7, press protein and polypeptide and 1: 1 weight ratio of protease adding protease in the Fructus Melo seed crude extract then; 5. stir the rapid reactant liquor 1 ± 0.1h of previous step under 55 ± 0.5 ℃, the condition of 1500～1800r/min, keeping reacting liquid pH value in the whipping process is 6～7; 6. reactant liquor heats 3～5min after-filtration, centrifugal for 100 ± 5 ℃, and the supernatant of collection carries out 10KD, 6KD successively and filters, and obtains Fructus Melo seed polypeptide extracting solution;
(C) prepare oral peptide composition: 1. the polypeptide in the Os Cervi polypeptide extracting solution mixes by 4: 1～1: 4 weight ratio with polypeptide in the Fructus Melo seed polypeptide extracting solution; 2. add the ratio of weight and number adding excipient of 5～95 parts of excipient by 5～95 parts of polypeptide, promptly obtain Orally administered cervus and cucumis polypeptide composition.
2. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 1 is characterized in that fresh deer bone powder is broken into the osseous granules of particle diameter less than 1cm.
3. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 1, it is characterized in that protease is made up of in papain, bromelain, ficin, cysteine proteinase, the soybean protein enzyme one or more, enzyme work is 8 * 10
5～11 * 10
5U/g.
4. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 1 is characterized in that adopting hydrochloric acid and/or sodium hydroxide to regulate pH value.
5. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 1 is characterized in that adopting micro-Kjeldahl, biuret method, forint phenol reagent process or Coomassie brilliant blue method to measure the content of protein and polypeptide in Os Cervi polypeptide crude extract and the Fructus Melo seed polypeptide crude extract.
6. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 1 is characterized in that protease is papain, and enzyme work is 9 * 10
5～10 * 10
5U/g.
7. the preparation method of Orally administered cervus and cucumis polypeptide composition according to claim 4 is characterized in that the Orally administered cervus and cucumis polypeptide composition of preparation is made oral liquid, tablet, capsule, dispersible tablet or suspensoid.
8. Orally administered cervus and cucumis polypeptide composition, it is characterized in that Orally administered cervus and cucumis polypeptide composition comprises 5～95 parts of polypeptide and 5～95 parts of excipient by ratio of weight and the number of copies, wherein polypeptide is the described Fructus Melo seed of a B step polypeptide in the described Os Cervi polypeptide of A step and the claim 1 in the claim 1, the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 4: 1～1: 4, Os Cervi polypeptide and Fructus Melo seed polypeptide molecular weight≤6KD.
9. Orally administered cervus and cucumis polypeptide composition according to claim 8 is characterized in that excipient is made up of in Radix Glycyrrhizae, sucrose, simple syrup, saccharin sodium, sodium carboxymethyl cellulose, dextrin, tween 80, magnesium stearate, carboxymethyl starch sodium, hyprolose, calcium hydrogen phosphate, the starch one or more.
10. Orally administered cervus and cucumis polypeptide composition according to claim 8 is characterized in that the weight ratio of Os Cervi polypeptide and Fructus Melo seed polypeptide is 3: 1～1: 3.
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| CN101461834B (en) * | 2009-01-09 | 2012-01-04 | 哈尔滨誉衡药业股份有限公司 | Cervus and cucumis polypeptide medicament composition oral preparation and preparation method thereof |
| CN102397447B (en) * | 2011-11-16 | 2013-08-21 | 金光 | Medicinal composition and granular medicine and preparation method and application thereof |
| CN105688182A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A Lugua polypeptide injection |
| CN105688181A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | Injection preparation prepared from beer bones and muskmelon seeds |
| CN107674113A (en) * | 2017-10-20 | 2018-02-09 | 广东医科大学 | The preparation method and its purposes of the transdermal patch of a kind of small active peptides and preparation method thereof and use active peptide |
| CN107988302B (en) * | 2018-01-23 | 2020-11-13 | 吉林省吉诺生物工程有限责任公司 | Preparation method of cervus and cucumis polypeptide and application of cervus and cucumis polypeptide in preparation of food with special medical application |
| CN108576350A (en) * | 2018-04-17 | 2018-09-28 | 广东宏鸣生物科技有限公司 | A kind of Animal Bone melon seeds Gly-His-Lys pressed candy |
| CN108671221A (en) * | 2018-07-26 | 2018-10-19 | 广东羲准生物科技有限公司 | Gugua polypeptide composition and its application |
| CN110339335B (en) * | 2019-07-01 | 2023-03-24 | 哈尔滨誉衡制药有限公司 | Lugua polypeptide injection |
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| CN1579542A (en) * | 2004-03-04 | 2005-02-16 | 江卫世 | Lugua polypeptides for injection and its preparing process |
| CN1742778A (en) * | 2005-10-09 | 2006-03-08 | 黑龙江迪龙制药有限公司 | Cervus and cucumis polypeptide injection composition and preparing method |
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| CN1579542A (en) * | 2004-03-04 | 2005-02-16 | 江卫世 | Lugua polypeptides for injection and its preparing process |
| CN1742778A (en) * | 2005-10-09 | 2006-03-08 | 黑龙江迪龙制药有限公司 | Cervus and cucumis polypeptide injection composition and preparing method |
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