WO2005115460A1

WO2005115460A1 - A method of producing polypeptides from plant protein and use of polypeptides

Info

Publication number: WO2005115460A1
Application number: PCT/CN2004/001051
Authority: WO
Inventors: Zhen Sun; Zhentian Sun
Original assignee: Zhen Sun; Zhentian Sun
Priority date: 2004-05-26
Filing date: 2004-09-16
Publication date: 2005-12-08
Also published as: CN2712931Y

Abstract

The invention provides a method of producing polypeptides from plant protein by using alternating high frequency high voltage electromagnetic field. The method comprises: making plant materials move in processing cavity, applying alternating high frequency high voltage electromagnetic field to two parallel stainless steel sheets, and obtaining the invented polypeptide solution and polypeptide powder. The invention also provides the pharmaceutical use of plant polypeptides produced by the above-mentioned method.

Description

FIELD OF THE INVENTION The present invention relates to a method for preparing a polypeptide from a vegetable protein, and more particularly to a method for extracting a polypeptide in a vegetable protein by using an alternating high frequency and high voltage electromagnetic field ; and the application of peptide products. BACKGROUND OF THE INVENTION There are two methods for preparing polypeptides from traditional plant proteins: one is to "cleave" a protein into a polypeptide by bioengineering techniques; the other is a chemical technique to "chemically degrade" a protein into a polypeptide. The conventional plant protein preparation methods have the following disadvantages:

1. The peptide product is not a pure natural product because of the need to add additives and catalysts during the production process;

2. The investment in production equipment is large, the process of controlling product quality is complicated, and the production cost is high. SUMMARY OF THE INVENTION The method for preparing a polypeptide from a vegetable protein according to the present invention can solve the technical problem of extracting a pure natural polypeptide product by using a simple production equipment and a production process, and discharging no waste gas, waste water and waste in the production process.

The invention also solves the application of the jujube polypeptide in antitumor drugs

The invention also solves the use of the soybean polypeptide in the treatment of eczema, acne and athlete's foot.

The invention also solves the use of the soybean polypeptide in the treatment of chronic hepatitis B.

The invention also solves the application of soybean polypeptide in medicine for treating gastric ulcer

The invention also solves the application of the licorice polypeptide in the medicine for treating AIDS. The method for preparing a polypeptide from the plant protein of the present invention is as follows:

1. Using a spiral propelling feeder or a conveyor feeder, the plant material that has been cleaned to remove impurities is fed into a processing chamber formed by two stainless steel plates with a relatively uniform distance;

2. Applying a high-frequency high-voltage alternating power source to the electrodes formed by the two stainless steel plates to generate an alternating electromagnetic field in the plant material filled between the two stainless steel plates, and the applied high-frequency alternating current source voltage is lower than The breakdown threshold voltage that the filled material can withstand;

3. Monitor the temperature of the material in the processing chamber. When the temperature of the lysate reaches 90 °C ~ 120 °C, collect the vapor of the polypeptide released in the form of water vapor, and then condense to become the polypeptide liquid; After pulverization, the mixture is immersed in water and ethanol, and the resulting mixture is precipitated and filtered to obtain a polypeptide liquid, which is then freeze-dried or dried to prepare a polypeptide powder.

A method for producing a polypeptide from a vegetable protein as described above, wherein the electromagnetic field generator generates a frequency in the processing chamber of from 200 kHz to 2500 MHz, and a voltage between the two plates of from 1000 volts to 20,000 volts.

A method for preparing a polypeptide from a vegetable protein as described above, wherein the equivalent frequency of the processing chamber is controlled by the matcher to be the same as the frequency of the electromagnetic field generator.

The method for preparing a polypeptide from the vegetable protein of the invention is directly introduced into the protein molecule (dipole molecule) according to the energy of the alternating high frequency and high voltage electromagnetic field, so that the molecular chain is interrupted, that is, the protein molecule is subjected to the high frequency and high voltage electromagnetic field. Energy is cleaved into small molecules (polypeptides) of different orders of magnitude. The preparation effect of the present invention takes soybean as an example, and samples of the extracted polypeptide cooling liquid and the polypeptide material extract sample are analyzed as follows:

1. Analysis of water vapor peptide samples: (sample appearance, light yellow transparent liquid)

(1) Analysis purposes and items: separation of peptide components;

(2) Analytical method - isoelectric focusing

TriscpTM2010GV pressurized electrochromatograph

Isoelectric focusing: 50 μ ιη inner diameter with capillary tube, polyacrylamide coated wall, total length: 30cm, effective length: 22cm,

Anode buffer: 25 mM phosphoric acid: Cathode buffer: 25 mM sodium hydroxide Focusing voltage: 10kv/10s

Migration. · Anode lift 6cm

Detection: 280nm UV detection

(3) Analysis results:

The sample contains a variety of peptide components, as shown in the spectrum shown in Figure 1. 2. Sample analysis of material leaching polypeptide: (sample appearance, light yellow turbid liquid)

(1) Analysis purposes and items: separation of peptide components

(2) Analysis method:

Isoelectric focusing

TriSepTM2010GV pressurized electrochromatograph,

Isoelectric focusing: 50 u m is a radial quartz capillary, coated with polyacrylamide, total length: 40 cm, effective length: 30 cm,

Anode buffer: 25 mM phosphoric acid: Cathode buffer: 25 mM sodium hydroxide, focusing voltage: 12KV/60S,

Migration: anode lift height 9cm,

Detection: 280nm UV detection;

(3) Analysis results: The sample contains a variety of peptide components, as shown in the spectrum shown in Figure 2.

The polypeptides produced by the methods of the invention have novel uses for the treatment of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an analytical spectrum of a sample of a polypeptide cooling liquid prepared by the present invention;

2 is a chromatogram of the sample preparation of the polypeptide material obtained by the present invention; and FIG. 3 is a flow chart of the process for preparing the polypeptide of the present invention. I. The preparation of the polypeptide of the present invention is shown in Figure 3. The plant protein is washed by the cleaning machine 1 to remove impurities, sent to the electromagnetic cracking processing chamber 2 to perform electromagnetic field cracking, and the plant is moved in the processing chamber. Plants with irregular geometric shapes need to be equipped with a conveyor belt in the processing chamber to move evenly. The conveyor belt is made of high-frequency resistant materials. If the grain-shaped plants are used, a double-shaped machining chamber can be used, and the material flow is completed by the speed of the cage. External radiation from the processing chamber not only harms the health of the operator, but also interferes with broadcasting, communication, radar, remote control, navigation, etc., so the processing chamber is strictly shielded. In the processing chamber, a metal plate and a grounding wire system are grounded as a cathode, and the other anode plate is completely covered with an aluminum plate, and the distance from the anode plate is not discharged and fired, and is firmly connected to the annular ground wire.

The frequency and voltage generated by the electromagnetic generator 4 are fixed, and the resonance frequency generated by the plants in the processing chamber is the same as the fixed frequency of the electromagnetic field generator, and the resonance frequencies of different plants in the same cavity are different, for any Plants can generate resonant frequencies in the cavity, so in the processing chamber and electromagnetic field generator A matching device 3 is provided, and the adjustable series-parallel capacitor of the matching device is used in parallel with the equivalent capacitance of the processing cavity, so that the equivalent frequency of the processing cavity is the same as the frequency of the electromagnetic field generator, and the high-frequency high-voltage electromagnetic field can be generated in the processing cavity. The plant protein is subjected to "electromagnetic cleavage" into a polypeptide. Therefore, the series-parallel capacitor of the matching device 3 is continuously adjustable, the matching device and the electromagnetic field generator are strictly shielded at the same time, and the shielding shell and the grounding system are strictly grounded, and the connection is firm.

The degree of cracking of plant proteins by high-frequency electromagnetic fields is determined by the temperature and electromagnetic field strength. The complete cracking temperature of different materials is also different. The control standard is based on the control temperature. The material temperature is determined by the residence time in the processing chamber. The residence time is long, the temperature is high, and the residence time is short. The temperature is low. In order to ensure the quality of the product, it is necessary to maintain a constant temperature. Maintaining the constant temperature is to adjust the residence time of the material in the processing chamber. Completed, that is, adjusting the speed of the plant moving within the processing chamber to complete.

Preparation method of pharmaceutical dosage form of the invention:

The polypeptide which is finally lysed in the processing chamber of the plant is partially released in the form of water vapor according to the internal water-containing molecule, and becomes a polypeptide liquid through the condenser 5. Most of the polypeptides are still in the plant. The plant protein is pulverized by the pulverizer 9 and placed in the soaking tank 10. The plant polypeptide solution is extracted by conventional extraction of the traditional Chinese medicine method, soaked in water or ethanol for 6 hours and 12 hours, and then the mixture is placed. In the centrifuge 1 1 , thousand, its aqueous solution or alcohol solution is put into the polypeptide liquid tank 12, precipitated for 6 hours and 12 hours, the supernatant is introduced into the filter 13, filtering, and the filtered polypeptide liquid enters the filling line, and is completed. Fill and sterilize 14% of qualified products and store them in the warehouse 15 . The polypeptide liquid 12 enters the vacuum low-temperature concentrator 16 and is concentrated, and then enters the lyophilizer 17 to be dried into a dry powder or dried to form a dry powder, which is then tableted or capsuled into a storage room by a tableting machine.

Several examples of common plant cracking processes are given below:

1. Soybean, choose a high frequency of 27.12 MHz, and the voltage between the two plates is 10,000 volts. According to the above voltage, the distance between the two plates is set to 25 cm. The material moves slowly in the machining chamber and its speed is controlled at 100 °C.

2, jujube, choose high frequency frequency 200 kHz, in order to avoid the broadcast frequency band, also choose industrial frequency 251.8 kHz, the voltage between the two plates is 1000 volts, the distance between the two plates is 20 cm, the material is in the processing chamber Slowly moving inside, the speed is controlled when the material reaches 90Ό.

3, licorice: 'Select high frequency frequency is 2500 MHz, if you consider the industrial frequency, you can choose 1735.6 MHz, the voltage between the two plates is 20,000 volts, the plate distance is 30 cm, the material moves slowly in the processing cavity, speed control The material can reach 120 °C. Second, the application of the polypeptide product of the invention:

(1) The use of the jujube polypeptide of the invention in an antitumor drug;

The applicant entrusted the Chinese Academy of Medical Sciences and Medicine to conduct a preliminary observation test on the anti-tumor effect of jujube peptide lysate. The contents are as follows:

A preliminary observation was made on the antitumor effect of the provided jujube polypeptide lysate. The MTT method was used to observe the cell killing experiment in 11 human tumor cell lines. The human leukemia cell line HL-60 was used to observe the differentiation induction effect. The animal transplanted tumor mouse sarcoma S 180, cervical cancer U14, Lewis cancer were used. And the mouse liver cancer H22 completed the anti-tumor effect observation.

The results showed that the jujube peptide liquid sample showed certain killing effect on the selected tumor cells, but because the sample content was unclear, the IC50 calculation could only be used to sample the jujube peptide liquid sample liquid per ml of cell culture solution, and its IC50 was less than 10 μ 1/ml o

There was no differentiation induction effect on HL-60 cells at the concentration of 5 l/ml in the experimental sample of jujube polypeptide.

The jujube peptide liquid sample was taken at a high dose of 0.4 ml/20 g of the original solution, and once per day, it had no obvious inhibitory effect on the growth of the transplanted tumor S 180, Lewis lung cancer and U14, and showed a certain inhibitory effect on the growth of liver cancer H22. , but the reproducibility is not satisfactory.

Sample:

Jujube peptide lysate, brownish red, with a small amount of sediment.

Drug usage design:

For the unknown content, all experiments were expressed as the stock solution. The in vitro cell assay was used for no more than 100 μl per ml of medium, and the highest dose in animal experiments was 0.4 ml/20 g of mice.

Experimental summary:

1. In vitro anti-tumor activity test

Drugs and Reagents: Jujube Peptide Liquid, Brown Liquid, sterilized by microfiltration membrane filtration during the experiment. Positive control drug, fluorouracil injection 10ml: 0.25g, is the product of Shanghai Xudong Haipu Pharmaceutical Co., Ltd., batch number 000612. The RPMI1640 is a GIBCO product. Calf serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd. MTT is a Bebco product.

Cell lines: A2780 (human ovarian cancer cells), HCT-8 (human colon cancer cells), Bel-7402 (human liver cancer cells), BGC803 (human gastric cancer cells), MCF-7 (human breast cancer cells), CaEs-17 (human esophageal cancer cells), 431 (human epidermis Cancer cells), EJ (human bladder cancer cells), U25 human glioma cells), PC-3M (human prostate cancer cells), KB (human oral epithelial cancer cells).

Instrument: BIORAD550 type microplate reader.

Experimental method: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide, MTT] tetrazolium salt reduction method.

Collect well-growing tumor cells, prepare lxl oVml cell suspension with RPMI1640 medium containing 10% calf serum, inoculate in 96-well culture plate, each well Ι μ Ι (containing 1000 tumor cells), set After 37 hours of incubation in a 37 ° C, 5% CO ₂ incubator, the drug was administered. The experiment was set up with a blank control. The positive control drug was fluorouracil. The test samples were set to 4 concentrations, 3 parallel holes per concentration, placed in a 37 ° C 5% C0 ₂ incubator, and cultured for 4 days. The culture solution was discarded, and MTT solution (0.4 mg/ml, prepared by RPMI1640) was added to each well, and incubated at 37 ° C for 4 hours. The supernatant was discarded, and DMSO 150 w was added to each well. The Fomazan particles were dissolved, and after gently shaking, the OD value was measured with a 550-type microplate reader at a detection wavelength of 540 nm and a reference wavelength of 450 nm.

Calculation of results: A dose response curve was obtained by plotting the different concentrations of the drug and the inhibition rate of the cells, and the half-inhibitory concentration (IC ₅ ) was determined therefrom.

Result

The results showed that the jujube peptide solution had a certain cell killing effect on the selected tumor strain in vitro, and in the case of eliminating the color interference of the sample, it was _{5 5 5} ο<1 l/ml (see Tables 1, 2).

in conclusion

The jujube peptide solution has a certain killing effect on the selected 11 cancer cell lines, indicating that it has certain anti-tumor activity in vitro.

2. Observation on the differentiation induction of human promyelocytic leukemia cell HL-60 Drugs and reagents: cAMP, same as before, positive control drug, all-trans retinoic acid (ATRA) is the product of Shanghai No. 6 Pharmaceutical Factory. Nitroblue tetrazolium (NBT) and phorbol ester (TPA) are products of Sigma. RPNI1640 is a GIBCO product. Calf serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.

Cell line: Human acute promyelocytic leukemia NB ₄ cells, a gift from Professor S. Waxman of the United States. The cells were cultured in RPMI1640 medium containing 10% calf serum at 37 ° C in a 5% CO ₂ incubator.

experimental method:

(1) Determination of NBT reducing ability, cells and control cells for 6 days of drug administration were centrifuged at 1 °C for 5 min at 1 rpm, and the supernatant was discarded, and 0.1 °/ was added to each tube. 0.5ml of NBT solution, containing TPA100ng, placed in a 37 ° C incubator for 3 small Time. l Centrifuge at OOOrpm for 5 min. Discard the supernatant, take a cell pellet and stain with Wright-Giemsa for 10 min. Under the oil microscope, 200 cells were observed per slide, and those with blue-black nail particles in the cytoplasm were NBT-positive cells, and the percentage of NBT positive was calculated.

(2) Morphological observation of the cells: The cells treated with the drug for 6 days and the control cells were centrifuged at 10,000 rpm for 5 min at 4 ° C, the supernatant was discarded, and the cell pellet was smeared, stained with Wright-Giemsa, and observed under an oil microscope. Morphological change. 200 cells were observed per slide, and the cells were counted by cell sorting to calculate the percentage of differentiated cells.

o o o

Table 1 Killing effect of cAMP on human cancer cells (1)

Inhibition rate (%) IC ₅₀

Tumor strain concentration cAMP concentration Fu οΑΜΡ(μ1/ηιΙ) Fu( g/ml)

(μΐ/ml) 1 2 3 ( g/ml) 1 2 3 1 2 3 1 2 3

MCF-7 2.5 27 11 10 0.1 65 '59 64

(human breast cancer cells) 5 60 42 34 1 90 88 86

10 80 76 77 10 94 94 93

20 97 97 97 <0.1

A2780 2.5 14 12 14 0.1 7 0 0

(Human ovarian cancer) 5 45 35 36 1 72 68 67

10 87 91 88 10 93 91 92

20 90 93 97 5.975 6.226 6.237 0.717 0.761 0.772

HCT-8 2.5 6 11 6 0.1 8 13 8

(human colon cancer cells) 5 30 42 32 1 81 83 80

10 68 75 67 10 89 88 89

20 88 89 89 7.683 6.687 7,688 0.618 0.576 0.625

KB 2.5 0 0 0 0.1 8 0 0

(Human oral epithelial cancer cells) 5 0 0 0 1 74 72 66

10 91 92 86 10 85 83 83 -

20 95 97 86 0.673 0.725 0.782

V

V To

o

Killing effect of cAMP on human cancer cells (2)

Inhibition rate (%) IQo

Tumor strain concentration cAMP concentration Fu οΑΜΡ(μ1/η il) Fu^g/ml)

(μΐ/ml) 1 2 3 1 2 3 1 2 3 1 2 3

Bel 7402 2.5 19 17 15 0.1 25 16 17

(Human liver cancer cells) 5 40 29 31 1 84 80 81

10 69 61 67 10 90 89 90

20 83 82 85 6.957 8.305 7.571 0.481 0.578 0.564

431 2.5 0 0 0 0.1 53 47 51

(Human epidermoid cells) 5 0 0 0 1 93 93 92

10 83 87 91 10 98 98 98

20 96 95 94 8.012 7.874 7.616 <0.1 0.159 <0.1

U251 2.5 14 7 12 0.1 27 3 4

(human glioma cells). 5 28 0 19 1 64 53 57

10 65 66 60 10 72 76 78

20 91 94 93 7.973 8.788 8.780 0.660 0.945 0.881

CaEs-17 2.5 2 16 5 0.1 0 6 0

(Human esophageal cancer cells) 5 30 0 8 1 76 75 72

10 81 61 70 10 91 89 90

20 - 92 91 ' 7.007 9.098 8.261 0.692 0.674 0.725

o

Table 1 Killing effect of cAMP on human cancer cells (3)

Inhibition rate (%) ICso

About

Trace concentration cAMP concentration Fu οΑΜΡ(μ!/ιη1) Fu( g/ml)

(μΐ/ml) 1 2 3 1 2 3 1 2 3 1 2 3

BGC-803 2.5 4 5 3 0.1 2 0 0

(Human gastric cancer cells) 5 13 9 10 1 60 52 54

10 63 93 56 10 89 91 91

20 92 - 90 8.659 7.049 9.459 0.845 0.965 0.933

PC-3M 2.5 11 9 6 0.1 17 5 3

(human frontal female cells) 5 0 1 2 1 52 44 42

10 66 68 56 10 75 75 72

20 74 90 88 8.676 8.657 9.444 0.948 2.778 3.434

EJ 2.5 8 16 8 0.1 17 9 6

(human bladder cells) 5 20 30 26 1 57 48 43

10 51 59 61 10 86 86 87

20 93 93 94 9.839 9.407 8.380 0.843 1.481 2.426

Killing effect of cAMP on human cancer cells (X Shi SD) Drug IC 50

Tumor strain, οΑΜΡ(μΙ/ιηΙ) Fu(^g/ml)

MCF-7 5.940 ±1.4752 <0.1

A2780 6.146±0.1482 0.750 ±0.0291

HCT-8 7,353 ±0.5765 0.606 ±0.0265

KB 7.790 ±0.1021 0.727 ±0.0545

Bel 7402 7.625 ±0.6542 0.541 ±0.0524

431 7.834 ±0.2010 <0.120

U251 8.514 ±0.4682 0.829 ±0.1495

CaEs-17 8.120±1.0561 0.697 ±0.0259

BGC-803 8.389 ±1.2275 0.914 ±0.0621

PC-3M 8.926 ±0.4490 2.387 ±1.2884

EJ 9.209 ±0.7494 1.583 ±0.7964

A549 13.470 ±0.9374 0.254 ±0.0715 result

The effect of jujube peptide solution on the reducing ability of NB ₄ cells (see Table 3).

The effect of jujube peptide solution on the morphology of NB ₄ cells (see Table 4). Table 3 Effect of cAMP on NBT reducing ability of NB ₄ cells

ΝΒΤreduction reaction

Group concentration

Positive cell negative cell positive cell%

Control 0 200 0

AT A ΙΟ^Μ 170 30 85 cAMP Ιμΐ/ml 0 200 0

Δμΐ ml 0 200 0

Table 4 Effect of cAMP on the morphology of NBT in NB ₄ cells

Cell morphology

Group concentration

Promyagules, young seeds, late granules, rod-shaped nucleus, lobular nucleus, differentiation

Control 200 0 0 0 0 0

ATRA ΙΟ^Μ 9 163 20 7 1 95.5 cAMP Ιμΐ/ml 199 1 0 0 0 0.5

2.5μΙ/πιΙ 200 0 0 0 0 0

5μ1 1 196 4 0 0 0 2

Conclusion: jujube polypeptide ml concentration were at 1, 2.5, 5 i / no significant effect on induction of differentiation ΝΒ ₄ cells.

3. Effects on the growth of transplanted tumors in animals

Tumor strain: Mouse liver cancer Η22, mouse cervical cancer U14, mouse Lewis lung cancer, mouse sarcoma S 180 This laboratory self-passage preservation.

Animals: Kunming mice, C57 mice, 18-22 g, used as early as sputum. Provided by the Laboratory Animal Institute of the Chinese Academy of Medical Sciences.

Control drug: cyclophosphamide injection, produced by Shanghai Hualian Company.

experiment procedure:

The tumor-bearing mice with good growth status were taken as ascites or tumor homogenate, diluted with sterile saline 1:5, and 0.2 ml of each mouse was inoculated into the underarm. The next day, After inoculation the animals were randomly divided into groups of ¹⁰ rats jujube polypeptide Lysates

(CAMP) According to the original solution, 3-fold dilution, 9-fold dilution, 3 doses were administered once daily in a volume of 0.4 ml/20 g for 10 or 14 days until the end of the experiment. The cyclophosphamide control group was intraperitoneally administered once at a dose of 100 mg/kg. The negative control group naturally produced ^:.

When the tumor grows to a certain volume, the mouse cervical vertebrae are dislocated and weighed. The tumor was removed and weighed. The mean values of the tumors in each group were calculated, and the t test was used to compare the statistical differences, and the tumor inhibition rate was calculated. The formula is:

The average tumor weight of the control group was the average tumor weight of the treatment group. The tumor inhibition rate (°/.) =

—X 100

Average tumor weight of the control group

Experimental results:

The results of the inhibitory effect of jujube peptide lysate on the growth of transplanted tumor in mice are shown in Tables 5, 6, 7, and 8.

Effect of 5 jujube peptide lysate (cAMP) on tumor growth of mouse liver cancer H22

Dosage number of animals (only) body weight (g) tumor fi(g) inhibition rate group

Ml/kg

Start End End End X+SD (%) Batch 1

Negative control 9 9 18.8±3.69 27.7+1.87 1.77±0.771

Cyclophosphamide 9 9 17.7±1.12 24.0±1.4 ί 0.38+0.139# 78.5 cAMP stock solution 10 9 17.6+0.92 23.5±2.33 0.88±0.311# 50.3

1/3 liquid 10 8 18.0±0.87 19.9±3.52 1.23±0.301 30.5

1/9 liquid 10 9 18.0+1.00 22.0+2.55 1.60+0.324 9.6

Batch 2

Negative control 10 9 27.0±1.25 36.4+2.51 2.38±0.900

cAMP stock solution 10 9 27.0± ί .% 32.3+3.35 1.73±1.420 37.2 Batch 3

Negative control 10 10 20.9±1.20 28.8+2.60 1.48+0.390

Cyclophosphamide 10 10 21.2 soil U2 26.2+2.15 0.42±0.479# 76.5 cAMP stock solution 10 9 20.5±0.71 24.5+4.20 0.9410.479 27.0

1/3 liquid 10 8 20.7±0.67 28.9+4.02 1.24+1.039 21.9

1/9 liquid 10 9 20.9il.86 29.0±5.00 1. '29+0.592 12.8

#: p 〈0.05 compared with the negative control group Effect of 6 jujube peptide lysate (cAMF) on tumor growth of mouse cervical cancer. Number of animals (only) Body 3⁄4(g) Tumor weight (g) Inhibition rate group

Ml/kg

Beginning to end X±SD (%) Negative control 9 9 20.3±1.22 23.8±1.86 1.89+1.194 Cyclophosphamide 9 9 19.9±1.45 20.3±1.00 0.21±0.078# 88.9 cAMP stock solution 10 9 20.4±0.97 20.8+0.92 1.20±1.054 36.5

1/3 liquid 10 8 20.7±1.16 23.3+1.70 1.23+0.638 32.8

1/9 liquid 10 9 20.5±1.18 24.2+2.44 1.39+0.565 26.5

#: p 〈0.05 compared with the negative control group

Effect of 7 jujube peptide lysate (cAMP) on the growth of mouse sarcoma S180

Dosage number of animals (only) body weight (g) tumor weight (g) inhibition rate group

Tnl/kg

Start of the beginning of the knot. X±SD (%) Negative control 9 9 19.8±1.32 32.0±2.40 1.96±1.175 Cyclophosphamide 9 9 20.2±1.23 19.0±1.89 0.32±0.239# 83.7 cAMP stock solution 10 9 19.9+1.83 23.5±1.27 1.3410.676 31.6

1/3 liquid 10 8 22.1+1.45 26Λ+2Α2 1.35+0.525 31.1

1/9 liquid 10 9 21.4±1.90 25.7±2.26 1.50+0.924 23.5

#: p 〈0.05 compared with the negative control group

Effects of 8 jujube peptide lysate (cAMP) on the growth of mouse lc and vis

Ml/kg

Start and end of X±SD ( ) Negative control 10 10 17.6±1.35 20.8+1.99 1.50±0.447 Cyclophosphamide 9 9 18.5±1.35 18.4+1.17 0.13±0.082# 91.3 cAMP stock solution 10 9 183±1.89 15.9±1.62 1.06± 0.336# 29.3

1/3 liquid 10 8 17.8+1.32 17.0±1.76 1.35±0.536 10.0

1/9 liquid 10 9 18.±1.35 17.0±1.49 1.38+0.592 8.0

#: p 〈0.05 compared with the negative control group in conclusion·

The results of the first batch of jujube peptide lysate at the selected dose showed a certain inhibitory effect on the growth of H22 xenografts, but the reproducibility was not good. There is no obvious growth inhibition effect on cervical cancer U14, SI80 and Lewis lung cancer.

Experimental design: Han Rui

Responsible for the experiment, experimenter: Liu Hongyan, Fu Zhaozhen Experimental date: September 12, 2002 Source preservation: Institute of Pharmaceutical Research, Chinese Academy of Medical Sciences

Contact: Fu Zhaoyu

(B) the application of soy peptide liquid in the treatment of gastric ulcer drugs

Soybean Peptide Solution for 35 Cases

Clinical treatment of gastric ulcer in Jilin Province Institute of Traditional Chinese Medicine Wang Limei Zhuang Lingling Wang Yu

Fan Limei Shang Zubo Wang

Abstract: From October 2002 to April 2003, 35 cases of gastric ulcer were treated with soybean peptide solution. After one month of treatment, the total effective rate of the treatment group was 82.8%, and the extent of gastroscopy and pathological changes was large. The pathological changes after treatment are mild or moderate to mild, and its mechanism may be related to the solubilization and detoxification of soybean polypeptide liquid, spleen and dampness, promoting blood circulation and collaterals, improving gastric mucosal circulation and nutrient metabolism.

Gastric ulcer is one of the key research topics of scholars at home and abroad. In our hospital from October 2002 to April 2003, 35 cases of gastric ulcer were treated with soy peptide, and all of them achieved satisfactory results. The observation results are as follows-

normal information

Case source: 35 patients in this group were observed in the Jilin Traditional Chinese Medicine Research Institute, of which 69. 1% were outpatients and 30.9% were inpatients. Gender and age: Among 35 cases, 19 males accounted for 54.3%, 16 females accounted for 45.7%, 21-30 years old 4 cases, accounting for 25.7%, 31--40 years old 10 cases, accounting for 29 cases 6%, 41 --- 50 years old 10 cases, accounting for 29.6%, 51 --- 60 years old 4 cases, accounting for 11.4%, 61 years old and above 2 cases, accounting for 5.7%, the youngest 21 Aged, maximum 66 years old.

Job title: 18 cases of workers, accounting for 51.4%; 8 cases of cadres, accounting for 22. 9; farmers

9 cases, accounting for 25.7%.

Course of disease: 2 --- 5 years, 10 cases, accounting for 29.6%; 6 --- 10 years, 12 cases, accounting for 34.3%; 1 1 --- 15 years, 10 cases, accounting for 28.6%; 16 years The above 3 cases, accounting for 8.5%; the longest course of disease is 23 years.

Case selection: The patient has a history of more than two years, showing symptoms of stomach cramps, fullness of flatulence, recurrent or progressive aggravation, and a gastroscopy, a diagnosis of gastric ulcer, and no other serious complications. This group of observation objects.

Diagnosis of this article: According to the 1995 clinical guidelines for the development of new Chinese medicines for the treatment of gastric ulcers.

Western medicine diagnostic criteria (refer to the standards developed by the Chongqing National Gastritis Diagnosis and Treatment Symposium in 1982).

TCM syndrome (refer to the 1989 China Association of Integrative Medicine, Digestive System Diseases Professional Committee diagnostic test standard) is divided into five cards, including spleen and stomach dampness syndrome, gastric collateral syndrome, as the focus of this article, the specific content is slightly.

Efficacy criteria: According to the guidelines issued by the Ministry of Health in 1995 to issue new Chinese medicine treatment guidelines for gastric ulcer:

1 Clinical cure: clinical symptoms, signs disappeared, gastroscopic examination of mucosal chronic inflammation improved, intestinal metaplasia and dysplasia returned to normal or disappear. '

2 markedly effective: the main clinical symptoms, signs disappeared, gastroscopic examination of mucosal chronic inflammation improved, intestinal metaplasia and dysplasia returned to normal or reduced by 2 degrees. 3 Effective: The main symptoms and signs are obviously alleviated. The scope of mucosal lesions in gastroscopy is reduced by more than 1/2, the inflammation is reduced, the ulcer surface is reduced, and intestinal metaplasia and dysplasia are alleviated.

4 Invalid: Cannot reach the effective standard or worsen the worse.

treatment method

1. Random grouping: Randomly grouped under the condition that the patient's natural condition, age, and condition are consistent.

2 Use of drugs: Treatment of soy peptide liquid, secondary / daily, 20ml / per 0

: One month of treatment is a course of treatment. If the condition is severe, it can last for 2-3 months. During the observation period, all the drugs for strengthening the spleen and nourishing the stomach should be stopped.

4. Observation methods: Outpatients and inpatients were reviewed once before and after treatment, and the sphincter (photographed by conditional) and various physical and chemical examinations were recorded. The results were recorded in the case observation table. The location and number of pathological biopsy were performed as specified.

5 observation results

Table 1: Total Effectiveness of Soybean Peptide Solution in Treating Gastric Ulcer

The total number of cases is clinically effective

0 total efficiency

Treatment group 35 3 10 16 6 82. 8% As can be seen from Table 1, the total effective rate of soy peptide liquid in the treatment of ulcers was 82.8%, the effect was more significant.

Table 2 Improvement of clinical symptoms by soy peptide liquid Efficacy

Bu

Number of cases

Inch efficiency%

Stomach cramps 30 bloating 25

84. 0%

Staying less and eating less 28

As can be seen from Table 2, the soybean-treated peptide liquid has a significant effect on the improvement of clinical symptoms. Before and after treatment, the curative effect is obvious, and the recovery rate of each symptom is 71. 4% or more.

Total time of the period January - March 2 - March and above

Group treatment group 35 12

6 As seen in Table 3, compared with the time of onset of other drugs, it is significantly better than its i drug.

Discuss

Gastric ulcer Chinese medicine belongs to the category of stomach cramps and so on. The cause of the disease, Western medicine believes that the central activity disorder affects the internal organs, while the smooth muscle tendon causes the gastrointestinal secretion, dysfunction and malnutrition of the stomach wall to cause inflammation of the gastric mucosa or thinning of the gastric mucosa. Clinical manifestations of stomach cramps, swelling, nausea, tenderness, tenderness, etc., Chinese medicine has long been remembered Loaded. In this study, 35 patients received a total effect of 82.8% in the first quarter of treatment. In this group of 35 cases, diagnosed as gastric ulcer, most of the comminuted gastritis, reflux esophagitis, chronic gastritis, duodenal ulcer embolism, the mechanism of action is through the soybean peptide liquid containing 18 kinds of amino acids and 1 1 The high nutrient effect of vitamins on the gastric mucosa and the promotion of various metabolic effects of the body, thereby achieving blood circulation, improving circulation and regulating immunity, and supporting the body to correct the evil.

The drug has a good effect on improving appetite, eliminating fullness and relieving pain, and the clinical recovery rate is 71. 4% or more.

The total effective rate under the microscope was 82.8%. There were 5 cases of adverse reactions in this medicine. The symptoms such as nausea, vomiting, and eating less and worsened after eating were aggravated. The observation under the microscope was aggravated. The remaining patients were stable and reliable, and it is an ideal drug for treating gastric ulcer.

April 2003

28th

(3) Application of Soybean Peptide Liquid in the Treatment of Chronic Hepatitis B

Clinical Observation of Soybean Peptide Solution in Treating Chronic Hepatitis B Jilin Institute of Traditional Chinese Medicine

Wang Yumei, Shang Zubo, Wu Jun, Su Yan et al. Chronic hepatitis B is a common disease with strong infection, long course of disease and prolonged unhealed. At present, there is still no reliable health care treatment. Our hospital was entrusted by Sun Zhenzhi in July 2002. It uses soy peptide liquid in the treatment of chronic hepatitis B, including chronic persistent hepatitis, chronic active hepatitis and chronic gastritis, gallbladder. The digestive system diseases such as inflammation have achieved satisfactory results. The results of 30 observations are as follows:

clinical information

Case source: 30 patients in this group were observed in the Jilin Traditional Chinese Medicine Research Institute, including 22 outpatients, 73%, and 8 inpatients, accounting for 27%. Gender and age: Among the 30 patients in the treatment group, 23 were males, accounting for 77%; 7 females, accounting for 23%; 3 of them aged 19-29 years, accounting for 10%; 8 cases of 30-39 years old, accounting for 27%; 11 cases were 40-49 years old, accounting for 37%; 6 cases were 50-59 years old, accounting for 20%; 2 cases were over 60 years old, accounting for 6%, the oldest was 72 years old, and the youngest was 19 years old. Among the 30 patients in the control group, 14 were male, accounting for 47%; 16 were female, accounting for 53%; 11 of them were 19-29 years old, accounting for 37%; 8 were 30-39 years old, accounting for 27%; 40-49 5 years old, accounting for 17%; 4 years old, 50-59 years old, accounting for 13%; 2 cases over 60 years old, accounting for 6%; the minimum age is 19 years old, the largest is 67 years old.

Treatment: Among the 30 patients in the treatment group, 3 patients in 2 years, accounting for 10%; <20 years in 20 cases, accounting for 66.7%, <10 years in 5 cases, accounting for 16.7%, <10 years, 2 cases accounted for 6.6%. The disease duration is up to 25 years and the shortest is two years.

Among the 30 patients in the control group, 5 patients in 2 years, accounting for 17%; 17 patients in 5 years, accounting for 57%, 4 patients in 10 years, accounting for 13%, and 4 patients in 10 years, accounting for 13%. The shortest course is two years and the longest is 27 years.

Observation method

1. Diagnostic method: According to the “Guidelines for Clinical Research of New Drugs for Traditional Chinese Medicine” (1993) issued by the Ministry of Health of the People's Republic of China. (Specific content omitted)

2. Random grouping: In the case of the patient's age, gender, condition, and natural conditions, the two groups are divided into two groups:

The treatment group (30 cases) was given soybean polypeptide solution, 20 ml per oral administration, three times a day. The control group (30 cases) administered amino acid (animal blood) capsules, 3 capsules each time, 3 times a day.

3. Treatment: One month is a course of treatment. It is better for patients with chronic weakness and two months of treatment. It is also an ideal adjuvant treatment for patients with severe illness and wasting disease.

4. Treatment: All inpatients in the outpatient clinic are reviewed once before and after treatment. Symptoms, signs, liver function, immunoglobulin, blood, urine routine, various viral antigens and antibodies, liver and gallbladder spleen B Type ultrasound, etc., and then detail the contents according to the requirements in the observation table, in which the symptoms in the table are expressed by weight (+++), medium (++), light (+), and no (one).

5. Judgment of curative effect: According to the “Guidelines for Clinical Research of New Drugs for Traditional Chinese Medicine” (1998) issued by the Ministry of Health of the People's Republic of China. Basic cure - Conscious symptoms disappear, hepatosplenomegaly stabilizes or shrinks, no tenderness and cramps, liver function tests are normal, and virus detection turns negative. The above indicators are stable for more than one year.

Significant effect: The symptoms disappeared, the hepatosplenomegaly remained stable, and there was no obvious tenderness and pain. The liver function was obviously restored or normal, and lasted for more than half a year.

Effective: The main symptoms disappear or disappear, the hepatosplenomegaly is stable and no obvious Tenderness and snoring pain, liver function tests are normal or the original value is reduced by more than 50%, and lasts for three months.

Invalid: After the end of the treatment, there is no change or deterioration of the indicators.

Observation results

In this group of 30 patients, after taking 1-2 courses of soy peptide liquid, 5 cases were cured clinically, accounting for 16.6%; 8 cases were markedly effective, accounting for 26.7%; 14 cases were effective, accounting for 46.7%; 3 cases were invalid, accounting for 10%; Effective 90%.

Table 1 Comparison of the total course of treatment between the two groups

It is not difficult to see from Table 1 that the total effective rate of soybean polypeptide solution for treating chronic hepatitis B is 90%, and the total effective rate of amino acid capsules in the control group is 86.7%. There is no significant difference between the two groups in the treatment of P>0.05, but the therapeutic effect of the treatment group. Have higher than

More than 76.9%.

The trend of the control group.

Significantly better than the control group, statistically treated P <0.05.

Effects of two groups on type B virus (antigen)

Therapeutic group

Positive and negative negative positive negative negative positive negative after treatment Surface antigen 23 7 16 14 20 10 18 12 e antigen 18 12 13 27 16 14 15 15 As can be seen from Table 5, the surface antigen conversion rate of the treatment group can reach 30.4%, and the e antigen conversion rate reaches 27.8%. The group surface antigen conversion rate was 10%, and the e antigen conversion rate was 6.3%. The contrast between the two groups was statistically better than the control group (P<0.05).

Discuss

In this group of 60 patients, 30 patients in the treatment group, 30 patients in the control group, all were chronic hepatitis B, and most cases with cholecystitis, chronic gastritis, duodenal ulcer embolism, one third of patients with persistent liver function Abnormalities, cases with persistently positive surface antigens and untreated patients, after taking 1-2 days of treatment with soy peptide liquid or as an adjuvant, the total effective rate was 90%, and the surface antigen conversion rate was 30.4%. The negative conversion rate was 27.8%, which was significantly better than the control group, which is a considerable effect. The mechanism of action is to improve the circulation of the blood circulation and regulate the immunity through the high nutritional effect of the 16 polypeptides, 18 amino acids and 11 vitamins on the liver cells in the soybean polypeptide solution. Supporting the righteousness, to the purpose of sin.

Soybean peptide liquid has obvious effects on improving clinical symptoms and retracting hepatosplenomegaly. Its mechanism of action is to enhance the protein synthesis of RNA and DNA in the liver through the transfer of various polypeptides and various amino acids. The excellent protein necessary for the body can directly nutrients and promote cell repair and metabolism, which may be different from the animal blood amino acid capsules. Therefore, it has obvious effects on improving various clinical symptoms and signs.

The motherland medicine believes that the liver is just hidden, non-flexible, and the main blood of the blood. When the virus commits the liver, the liver loses its stagnation, causing the air to block, the blood stasis to stop, and the blood and blood are not smooth. Clinical manifestations such as liver pain, stomach pain, dyspepsia, fatigue, and other symptoms such as hepatosplenomegaly. Therefore, the treatment of this disease, especially the digestive system diseases, not only the nutrition to strengthen the mucosa to improve the metabolism, but also from the blood circulation, reconciliation and blood.

According to the five elements, the liver belongs to the wood, the spleen and stomach belong to the soil, and the two complement each other and restrict each other. The liver disease is most likely to hurt the spleen, causing the lesion of the spleen and stomach, and the spleen is weak and the evil, but can not be scattered in the liver, and the erotic color is in the tendon, thus making Hepatic dysfunction, so the function of the liver's spleen and gallbladder is closely linked to the spleen and stomach transport and absorption system.

Where the transmission of essence, the formation of qi and blood, the operation of the body fluid, the metabolism of the substance, in addition to the power of the spleen and stomach, the function of the liver is to be separated from the liver. For example, "There is a question about the whole form of life." Cloud: "The soil is wood and the wood is good," and it is suggested that we treat the liver and neglect the spleen, so that the blood is biochemically passive, resulting in invincible, virus. Long love, long-term illness, and soy peptide liquid is the medicine for the spleen and stomach, not only the essence of nutrition, but also the innate essence. It is the drug of choice for liver protection, spleen and kidney. "Golden Chambers" Cloud: "See the disease of the liver, know the liver and pass the spleen, when the first spleen, this is a cure for the liver."

At the same time, it has obvious effects on 4 patients with acne. (Internal or external rubbing), it disappears after only 2 weeks of acne. This is a variety of peptides and amino acids. Vitamins have the effect of regulating nutrition on skin metabolism. Soy contains flavonoids, which have the effect of regulating endocrine.

Soybean Peptide Liquid contains a variety of nutrients, which is the best quality of the body. Since the application of the drug, there has been no adverse reactions, and it is now welcomed by the majority of patients and ideal adjuvant therapy.

January 30, 2003

(IV) Application of licorice peptide liquid in the treatment of AIDS drugs

Summary of the preliminary clinical observation of licorice peptide capsule in the treatment of AIDS: Because my hometown is the hardest hit area of AIDS, at the request of Sun Zhen of Dalian Yixiang Biotechnology Co., Ltd., the first time for AIDS volunteers three times a day, one licorice peptide capsule orally, a total of 15 For AIDS patients, the time is 3 months.

The results were effective in 12 cases, with a total effective rate of 80%, ineffective in 3 cases, and a total ineffective rate of 20%.

Among the 12 cases, 3 cases were converted from fever to no fever, and 8 cases were relieved. There were 4 cases of fatigue reduction. There were 1 case of diarrhea and no diarrhea, and 2 cases of diarrhea. Shortness of breath, no shortness of breath is 1 case. There were 2 cases of shortness of breath. There were 2 cases with headaches and 2 cases with headache reduction. There were 2 cases of weight gain.

It can be seen that licorice peptide capsule has a certain therapeutic effect on AIDS patients, and no toxic side effects have been found. Our hospital is willing to further increase the dose of licorice capsules for clinical observation, and record the preclinical CD ₄ values and changes after treatment, so as to scientifically see the therapeutic effect, and record the weight, fatigue, fever, diarrhea and phlegm in more detail. , shortness of breath, rash, etc. Shaodian Township Shifo Aifang Hospital Hospital Leader: Li Tieliang Observing Doctor: Li Tieliang

August 26, 2004

Claims

Claim

1. A method for preparing a polypeptide from a vegetable protein, the steps of which are as follows:

(1) using a spiral propelling feeder or a conveyor feeder to feed the plant material that has been cleaned and removed of impurities into a processing chamber formed by two stainless steel plates at a relatively uniform distance;

(2) Applying a high-frequency high-voltage alternating power source on the electrodes formed by the two stainless steel plates to generate an alternating electromagnetic field in the plant material filled between the two stainless steel plates, and the voltage of the high-frequency alternating power source applied Lower than the breakdown threshold voltage that the filled material can withstand;

(3) monitoring the temperature of the material in the processing chamber. When the temperature of the lysate reaches 90 ° C to 120 ° C, the collection of the polypeptide vapor released in the form of water vapor is condensed to become a polypeptide liquid; The material is pulverized and immersed in water and ethanol, and the resulting mixture is subjected to centrifugation and drying, and then precipitated and filtered to prepare a polypeptide liquid, which is then freeze-dried or dried to prepare a polypeptide powder.

2. A method for preparing a polypeptide from a vegetable protein according to claim 1, wherein: said electromagnetic field generator generates a frequency in the processing chamber at a frequency of from 200 kHz to 2500 MHz, and the voltage between the two plates is between 1000 volts ~ 20,000 volts.

3. A method of preparing a polypeptide from a vegetable protein according to claim 1, wherein the equivalent frequency of the processing chamber is controlled by the matching device to be the same as the frequency of the electromagnetic field generator.

4. The use of the jujube polypeptide prepared by the method of claim 1 in an antitumor drug.

5. The use of the soybean polypeptide prepared by the method of claim 1 for the treatment of eczema, acne, and athlete's foot.

6. The use of the soybean polypeptide prepared by the method of claim 1 for the treatment of chronic hepatitis B

7. The use of the soybean polypeptide prepared by the method of claim 1 for the treatment of gastric ulcer

8. The application of the licorice polypeptide prepared by the method of claim 1 in the treatment of AIDS