AU2019101528A4 - Ace inhibitory peptide with cell repairing, nourishing and activating effects and preparation method thereof - Google Patents

Abstract

Abstract The present invention relates to an ACE inhibitory peptide with cell repairing, nourishing and activating effects, and the preparation method. 4700ReflectorSpec#1[BP=967.6,6461] £0 10 - N R - 59.0 840.6 1082.2 1323.8 1565.4 1807.0 Mass (mhz Fig. 1 regulation,0.0121 renin inhibitor,0.0242 opioid peptide,0.004 stimulation,0.0403 neuropeptide,0.0141 angiotensin converting immunostimulation,0.002 enzyme inhibitor,0.4617 activation of ubiquitin dipeptide-kininase -mediated proteolysis,0.0081 IV inhibitor,0.6512 a-glucosidase inhibitor,0.002 anti- anti-forgetting,0.0081 thrombosis, dipeptidyl peptidase 0.0101 anti-oxidation,0.0847 III inibitor,O.0766 bacterial permease ligand,0.002 diesterase inhibitor,0.0101 Fig. 2

Description

2019101528 05 Dec 2019

ACE INHIBITORY PEPTIDE WITH CELL REPAIRING, NOURISHING AND ACTIVATING EFFECTS AND PREPARATION METHOD THEREOF

TECHNICAL FIELD

The present invention provides an ACE inhibitory peptide with cell repairing, nourishing and activating effects, which is a small-molecular peptide with cell repairing and activating effects, and further discloses a preparation method of the small-molecular peptide, belongingto the field of medicine technology.

BACKGROUND ART

At present, the health-care and health-preserving effects of black foodsare more and more recognized and favored by people. Many kinds of black foodsare used in this invention, such as black peanuts, black sesames, black rice, black soya beans, and black wolfberries, each withdistincteffects and ediblemethods. The task of the present invention is to provide a small-molecular peptide extracted from the above foods, which has the effects of repairing cell damage, activating cells and the like.

A peptide is a biochemical substance between an amino acid and a protein, and has a molecular weight less than that of the protein, and more than that of the amino acid. Peptides are chains of amino acids linked by peptidebonds. The peptidesmay be divided into polypeptides, oligopeptides and small-molecular peptides according to the compositions of amino acids. The polypeptide refers to a chain of 10 or moreamino acids with a molecular weight of more than lOkDa, such as soy protein having a molecular weight of several tens kDa, and collagen having a molecular weight of 100 to 300kDa, etc. The oligopeptide refers to a chain of 2 to 9 amino acids with a molecular weight of less than IkDa. In comparison to polypeptides and oligopeptides, the small-molecular peptide (OCO) is an oligopeptide with ultralow molecular weight, typically of 180 to 480 Da, and is a highly active peptide consisting of only 2 to 4 amino acids. Collagen ofsmall-molecular peptide refers to dipeptide, tripeptide or tetrapeptide with an average molecular weight of 300 Da. The well-known small-molecular hyaluronic acid is also one of the small peptide collagens.

Peptides are basic active substances essential to the human body. The discovery of peptides has deciphered the codes of the birth, senility, illness and death of human beings, and has also opened a new era of disease treatment. According to the present researches, the peptides have multiple i

2019101528 05 Dec 2019 positive effects on the human body, such as inhibiting, activating, repairing and promoting effects. In addition, the small-molecular peptides can further supplement nutritional requirements, repair defects and improve functional states. The small pep tideshave the characteristic of being preferentially absorbed by the human body, and can protect amino acids from being damaged when absorbed by the human body. The mixture of the small peptides and the amino acids presents an optimal absorption mechanism for the human body to absorb proteins. The small peptides also bear the mission of a carrier in the human body, to adsorb, adhere and load nutrient substances commonly taken by the human body, especially trace elements beneficial to the human body such as calcium, ontoit. Then, nutrient substances are transported by the small peptidesfunctioning as a means of transportto various cells, organs and tissues of the human body, and are absorbed and utilized by the human body together with the small peptidesto play their respective functional roles. After being absorbed by the human body, the small peptides serve as neurotransmitters to transmit information, so that various organs, systems and tissues of the human body play theirrespective and integral roles. Focusing on repairing denatured cells in the human body and solving the problems of absorption and utilization in the human body,the small-molecular peptideshave more advantageous effects on the human bodycompared with polypeptides and oligopeptides. In case of common chronic diseases such as hypertension, diabetes, gout, cardiovascular and cerebrovascular diseases, hyperthyroidism, arthritis, gastritis, insomnia, cancers and the like, medicines can only control these diseases to some extent, while the small-molecular peptides can achievenot only nutrition and health care, butalso diseaseprevention owning to their bidirectional regulation function.

The small-molecular peptide plays a key role in the field of cell activation. Applicants of theinventionhavefound two small-molecular peptides aseffective inhibitors with relatively high content in proteins extracted from black foods: an ACE inhibitory peptide (which is antihypertensive) and a DP P-IV inhibitory peptide (which is antidiabetic). Based onsuch findings, we continued to study the activation of tumor-infiltrating dendritic cells (TIDCs) induced by melanoma cells byone of the small-molecular peptides. Dendritic cells (DCs) are professional antigen presenting cells, which have the abilities to capture, process and present antigens. Mature DCs can induce T lymphocytes to be activated into tumor-specific cytotoxic T lymphocytes (CTL), and can secrete costimulatory molecules (CD80, CD86), major histocompatibility complex Π (MHC Π ), cytokine IL-12 and the like, and play an immunomodulatory role. Cytokines such as CD80, CD86, MHCII and IL-12can only be highly expressed in mature DCs, so they are called

2019101528 05 Dec 2019 maturation marker molecules of DCs. A large number of DCs are infiltrated in tumor tissues to obtain tumor-infiltrating dendritic cells (TIDCs).However, tumor cells and tumor microenvironment release factors will inhibit or reverse the maturation and normal functions of DCs, resulting in immatureTIDCs in melanoma tissues which can only express low levels of CD80, CD86, IL-12 and transmembrane glycoprotein CDllc. DCs can also secrete immunosuppressive cytokine IL-10. Immunoescape in tumor mainly results from insufficient activation of tumor-specific MHC-I-restricted CTL, while lowly-expressed costimulatory molecules on immature TIDCs may be the main reason for the failure of CTL activation. Therefore, TIDCs have become one of the most promising targets for controlling tumor escape. The small-molecular peptideseparated and extracted fromblack foodsin this invention can enhance the inhibited immune functions of chemotherapy or radiotherapy patients. It has been found in studiesthat this small peptidecan induce the maturation of bone marrow-derived dendritic cells (BMDCs) and promote the expression of maturation marker molecules on the surfaces of the BMDCs.

The small-molecular peptidesplay a crucial role as well in the field of cell repair. Studies have shown that hyperoxiamaycause damages to body’s cells, as well as acute lung injury, pneumonia, lung cancer and other diseases. Some common antioxidants (such as vitamin E) available on the market can remove active oxygen, but have a weak repairing effect on damages that have been caused, and can produce side effects. Experiments have showed that the small-molecular peptideof this inventionhave a powerfulantioxidationeffect.

SUMMARY OF THE INVENTION

In order to solve the problems in the prior art, the present invention provides an ACE inhibitory peptide with cell repairing, nourishing and activating effects, which is a small-molecular peptide separated and extracted from black foods, and studies its cell repairing and activating effects.

A preparation method of the ACE inhibitory peptide with cell repairing, nourishing and activating effects of this invention comprises the following steps:

weighing the following materials in parts by weight: 200-300 parts of black soya beans; 200-300 parts of black peanuts; 100-500 parts of black sesames; 100-150 parts of black rice; 100-150 parts of black oats; 80-125 parts of wolfberries; 100-150 parts of black mulberries; 30-80 parts of blackberries; and 80-125 parts of brown sugar;

2019101528 05 Dec 2019

A. fully disinfecting a fermentor by using alcohol, and blanching with boiling water for sterilizing;

B. cleaning the above materials with water, and drying to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, and subsequently pouring the materials into the fermentor;

C. adding boiling water into the fermentor, followed by uniformly stirring, wherein a volume ratio of the materials to water is 1: 4;

D. when the temperature in the fermentor is reduced to about room temperature, adding fermentative bacteria powder into the fermentor, and sealing for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria is 1: 1;

E. hydrolysis: putting fermentation broth into a mixed enzymolysisfermentor, and rising temperature to 53°C, followed by the addition of 0.1% W of cellulase, controlling the reaction temperature to 45-65°C and the pH value to 6.0 or so, and performing a reaction for 1 h;

F. adding ferment inactivated with cellulase at a high temperature into 0.1% W of papain;

G. controlling the following reaction conditions: pH of 6.0 or so, temperature of 53 °C, and dosage of 0.2% w;

H. controlling reaction time to 30-60 min; and

I. putting the reactant into a centrifuge and centrifuging at 5000 r/min for 20 min, storing supernatant and precipitate separately, filtering the supernatant, and drying the precipitate in a drying box for later use, thereby obtaining a food small-molecular peptide named as: ACE antihypertensive inhibitory peptide.

[Usage and dosage] oral administration; 2 times/day; 50 ml/time.

The pharmacological properties of the present invention are as follows:

blackpeanuts are of a new variety containing nutrients such as calcium, potassium, copper, zinc, iron, selenium, manganese, 8 vitamins, and 19 amino acids essential to the human body, which is also rich in trace elements such as selenium, iron and zinc, and melanin. Black peanuts are rich in 18 amino acids, the total amount of which is 27.57%, after black soya beans, where essential amino acids accounts for 22.90%.

Black soya beans are black seeds of soybeans,a species of legume. The black soya beans have a protein content of 36%, are easy to digest, and have an important significance for meeting the requirements of the human body on proteins. The black soya beans have a fat content of 16%,

2019101528 05 Dec 2019 mainly including unsaturated fatty acids, the absorption rate of which is up to 95%, and further have the effect of reducing cholesterol in blood in addition to meeting the requirements of the human body on fat. The black soya beans are rich in vitamins, vitellin, melanin, lecithin and other substances, wherein vitamin B and vitamin E are very high in content, and achieve nutrition and health care effects. The black soya beans also contain rich trace elements, which are essential to maintain complete body's functions, delay the aging of the body, reduce the blood viscosity, and meet the brain's demand for trace substances. However, the black soya beans itself have some disadvantages, for example:

(1) the black soya beans are ingested to enter kidney yin, so people who are afraid of cold in winter and suffer kidney yang deficiency are suggested not to eat too much;

(2) afterfried black beans are eaten, constipation can be caused.

Therefore, black rice is appliedin the present invention. The black rice has rich nutrition, as well as high edible and medicinal values. Meanwhile, the black rice can solve the problemthat wall-broken peanuts and black soya beans contain too much oil, harmonize the characteristics of Yin in nature and Yang deficiency of the black soya beans, and achieve the purposes of exerting the advantages of various ingredients and eliminating various defects. In order to overcome the defect ofthe black soya beans that easily cause constipation, semen sesame is added. The semen sesame contains a large amount of fat and proteins, which can help achieve the effect of relaxing bowels, andnutritional ingredientssuch as sugars, vitamin A, vitamin E, lecithin, calcium, iron, and chromium. The semen sesame has the effects of invigorating stomach, protecting liver and promoting red blood cell growth, and meanwhile can increase melanin in vivo, which is beneficial to hair growth.

In addition to the above black foods, black wolfberries, medlar, black oats, black mulberries, blackberries and brown sugar are also applied in the present invention. The black wolfberries contain a large amount of anthocyanins, and have the effects of delaying senescence, maintaining beauty and keeping young, promoting the production of body fluid and improving eyesight, improving the ability of skin to absorb oxygen, protecting liver function, tonifying kidney function, improving immunity, resisting fatty liver, preventing and resisting cancers, resisting fatigue and the like. The medlar is a mature fruit of wolfberries in eggplant plants, has various health-care effects, and is of a medicinal and edible dual-purpose food approved by the Ministry of Health. The medlar is beneficial to health in case of moderate ingestion, achieves the main edible benefits of nourishing

2019101528 05 Dec 2019 essence and improving eyesight, and nourishing the liver and kidney, and are suitable for crowd who suffers from consumptive disease and deficiency of kidney essence, soreness of waist and knee joint, impaired visual function, as well as vertigo and tinnitus.

The advantageous effects of the inventionare as follows:

Provided is a small-molecular peptide from black foods,whichisfound through component identification (it is proved to be a small molecule peptide based on the provided identification data or spectra !)tofunctionas an ACE (antihypertensive) inhibitor. Experiments have proved that such small-molecular peptide has an activating effect on TIDCs induced by melanoma cells and a repairing effect on A549 cell damage caused by hydrogen peroxide.Medicaments prepared from the small-molecular peptide have the characteristics of remarkable clinical effect,as well as small toxic and side effects. Thepreparation method of the extracted food small-molecular peptide of the present invention is simple and practical.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows component identification assay of a small-molecular peptide of the present invention;

Fig. 2 shows a frequency of occurrence of active peptides in soy protein of the present invention;

Fig. 3 shows a frequency of occurrence of an ACE inhibitory peptide obtained fromcleavage of a soy protein sequence by single enzyme/enzyme combination of the selected five enzymes, wherein the frequency of occurrence of the ACE inhibitory peptide obtained fromcleavage by two enzymes proteinase K+papain combination is highest; and

Fig. 4 shows a frequency of occurrence of a DPP-IV inhibitory peptide obtained from cleavage of a soy protein sequence bysingle enzyme/enzyme combination ofthe selected five enzymes, wherein the frequency of occurrence of the DPP-IV inhibitory peptide obtained fromcleavage by two enzymes proteinase K+papain combination is highest.

DETAILED DESCRIPTION OF THE INVENTION

To make the objectives, technical solutions, and advantages of the present invention clearer, the examples of the present invention will be described in further detail below with reference to the

2019101528 05 Dec 2019 accompanying drawings.

Example 1

200g of black soya beans, 200g of black peanuts, lOOg of black sesames, lOOg of black rice, lOOg of black oats, 80g of wolfberries, lOOg of black mulberries, 30g of blackberries, and 80g of brown sugar were weighed in parts by weight;

A. a fermentor was fully disinfected by using alcohol, and blanched with boiling water for sterilizing;

B. the above materials were cleaned with water, and dried to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, andthe materials were subsequently poured into the fermentor;

C. boiling water was added into the fermentor, followed by uniformly stirring, wherein a volume ratio of the materials to water wasl: 4;

D. when the temperature in the fermentor was reduced to about room temperature, fermentative bacteria powder was added into the fermentor and sealed for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria was 1:1;

E. hydrolysis: fermentation broth was put into a mixed enzymolysisfermentor, and heated to 53°C, followed by the addition of 0.1% W of cellulase; the reaction temperature was controlled to 45-65°C and the pH value to 6.0 or so; a reaction was performed for 1 h;

F. ferment inactivated with cellulase at a high temperature was added into 0.1% W of papain;

G. the following reaction conditions were controlled:pH of 6.0 or so, temperature of 53 °C, and dosage of 0.2% w;

H. a reaction time of 30-60 min was controlled; and

I. the reactant was put into a centrifuge and centrifuged at 5000r/min for 20 min; supernatant and precipitate were stored separately, wherein the supernatant was filtered, and the precipitate was dried in a drying box for later use, thereby obtaining a food small-molecular peptide (it is preferable to provide proofs to demonstrate that it is asmall-molecular peptide!) named as: ACE antihypertensive inhibitory peptide.

Example 2

300g of black soya beans, 300g of black peanuts, 150g of black sesames, 150g of black rice, 150g of black oats, 125g of wolfberries, 150g of black mulberries, 80g of blackberries, and 125g of brown sugar were weighed in parts by weight;

2019101528 05 Dec 2019

A. a fermentor was fully disinfected by using alcohol, and blanched with boiling water for sterilizing;

B. the above materials were cleaned with water, and dried to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, andthe materials were subsequently poured into the fermentor;

C. boiling water was added into the fermentor, followed by uniformly stirring, wherein a volume ratio of the materials to water wasl: 4;

D. when the temperature in the fermentor was reduced to about room temperature, fermentative bacteria powder was added into the fermentor and sealed for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria was 1:1;

E. hydrolysis: fermentation broth was put into a mixed enzymolysisfermentor, and heated to 53 ° C, followed by the addition of 0.1% W of cellulase; the reaction temperature was controlled to 45-65 ° C and the pH value to 6.0 or so; a reaction was performed for 1 h;

F. ferment inactivated with cellulase at a high temperature was added into 0.1% W of papain;

G. the following reaction conditions were controlled: pH of 6.0 or so, temperature of 53 ° C, and dosage of 0.2% w;

H. a reaction time of 30-60 min was controlled; and

I. the reactant was put into a centrifuge and centrifuged at 5000r/min for 20 min; supernatant and precipitate were stored separately, wherein the supernatant was filtered, and the precipitate was dried in a drying box for later use, thereby obtaining a food small-molecular peptide named as: ACE antihypertensive inhibitory peptide.

Example 3

250g of black soya beans, 250g of black peanuts, 125g of black sesames, 125g of black rice, 125g of black oats, lOOg of wolfberries, 125g of black mulberries, 50g of blackberries, and lOOg of brown sugar were weighed in parts by weight;

A. a fermentor was fully disinfected by using alcohol, and blanched with boiling water for sterilizing;

B. the above materials were cleaned with water, and dried to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, andthe materials were subsequently poured into the fermentor;

C. boiling water was added into the fermentor, followed by uniformly stirring, wherein a

2019101528 05 Dec 2019 volume ratio of the materials to water was 1: 4;

D. when the temperature in the fermentor was reduced to about room temperature, fermentative bacteria powder was added into the fermentor and sealed for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria was 1:1;

E. hydrolysis: fermentation broth was put into a mixed enzymolysisfermentor, and heated to 53°C, followed by the addition of 0.1% W of cellulase; the reaction temperature was controlled to 45-65°C and the pH value to 6.0 or so; a reaction was performed for 1 h;

F. ferment inactivated with cellulase at a high temperature was added into 0.1% W of papain;

G. the following reaction conditions were controlled: pH of 6.0 or so, temperature of 53 °C, and dosage of 0.2% w;

H. a reaction time of 30-60 min was controlled; and

I. the reactant was put into a centrifuge and centrifuged at 5000r/min for 20 min; supernatant and precipitate were stored separately, wherein the supernatant was filtered, and the precipitate was dried in a drying box for later use, thereby obtaining a food small-molecular peptide named as: ACE antihypertensive inhibitory peptide.

Experimental Examples

The black foods prepared inExamples 1-3 weresubjected to protein extraction. Component identification is firstly performed on the extracts, see Fig. 1. The frequency of occurrence of active peptides in soy protein was then measured using BIOPEP, and the graph was shown in Fig. 2. Five enzymes were then selected, and the extracted protein sequence was subjected to cleavage by single enzyme/enzyme combination, thereby obtaining the frequency of a first small-molecular inhibitory peptide, see Fig. 3. Five enzymes were then selected, and the extracted protein sequence was subjected to cleavage by single enzyme/enzyme combination, thereby obtaining the frequency of a further small-molecular inhibitory peptide, see Fig. 4.

Experimental Example 1

Component identification was performed on the extracted small-molecular peptide to obtain the distribution frequency of the inhibitory peptide.

The results are shown in Figs. 1, 2, 3 and 4.

Experimental Example 2 Activation Effect of Small-molecular Peptides in Black Foods on Cells

BALB/c mice were purchased from Beijing Charles River Laboratory Animal Co., Ltd., and

2019101528 05 Dec 2019 fed at room temperature in a quiet environment, wherein the sunshine time was from 7:00 to 19: 00 each day. Mice were supplied with sufficient food and water daily by professional animal breeders.

The used reagents DMSO and trypsin, serum, culture medium, ELISA kit, PE-CD86, APC-MHC II, FITC-CDllc, IL-4, recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF), murine IL-12 and IL-10 ELISA kit were purchased from Nanjing Jiancheng Bioengineering Company.

1. Induced culture and identification of TIDCs

Melanoma cells B16 in the logarithmic growth phase were cultured in a serum-free medium for 24 h. Supernatant was collected to obtain a B16 cell conditioned medium, whichwas filtered with a 0.22 pm filter membrane, then sub-packaged and stored at -80°C for later use. A BALB/c mousewas sacrificed by decapitation. The intact femur was dissected out. A cell suspension was collected from the femur and cultured in an RPMI 1640 medium withIL-4 and recombinant murine GM-CSFadded. Non-adherent cells were removedafter 24h of culture at 37 °C. The RPMI 1640 medium containing IL-4 and recombinant murine GM-CSF was added again, and was changed every other day. When the induced culture was performed for 4 days, a 50% B16 cell conditioned medium was added, and then the induced culturecontinuedfor 3 days. TIDCs were identified by photographing with an inverted phase contrast microscope.

2. Method for detecting CDllc, CD86, MHC II, IL-12 and IL-10 in TIDCs

Afterthe culture and identification, the obtained TIDCswere inoculated into 96-well plates. 0pmol/L, 15pmol/L, 25pmol/L and 50 pmol/L of ACEs were added into the TIDCs separately, which were recorded as a 0 pmol/L group, a 15 pmol/L group, a 25 pmol/L group, and a 50 pmol/L group, followed by incubating at 37 °C for 48 h and separating TIDCs cells and supernatant therefrom. The collected TIDCs were washed twice with PBS, then added with 100 pL of PBS and 10 pL of rat ChaoXing serum precooled with iceand blockedat 4 °C for 40 min,and then addedwith fluorescence labeled CDllc, CD86 and MHCII antibodies and incubatedat 4 °C for 40 min,and washed twice with PBS and detected by a flow cytometer. The expression levels of CDllc, CD86 and MHC Π on the surfaces ofTIDCs were expressed by mean fluorescence intensity values. The TIDCs cell supernatant was collected, and IL-2 and IL-10 in the TIDCs were measured according to an ELISA kit specification.

3. Observation of binding of TP5 to TIDCs.200 pL of 1 pg/mL FITC labeled TP5 solution was added to a TIDCs cell culture dish, and incubated at 37 °Cfor 3 h; washed with PBS and io

2019101528 05 Dec 2019 immobilized with a 4% neutral paraformaldehyde solution, andthen added with cell nucleus dye Hoechst 33342, stained at room temperature for 10 min,washed with PBS, and then added with 200 pL of anti-fluorescence quencher. The binding of ACE to TIDCs was observed with a laser confocal microscope.

4. Statistical methods.SPSS 20.0 statistical software was used. Metrological data were expressed as x ± s. Comparisons between groups were performed using one-way analysis of variance or analysis of repeatedly-measured variance. P<0.05 was statistically significant in difference.

See Appendix Table 1 for results:

Table 1. Comparison of expression levels of CD 11c, CD86, MHC II, IL-12, and IL-10 in TIDCs (x ± s)

	CDllc (%)	CD86 (%)	MHCII (%)	IL-12 (pg/mL)	IL-10 (pg/mL)
50 pmot/L group	18. 35 + 0.15*	42.27 + 0.14	49. 85 + 0.27	317.0+1.0	105.0+1.0
25 pmot/L group	15. 85 + 0.22	37. 02 + 0.21*	39. 26 + 0.24*	282.0+ 1.5	131.0+1.5
15 pmoI/L group	16. 37 + 0.12	21.03 + 0.18	42. 28 + 0.29*	290.0+ 1.4	136.0+1.3
0 pmoI/L group	13. 35 + 0.16	12. 54 + 0.19	26. 08 + 0.26	222. 0 ± 1. 2	145.0+1.1

Note:compared to the 0 pmol/L group, *P <0.05; compared to 10 pmol/L group, #p <0.05; compared to the 20 pmol/L group, ΔΡ <0.05.

Conclusion:

The ability of TIDCs subjected to ACE action to express costimulatory molecules was increased significantly and the secretion amount of IL-12 was increased gradually, indicating that TIDCs were activated by ACE. In contrast, the secretion amount of IL-10 decreased with the increase of ACE concentration, probably because ACE promoted IL-12 secretion, which in turn inhibits IL-10 secretion.

The results showed that ACE could promote the expression of maturation marker molecules CDllc, CD86 and MHC Π on the surfaces of TIDCs, promote TIDCs to secret IL-12 and inhibit the secretion of IL-10, indicating ACE could activate the antigen presenting function of TIDCs whose immune activity was inhibited, and play an important role in specific immune response.

Experimental Example 3

2019101528 05 Dec 2019

Effects of small peptides in black video on cell repair

A 549 cell line was cultured: the 549 cell line was recovered and then cultured in a modified Eagle (DMEM) high glucose medium containing caff serum in percentage by volume of 10%, penicillin (100 mg/L) and streptomycin (100 mg/L), aseptically cultured in a CO2 incubator in percentage by volume of 5% at 37 °C, and treated with trypsin in percentage by volume of 0.25% for digestive passage.

Cells were stimulated with 500 pmol/L of H2O2 to produce injuries. Cells were divided into a blank control group, a H2O2 group (injured with 500 pmol/L of H2O2 for 24 h), an ACE inhibitory peptide pretreatment group (incubated with 10, 20, 30, 50 mg/mL of ACE for 24 h), and an ACE posttreatment group (injured with 500 pmol/L of H2O2 for 24 h, and then incubated with 10, 20, 30, 50 mg/mL of ACE for 24 h). The cells of the positive control group were treated with antioxidant N-acetylcysteine (NAC), i.e. the positive control pretreatment group was incubated with 6 mmol/L of NAC for 24 h, and then injured with 500 pmol/L of H2O2 for 24 h. The positive control group was injured with 500 pmol/L of H2O2 for 24 h and then incubated with 6 mmol/L of NAC for 24 h.

Cells were cultured in a culture dish having a diameter of 10cm, treated with medicaments,then collected, and washed three times with a 4 °C phosphate buffer solution (PBS). The precipitate was lysed with cell lysate, and centrifuged under 1600^xg at 4 °C for 15 min. The supernatant was taken as a sample to be tested. MTT colorimetric method was used to measure the cell viability. The MDA content, SOD activity, and CAT activity T-AOC were measured strictly according to the specification.

The protein concentrations of cells in different groups were determined by using a protein concentration kit, and the levels of MDA, SOD, CAT and T-AOC within the cells were corrected.

Experimental data were expressed as x ± s, respectively. SPSS 19.0 software was used for statistical analysis. The comparisons between groups were subjected to one-way analysis of variance, and pairwise comparisons were performed using Dunnett t test. Test level a = 0.05.

The small-molecular peptide provided by the present invention has an activating effect on TIDCs induced by melanoma cells and a repairing effect on A 549 cell damage caused by hydrogen peroxide. A medicament prepared from the small-molecular peptide has the characteristics of remarkable clinical effect, as well as small toxic and side effects. The preparation method of the extracted food small-molecular peptide of the present invention is simple and practical.

The sequence numbers of the foregoing examples of the present invention are merely for a

2019101528 05 Dec 2019 descriptive purpose, and not intended to represent the advantages and disadvantages of the examples.

The above are only preferred examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements and the like made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (3)

2019101528 05 Dec 2019 WHAT IS CLAIMED IS:

1. An ACE inhibitory peptide with cell repairing, nourishing and activating effects, wherein it is prepared by the following method:

weighing the following materials in parts by weight: 200-300 parts of black soya beans, 200-300 parts of black peanuts, 100-500 parts of black sesames,100-150 parts of black rice, 100-150 parts of black oats, 80-125 parts of wolfberries, 100-150 parts of black mulberries, 30-80 parts of blackberries, and 80-125 parts of brown sugar;

A. fully disinfecting a fermentor by using alcohol, and blanching with boiling water for sterilizing;

B. cleaning the above materials with water, and drying to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, and subsequently pouring the materials into the fermentor;

C. adding boiling water into the fermentor, followed by uniformly stirring, wherein a volume ratio of the materials to water is 1: 4;

D. when the temperature in the fermentor is reduced to about room temperature, adding fermentative bacteria powder into the fermentor, and sealing for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria is 1: 1;

E. hydrolysis: putting fermentation broth into a mixed enzymolysisfermentor, and rising temperature to 53°C, followed by the addition of 0.1% W of cellulase, controlling the reaction temperature to 45-65°C and the pH value to 6.0 about; performing a reaction for 1 h;

F. adding ferment inactivated with cellulase at a high temperature into 0.1% W of papain;

G. controlling the following reaction conditions: pH of 6.0 about, temperature of 53 °C, and dosage of 0.2% w;

H. performing the reaction under a reaction time of 30-60 min; and

I. putting the reactant into a centrifuge and centrifuging at 5000 r/min for 20 min, storing supernatant and precipitate separately, filtering the supernatant, and drying the precipitate in a drying box for later use, thereby obtaining a food small-molecular peptide named as: ACE antihypertensive inhibitory peptide.

2. The ACE inhibitory peptide with cell repairing, nourishing and activating effects according i

2019101528 05 Dec 2019 to claim 1, wherein, a preferred ratio of each raw material is: 250g of black soya beans:250g of black peanuts:125g of black sesames: 125g of black rice:125g of black oats:100g of wolfberries: 125g of black mulberries:5Og of blackberries: lOOg of brown sugar.

3. A preparation method of the ACE inhibitory peptide with cell repairing, nourishing and activating effects comprising the following steps:

weighing the following materials in parts by weight: 200-300 parts of black soya beans, 200-300 parts of black peanuts, 100-500 parts of black sesames, 100-150 parts of black rice, 100-150 parts of black oats, 80-125 parts of wolfberries, 100-150 parts of black mulberries, 30-80 parts of blackberries, and 80-125 parts of brown sugar;

A. fully disinfecting a fermentor by using alcohol, and blanching with boiling water for sterilizing;

B. cleaning the above materials with water, and drying to remove moisture, followed by wall-breaking crushing by using a wall breaking machine, and subsequently pouring the materials into the fermentor;

C. adding boiling water into the fermentor, followed by uniformly stirring, wherein a volume ratio of the materials to water is 1: 4;

D. when the temperature in the fermentor is reduced to about room temperature, adding fermentative bacteria powder into the fermentor, and sealing for 7 days for fermentation, wherein a ratio of yeast to lactic acid bacteria is 1: 1;

E. hydrolysis: putting fermentation broth into a mixed enzymolysisfermentor, and rising temperature to 53°C, followed by the addition of 0.1% W of cellulase, controlling the reaction temperature to 45-65°C and the pH value to 6.0 or so; performing a reaction for 1 h;

F. adding ferment inactivated with cellulase at a high temperature into 0.1% W of papain;

G. controlling the following reaction conditions: keeping pH of 6.0 about, temperature of 53 °C, and dosage of 0.2% w;

H. controlling reaction time to 30-60 min; and

I. putting the reactant into a centrifuge and centrifuging at 5000r/min for 20 min, storing supernatant and precipitate separately, filtering the supernatant, and drying the precipitate in a drying box for later use, thereby obtaining a food small-molecular peptide named as: ACE

2019101528 05 Dec 2019 antihypertensive inhibitory peptide.