CN114805521B - American ginseng polypeptide and composition thereof and application of American ginseng polypeptide and composition thereof in lowering blood pressure and improving immunity - Google Patents
American ginseng polypeptide and composition thereof and application of American ginseng polypeptide and composition thereof in lowering blood pressure and improving immunity Download PDFInfo
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Abstract
The application discloses American ginseng polypeptide, a composition thereof and application of the American ginseng polypeptide in reducing blood pressure and improving immunity. The American ginseng polypeptide and the composition thereof comprise at least one of the polypeptides shown in SEQ ID NO. 1-8. The American ginseng peptide 1-8 has obvious antioxidant performance. In vivo experiments prove that the American ginseng peptides 1-8 not only have the blood pressure reducing effect equivalent to that of captopril, but also specifically realize the blood pressure reducing effect by improving RAAS imbalance, and have potential application prospects in the fields of improving secondary hypertension caused by renal hypertension, developing diastole with waist soreness and back pain and the like.
Description
Technical Field
The application relates to the technical field of American ginseng, in particular to American ginseng polypeptide, an American ginseng polypeptide composition and application of the American ginseng polypeptide composition in blood pressure reduction and immunity improvement.
Background
American ginseng (American ginseng) is a plant of the genus Panax of the family Araliaceae, is native to Canada and the United states, is successfully introduced in China, and is popular with people of all countries in the world because of wide biological activity and unique pharmacological action. To date, there are 3 aglycones that have been isolated and identified as saponin components from American ginseng, namely Dammarane type (Dammarane), oleanane type (Oleanane), and Ocotillol type (Ocotillol), and nearly 40 types of isolated ginsenosides.
Modern clinical pharmacological experiments prove that the American ginseng has the effects of protecting cardiovascular cells, enhancing nerve cells, enhancing the immunity of the organism, conditioning endocrine and the like. However, further deep digging of active ingredients in American ginseng and secondary resource development and utilization of the active ingredients still have very important significance.
Disclosure of Invention
In view of the above, the present application aims to provide an active ingredient of panax quinquefolium different from the active ingredients of panax quinquefolium disclosed in the prior art, so as to fully explore and develop a utilization method of the active ingredient of panax quinquefolium.
In a first aspect, the embodiments of the present application disclose an American ginseng polypeptide and a composition thereof, comprising at least one of the polypeptides shown in SEQ ID No. 1-8.
In a second aspect, the present application discloses a pharmaceutical composition comprising the polypeptides of the first aspect and compositions thereof, and a pharmaceutically acceptable carrier or diluent.
In embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable salt of the american ginseng polypeptide of the first aspect, and optionally a buffer, a tonicity modifier, a pharmaceutically acceptable vehicle, and/or a stabilizer.
In the examples of the present application, the pharmaceutically acceptable carrier or diluent is used to improve the tolerability of the composition and allows for better solubility and better bioavailability of the active ingredient.
In the present examples, the pharmaceutically acceptable carrier or diluent includes an emulsifier, a thickener, a redox component, a starch, an alcohol solution, a polyethylene glycol, or a lipid.
In a third aspect, the present application discloses methods for preparing the polypeptides of the first aspect and compositions thereof, comprising:
obtaining callus of American ginseng;
obtaining the clone of the American ginseng callus;
performing suspension culture in a liquid culture solution to obtain American ginseng cells with high protein yield;
collecting the American ginseng cells, and performing enzymolysis, gel chromatography purification and liquid chromatography purification on the American ginseng cells to obtain the American ginseng polypeptide and the composition thereof.
In the embodiment of the present application, the step of suspension culture specifically includes: performing dark culture on the American ginseng callus clone in a first suspension culture medium for 20 days, and adding a fresh second suspension culture medium to continue culture for 7 days to obtain the high-yield cells;
or alternatively
Carrying out dark culture on the American ginseng callus clone in a first suspension culture medium for 15 days, and then transferring the American ginseng callus clone to a fresh second suspension culture medium for culture for 15 days to obtain the high-yield cells;
wherein the first suspension culture medium is an MS culture medium containing at least one of 0.02-0.4 mg/L gibberellin A12, 0.02-0.4 mg/L gibberellin A13, 0.02-0.4 mg/L gibberellin A15, 0.02-0.4 mg/L gibberellin A19, and 0.02-0.4 mg/L gibberellin A23;
the second suspension culture medium is an MS culture medium containing at least one of 0.02-0.4 mg/L gibberellin A12, 0.02-0.4 mg/L gibberellin A13, 0.02-0.4 mg/L gibberellin A15, 0.02-0.4 mg/L gibberellin A19, 0.02-0.4 mg/L gibberellin A23, 0.02-0.4 mg/L gibberellin A24 and 0.02-0.4 mg/L gibberellin A34.
In the examples herein, the first suspension medium further comprises 1.5-5.4 wt% sucrose, 0.02-3.4 wt% D-galactose; the second suspension medium further comprising 1.5-5.4 wt% sucrose, 0.02-3.4 wt% D-galactose.
In the examples of the present application, the first suspension medium is an MS medium containing at least one of 0.1 to 0.3mg/L of gibberellin A12, 0.1 to 0.3mg/L of gibberellin A13, 0.1 to 0.3mg/L of gibberellin A15, 0.1 to 0.3mg/L of gibberellin A19, and 0.1 to 0.3mg/L of gibberellin A23;
the second suspension culture medium is an MS culture medium containing at least one of 0.1-0.3 mg/L gibberellin A12, 0.1-0.3 mg/L gibberellin A13, 0.1-0.3 mg/L gibberellin A15, 0.1-0.3 mg/L gibberellin A19, 0.1-0.3 mg/L gibberellin A23, 0.1-0.3 mg/L gibberellin A24 and 0.1-0.3 mg/L gibberellin A34.
In a fourth aspect, the present application discloses the use of the american ginseng polypeptide and the composition thereof of the first aspect, or the use of the american ginseng polypeptide and the composition thereof prepared by the preparation method of the second aspect in the preparation of drugs related to lowering blood pressure or enhancing immunity.
Compared with the prior art, the application has at least the following beneficial effects:
the method comprises the steps of preparing calluses of American ginseng seedlings, carrying out induction culture on the calluses to obtain clone of the calluses, carrying out large-scale suspension culture to obtain high-yield cell strains of the calluses, carrying out enzymolysis and purification on cell cultures of the cell strains, and obtaining 1-8 American ginseng peptides. In vitro experiments show that the American ginseng peptide 1-8 has obvious antioxidant performance. Finally, in vivo experiments prove that the American ginseng peptides 1-8 not only have the equivalent blood pressure lowering effect of captopril, but also specifically realize the blood pressure lowering effect by improving RAAS imbalance, and have potential application prospects in the fields of improvement of secondary hypertension caused by renal hypertension, progression of diastole with waist soreness and back pain and the like.
Drawings
FIG. 1 is a gel chromatography elution profile provided in examples 1 to 6 of the present application.
FIG. 2 is a gel chromatography elution profile provided in comparative examples 1 to 7 of the present application.
FIG. 3 is an electrophoretogram of F1 to F15 provided in the examples of the present application.
FIG. 4 is an electrophoretogram of F16 to F30 provided in the examples of the present application.
FIG. 5 is a RP-HPLC plot of F3, F4, F8, F9, F10, F11, F13, F14 and F15 as provided in the examples herein.
FIG. 6 is a RP-HPLC plot of F16, F17, F18, F21, F22 and F23 as provided in the examples herein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually specified in detail in this application are conventional and commercially available; methods which are not specified in detail are all customary experimental methods and are known from the prior art.
Induction and culture of American ginseng callus and establishment of suspension cell line
1. Materials and methods
1. Material
American ginseng seedling, longcounty Kangyuan medicinal material planting professional cooperative. Gibberellin A3, shenzhen zhen zhengqiang biotechnology, purity: 98 percent of; gibberellin A9, CAS:427-77-0, beijing Bailingwei science and technology Limited; gibberellin A12, shenzhen Zhenzhen Zhengqiang Biotechnology Limited, CAS:1164-45-0; gibberellin A13, shenzhen Shenqiang Biotechnology Limited, CAS:2922-24-9; gibberellin A15, shenzhen Shenzheng Biotech, CAS:13744-18-8; gibberellin A19, shenzhen Shenqiang Biotechnology Limited, CAS:6980-44-5; gibberellin A20, available from Bailingwei science and technology Co., ltd, beijing, CAS, 19143-87-4; gibberellin A23, shenzhen Shenqiang Biotechnology, inc., CAS:20134-29-6; gibberellin A24, shenzhen Shenqiang Biotechnology Limited, CAS:19427-32-8; gibberellin A34, shenzhen Shenqiang Biotechnology Limited, CAS:32630-92-5.
2. Callus induction and subculture
Soaking American ginseng root in alcohol for 3-5 times, washing with sterile water for 5 times after 30min each time, taking the middle section, cutting into slices, inoculating to callus induction culture medium, inoculating 4-5 slices in each culture bottle, placing in a biochemical incubator, dark culturing at 24 +/-1 ℃, and culturing for 28-30 days.
The callus culture medium used therein was a medium containing 1mg/L of 2,4-D, 3g/L of sucrose, 2.5mg/L of zeatin, 700mg/L of LH (lactoprotein hydrolysate, product No. L33040; purity: BR) and lmg/L of KT (cytokinin, sigma) and 0.7% agar concentration, pH5.8.
Observing the change of the American ginseng root explant callus in the culture process, selecting the callus with light yellow or tender white color, high growth speed, loose texture and good dispersibility according to the induction condition of the explant callus, transferring the callus into a fresh subculture medium, wherein the formula of the subculture medium is the same as that of the callus medium except that the subculture medium does not contain phytohormone, and the culture conditions are also the same.
3. Suspension culture of American ginseng to obtain high-yield cells
After the American ginseng callus is screened and subcultured for many times, the clone of the American ginseng callus with high growth speed, loose texture, light white and transparent is selected and transferred to a liquid culture medium for suspension culture.
Specifically, the implementation process of one embodiment 1 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension medium is an MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A3, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, and 0.2mg/L gibberellin A19. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
The implementation process of one embodiment 2 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension medium is an MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
The implementation process of one embodiment 3 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension medium is an MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, 0.2mg/L gibberellin A23, 0.2mg/L gibberellin A24, and 0.2mg/L gibberellin A34.
The implementation process of one embodiment 4 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in dark culture; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain the cells. Wherein the first suspension medium is an MS medium containing 2.5wt% of sucrose, 0.5wt% of D-galactose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 2.5wt% sucrose, 0.5wt% D-galactose, 700mg/L LH, 0.1mg/L KT, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
The implementation process of one embodiment 5 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain the cells. Wherein the first suspension medium is an MS medium containing 2.5wt% sucrose, 0.5wt% D-galactose, 700mg/L LH, 0.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was an MS medium containing 2.5wt% sucrose, 0.5wt% D-galactose, 700mg/L LH, 0.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
The implementation process of one embodiment 6 is as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 15 days in dark culture; transferring the culture medium into a 100mL triangular flask from 3mL to the second suspension culture medium, filling 30mL culture medium into the triangular flask, wherein the transfer amount is 10%, and continuously culturing for 15 days to obtain the cells. Wherein the first suspension medium is an MS medium containing 2.5wt% sucrose, 0.5wt% D-galactose, 700mg/LLH, 0.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 2.5wt% sucrose, 0.5wt% D-galactose, 700mg/L LH, 0.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
One example of a comparative example 1 was carried out: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in dark culture; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain the cells. Wherein the first suspension culture medium is MS culture medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.5mg/L gibberellin A3 and 0.5mg/L gibberellin A9. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.5mg/L gibberellin A3 and 0.5mg/L gibberellin A9.
One comparative example 2 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension culture medium is MS culture medium containing 3wt% of sucrose, 700mg/L of LH, 0.1mg/L of KT and 1.0mg/L of gibberellin A3. The second suspension culture solution is MS culture medium containing 3wt% of sucrose, 700mg/L LH, 0.1mg/L KT and 1.0mg/L gibberellin A3.
One comparative example 3 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension culture medium is MS culture medium containing 3wt% of sucrose, 700mg/L of LH, 0.1mg/L of KT and 1.0mg/L of gibberellin A12. The second suspension culture solution is MS culture medium containing 3wt% of sucrose, 700mg/L of LH, 0.1mg/L of KT and 1.0mg/L of gibberellin A12.
One comparative example 4 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension medium is an MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.01mg/L gibberellin A12, 0.01mg/L gibberellin A13, 0.01mg/L gibberellin A15, 0.01mg/L gibberellin A19, and 0.01mg/L gibberellin A23. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.01mg/L gibberellin A12, 0.01mg/L gibberellin A13, 0.01mg/L gibberellin A15, 0.01mg/L gibberellin A19, and 0.01mg/L gibberellin A23.
One comparative example 5 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain cells. Wherein the first suspension medium is an MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.5mg/L gibberellin A12, 0.5mg/L gibberellin A13, 0.5mg/L gibberellin A15, 0.5mg/L gibberellin A19, and 0.5mg/L gibberellin A23. The second suspension culture was MS medium containing 3wt% sucrose, 700mg/L LH, 0.1mg/L KT, 0.5mg/L gibberellin A12, 0.5mg/L gibberellin A13, 0.5mg/L gibberellin A15, 0.5mg/L gibberellin A19, and 0.5mg/L gibberellin A23.
One comparative example 6 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in dark culture; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain the cells. Wherein the first suspension medium is an MS medium containing 1.0wt% sucrose, 3.5wt% D-galactose, 700mg/L LH, 0.02mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 1.0wt% sucrose, 3.5wt% D-galactose, 700mg/L LH, 0.02mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
One comparative example 7 was carried out as follows: inoculating the clone of the American ginseng callus into a first suspension culture medium, adopting a constant temperature shaking table, rotating at 90-110rpm, filling 30mL of culture solution into a 100mL triangular flask, culturing at 25 +/-1 ℃ for 20 days in a dark culture medium; adding 10mL of fresh second suspension culture solution, and continuously culturing for 7 days to obtain the cells. Wherein the first suspension medium is an MS medium containing 5.5wt% sucrose, 0.01wt% D-galactose, 700mg/L LH, 1.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23. The second suspension culture was MS medium containing 5.5wt% sucrose, 0.01wt% D-galactose, 700mg/L LH, 1.5mg/L mannitol, 0.2mg/L gibberellin A12, 0.2mg/L gibberellin A13, 0.2mg/L gibberellin A15, 0.2mg/L gibberellin A19, and 0.2mg/L gibberellin A23.
3. Determination of Biomass
And (3) measuring the biomass of the American ginseng suspension cells, centrifuging the culture solution for 5min at 600r/min to obtain precipitates, namely the collected cells, freeze-drying and weighing.
4. Detection of cell viability of American ginseng
After American ginseng suspension cell fluid is taken and centrifuged for 5min at 600r/min, the supernatant is removed, 5mL of the prepared PBS buffer solution with the pH value of 7.0 and containing 0.2 percent triphenyltetrazolium chloride (TTC, shanghai-sourced leaf Biotech Co., ltd., CAS: 298-96-4) is added, and dark treatment is carried out for 14h at normal temperature. The supernatant solution was removed, the cells were washed with 5mL of distilled water, and after the cell pellet was allowed to stand still, the cells were washed again by aspirating the supernatant, and repeated 3 times in total. Decolorizing with 5mL of 95% ethanol, performing water bath at 60 ℃ for 30min, and shaking the test tube upside down gently every 5min to shake the test tube evenly. After the cells are precipitated, an ultraviolet spectrophotometer is used for measuring the OD value of the supernatant solution, and the OD value represents the cell activity.
5. Detection of content of total saponins in American ginseng cells
The content of total saponins in American ginseng cells is detected by referring to spectrophotometry for measuring the content of total soaps in food (T/AHFIA 004-2018).
Standard curve: accurately weighing 20.00mg of American ginseng saponin standard substance (Nanjing Zealand pharmaceutical science and technology Co., ltd.), dissolving with 70% ethanol, diluting to 20mL, preparing 2mg/mL mother liquor, diluting to 20mL each of gradient standard solutions of 0.002mg/mL, 0.004mg/mL, 0.008mg/mL, 0.01mg/mL and 0.015mg/mL, adding 0.20mL of 5% vanillin glacial acetic acid solution, dissolving the residue, adding 0.80mL of perchloric acid, mixing, heating in 60 deg.C water bath for 20min, cooling, adding 5.00mL of glacial acetic acid, shaking uniformly, and measuring absorbance at 560nm wavelength. And drawing a standard curve by taking the mass of the standard substance as a horizontal coordinate and the absorbance as a vertical coordinate to obtain a regression equation.
And (3) testing the sample: centrifugally collecting American ginseng suspension cells, freeze-drying, grinding into powder, accurately weighing 1g of powder, wrapping with filter paper, putting into a Sabourdon extractor, adding methanol for reflux extraction for 24 hours, degreasing with diethyl ether, extracting with n-butanol, recovering n-butanol under reduced pressure, dissolving residues with deionized water, freeze-drying, accurately weighing 1mg, preparing into 20mL solution, processing with reference to a treatment method of a gradient standard solution, measuring absorbance at a wavelength of 560nm, substituting into a regression equation to calculate the concentration and content of a sample, and calculating according to a formula, wherein the total saponin yield = saponin content multiplied by the dry weight (mg/L) of a cell suspension culture in unit volume.
6. Extraction of total protein of American ginseng root suspension cell
The suspension cells were collected, sonicated with pH7.0.05M Tris-HCl buffer for 30min, and then ammonium sulfate was added to 80% saturation while stirring was continued. Removing ammonium sulfate by dialysis, dissolving the precipitate in water, applying hollow fiber membrane, and lyophilizing the concentrated solution to obtain protein lyophilized powder of radix Panacis Quinquefolii suspended in a bar, and measuring the protein content in AGP by Bradford method; and calculated according to the formula, total protein yield = protein content x dry weight (mg/L) per volume of cell suspension culture.
7. Proteolysis of American ginseng root suspension cells
Taking 50mg of the extracted panax quinquefolium root suspension cell root protein freeze-dried powder, adding 500mL of 0.1M sodium carbonate-sodium bicarbonate buffer solution with pH =9.0, simultaneously adding alkaline protease (food grade, hefeihuang biological products Co., ltd.) to make the concentration of the solution be 5000/mL, treating at 55 ℃ for more than 6h, boiling for 5min to inactivate the enzyme, recovering to room temperature, centrifuging at 3500rpm at 4 ℃, and taking supernatant to obtain an enzymatic hydrolysate.
8. Purification by gel chromatography
Concentrating the prepared enzymolysis liquid under reduced pressure, loading the enzymolysis liquid to a gel chromatography column (10 mM multiplied by 600 mM) of Sephadex G-50 (cargo number S8150, solarbio), standing and adsorbing for 15min, eluting with 10mM sodium phosphate buffer solution (pH7.4) at the flow rate of 1mL/min, collecting peak value eluent at 280nm, collecting 2mL of each tube, and freeze-drying to obtain the polypeptide freeze-dried powder purified in the enzymolysis liquid.
9、SDS-PAGE
And (3) carrying out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on the American ginseng peptide obtained by gel chromatography purification by adopting 12.0% separation gel.
10. RP-HPLC separation and purification
And (3) dialyzing, concentrating and desalting the obtained freeze-dried coarse powder by using a dialysis bag MD34 (Beijing Solebao scientific and technological preference company) with the molecular weight cutoff of 7000, filtering by 0.22 mu m, purifying by HPLC preparative chromatography, collecting sufficient eluent according to peaks, freeze-drying, dissolving in 0.15% formic acid aqueous solution to serve as a test sample of RP-HPLC, loading the test sample on a C18 chromatographic column, collecting chromatographic peak eluent, concentrating, and freeze-drying to obtain the American ginseng peptide freeze-dried powder.
The conditions of the preparative chromatography were: liquid preparative chromatography column Supersil AQ-C18 (5 μm,10 mm. Times.200 mm, danexitt), agilent HPLC1200 series system (Agilent, waldburg, germany), diode Array Detector (DAD). The mobile phase is as follows: phase a 0.1% trifluoroacetic acid and phase B0.1% trifluoroacetic acid in acetonitrile; the gradient program was: 0 → 65min, linear gradient 1 → 34% phase B; 65 → 75min, linear gradient 34 → 45% B;75 → 80min,47 → 90% by weight B;80 → 90min, linear gradient 90% B. The flow rate was 0.6ml/min, the column temperature was 25 ℃, the injection amount was 10. Mu.L, and the DAD wavelength was set to 214nm.
11. American ginseng peptide sequence identification
Preparing a test solution: preparing a mixture containing acetonitrile, ultrapure water and trifluoroacetic acid in a volume ratio of 200:100:3 (V/V), adding sinapic acid to make the final concentration be 100mg/mL, and obtaining solution B; weighing an appropriate amount of 1 μ L of American ginseng peptide lyophilized powder, dissolving in 1 μ L of solution B to obtain a sample solution, and filtering with 0.45 μm filter membrane to obtain a product for MALDI-TOF-MS detection.
Chromatographic conditions are as follows: HPLC detection mode: ultraviolet light; the scanning range is 50-2000 m/z; capillary exit voltage: 166.0V, skimmer coupled system Voltage: 40.0V, oct 1DC 12.00V, oct 2DC 2.70V, ampl separation width: 4.0m/z, fragmenter voltage: 1.00Vt; ion polarity: a positive ion; type of ion source: ESI (electrospray ionization); drying temperature: 325 ℃, atomizer pressure: 15.00psi, dryer flow rate: 5.00L/min. Mass to charge ratios of the polypeptides and fragments of the polypeptides 10 fragment patterns were taken after each full scan. The original file was analyzed using the de navy algorithm in Mascot 2.3 software. The relevant parameters are Enzyme = none, variable modified-canon: oxidation (M), peptides tolerance:20ppm, MS/MS tolerance:0.1u, mascot results in a filter parameter FDR ≦ 0.01.
2. As a result, the
TABLE 1
| Detailed description of the preferred embodiments | Biomass (g/L) | Cell viability | Total saponin yield (mg/L) | Total protein yield (g/L) |
| Example 1 | 10.09±0.24b | 0.53±0.064a | 367.6±31.5b | 1.61±0.22a |
| Example 2 | 10.15±0.17b | 0.55±0.057a | 375.8±29.7ab | 1.62±0.17a |
| Example 3 | 10.23±0.15b | 0.57±0.054a | 378.6±32.6ab | 1.64±0.16a |
| Example 4 | 10.24±0.21b | 0.61±0.043a | 382.1±26.4ab | 1.66±0.22a |
| Example 5 | 10.25±0.18b | 0.64±0.035a | 386.4±19.7a | 1.68±0.15a |
| Example 6 | 10.28±0.20b | 0.67±0.023a | 390.3±25.4a | 1.67±0.16a |
| Comparative example 1 | 8.27±0.24c | 0.31±0.016b | 323.7±19.4c | 1.28±0.14b |
| Comparative example 2 | 8.15±0.17c | 0.32±0.018b | 325.4±22.3c | 1.25±0.17b |
| Comparative example 3 | 8.32±0.18c | 0.34±0.022b | 326.9±18.6c | 1.24±0.16b |
| Comparative example 4 | 7.86±0.11d | 0.29±0.021b | 321.8±26.4c | 1.19±0.19b |
| Comparative example 5 | 11.38±0.28a | 0.62±0.014a | 385.4±24.9a | 1.65±0.25a |
| Comparative example 6 | 10.04±0.21b | 0.54±0.012a | 372.5±30.7ab | 1.64±0.12a |
| Comparative example 7 | 10.07±0.18b | 0.55±0.013a | 374.8±33.2ab | 1.66±0.14a |
In the suspension culture process of the clone of the American ginseng root callus through the above examples and comparative examples, the cells are initially delayed on the 0 th to 4 th days of culture, the logarithmic growth phase is carried out after the 4 th day, and the mass propagation phase is carried out on the cells for 10 to 20 days, so that the biomass is maximized, and the total saponin yield and the total protein yield are maximized. As can be seen from Table 1, the biomass, cell viability, total saponin production and total protein yield of the suspension cells obtained by the clone of American ginseng root callus in examples 1 to 6 are significantly higher than those of comparative examples 1 to 4, thereby illustrating that the suspension culture process of the clone of American ginseng root callus provided by the example of the present application is more favorable for accumulating the biomass, saponin and protein and can maintain higher cell viability.
Furthermore, the application also carries out enzymolysis treatment on protein in suspension cells obtained by the clone of the American ginseng root callus in each example and comparative example, gel purification is carried out on the enzymolysis liquid, and a plurality of purified peaks are obtained by purifying in elution collecting pipes and 50 th pipes of the examples 1-6; while the elution collecting tubes of comparative examples 1 to 7 were more than those of collection tubes 1 to 50. And the collected components of examples 1 to 6 and comparative examples 1 to 7 were named as shown in table 2; since comparative examples 3 and 4 have a large number of OD280nm elution peaks concentrated in the 1 st to 20 th tubes, the elution and separation effects are poor, and they are not individually named.
TABLE 2
| Description of the preferred embodiment | Eluting the fractions |
| Example 1 | F1、F2、F3、F4、F5 |
| Example 2 | F6、F7、F8、 |
| Example 3 | F9、F10、F11 |
| Example 4 | F12、F13、F14 |
| Example 5 | F15、F16、F17、F18 |
| Example 6 | F19、F20、F21、F22、F23 |
| Comparative example 1 | F24、F25 |
| Comparative example2 | F26、 |
| Comparative example 3 | － |
| Comparative example 4 | － |
| Comparative example 5 | F27、F28 |
| Comparative example 6 | F29 |
| Comparative example 7 | F30 |
SDS-PAGE patterns of the eluted fractions referred to in Table 2 are shown in FIGS. 3 and 4. As a result, it was found that the bands of F3, F4, F8, F9, F10, F11, F13, F14, F15, F16, F17, F18, F21, F22 and F23 were preferable, and further separation and purification were continued by RP-HPLC as shown in FIGS. 5 and 6, and the elution peaks were collected and sequence-identified, and the results are shown in Table 3.
TABLE 3
In vitro antioxidant property
1. Test article
The American ginseng polypeptides (respectively named as American ginseng peptides 1-8) shown in SEQ ID NO. 1-8 are prepared into lyophilized powder and prepared into 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL and 2.5mg/mL.
2. Scavenging DPPH free radicals
Taking 1mL of test solution with each concentration, adding 4.5mL of 0.1mmol/L DPPH working solution into a test tube, fully shaking up, reacting for 30min in a dark place, measuring the absorbance under the wavelength of 515nm, and replacing the test solution with distilled water in a blank group. The DPPH free radical clearance rate of the test article is calculated according to the following formula: clearance (%) = (A0-a)/A0 × 100%; wherein, A0 and A1 represent the absorbance of blank and sample respectively.
3. Scavenging effect on hydroxyl free radical
Adding 1ml of sample solution (blank is replaced by distilled water) with each concentration into a test tube, adding 9mmo1/L salicylic acid-ethanol solution and 9mmo1/L FeSO 4 1ml of each solution was added, and 8.8mmo1/L H was finally added 2 O 2 The solution was reacted at 37 ℃ for 30min at 1m1, and the photometric value was measured at 510 nm. The clearance rate (%) of the test article to hydroxyl radical (%) = (A0-A)/A0 x 100% was calculated as follows; a0 and A respectively represent blank and sample reaction solution absorbance.
4. Scavenging effect on superoxide anion
Taking 1ml of a sample with each concentration, adding 5ml of Tris-HCl buffer solution with 0.05mol/L and pH =8.2, placing the sample in a water bath with the temperature of 25 ℃ for preheating for 20min, adding 0.4m1 of 4.5mmol/L pyrogallol, uniformly mixing, and measuring the absorbance values at the initial time and after 1min under the wavelength of 320 nm. Replacing the test sample with distilled water with the same volume in the blank, and calculating the clearance rate (%) of the test sample to superoxide anion (delta A0-delta A)/delta A0 x 100% according to the following formula; and delta A0 and delta A respectively represent the absorbance difference of the blank and the test sample for 1 min.
5. Statistical analysis
All test data are expressed as mean and standard deviation.
6. Results
TABLE 4DPPH clearance
TABLE 5 hydroxyl radical scavenging Rate
TABLE 6 superoxide anion removal
As shown in tables 4, 5 and 6, when the concentration of the American ginseng peptide 1-8 reaches 1.0mg/ml, the clearance rate of DPPH, hydroxyl free radicals and superoxide anions reaches more than 80 percent, and the American ginseng peptide has better in-vitro oxidation resistance.
In vivo assay
1. Experimental methods and materials
1. Laboratory animal
Wistar rat, jagsu esmoline; male and female, body constitution 200 + -20 kg, and normal diet.
2. Molding machine
Injecting 45mg/kg pentobarbital sodium solution into SD rat body via abdominal cavity, sterilizing the left skin of rat abdomen with 75% alcohol, cutting, 1.5-2.0 cm below left hypochondrium and 1.0cm away from spine, separating left renal artery with glass rod, ligating the left renal artery with suture, and suturing muscle and skin layer by layer after operation. Intraperitoneal injection of penicillin sodium 2X 10 after suturing 5 U prevents post-operative wound infection. A blank group of 10 SD rats was subjected to a sham operation without ligation of the left renal artery. After 4 weeks, the systolic blood pressure of the tail artery of the rat is measured, and the rat with the systolic blood pressure higher than 160mmHg can be regarded as successful renal hypertension model building.
3. Grouping experiment
The successfully modeled renal hypertension model rats are respectively taken as a model group, an administration group and a positive group, and 10 normal Wistar rat crop normal groups are taken. The administration group administers the American ginseng peptides 1 to 8 provided in the above examples at a dose of 40mg/kg body weight. Captopril (Teyi pharmaceutical group, inc., national drug Standard H44020939) was administered at the same dose to the positive group. The administration is continued for 12 weeks, 1 time per day, and equal volume of physiological saline is given to the blank group and the model group by intragastric administration every day.
4. Observation index
After 2h of the last administration, the blood pressure of each group of rats was measured, and the rats were sacrificed by cervical dislocation. Blood was taken, serum was separated, and the contents of angiotensin I (AngI), angiotensin II (AngII) and Aldosterone (ALD) (General Aldosterone (ALD), cat # ZY-ALD-Ge) in rat plasma were measured using ELISA kit (Husha organism). 0.1g of thoracic aortic tissue was excised, homogenized and Ang II content was determined.
5. Statistical analysis
All test data are expressed as mean and standard deviation, processed using SPSS13.0 software, and subject to multiple comparisons and marked for significant differences.
2. Results
TABLE 7
Table 7 significant difference comparison was performed on each data, and as can be seen from table 7, compared with the normal group, the blood pressure of the rats in the model group, the positive group and the administration group at the 4 th week after operation was significantly higher than that in the normal group, and the average systolic blood pressure was over 160mmHg, indicating that the molding was successful. The rats in the administration group and the positive group start to perform intervention treatment at the 4 th week after operation, and after the 6 th week after administration, the average systolic blood pressure of the rats is remarkably reduced relative to the 4 th week after operation, which indicates that the effect of remarkably reducing the blood pressure of the rats can be achieved when the rats in the renal hypertension model are subjected to the intervention treatment by captopril or American ginseng peptides 1-8 provided by the embodiment of the application, and indicates that the American ginseng peptides 1-8 provided by the embodiment of the application have the blood pressure reducing effect equivalent to that of the captopril.
TABLE 8
In table 8, multiple comparisons and significant difference labeling were performed for each column of data. As can be seen from Table 8, the amounts of AngI, angII and ALD in the serum of the rats in the model group are significantly higher than those in the normal group, indicating that the molding is successful. After the captopril dry prognosis is carried out on renal hypertension model rats, the content of AngII and the content of ALD are both obviously reduced compared with that of a model group, the AngI is not reduced, and the content of AngI after intervention treatment is even higher than that of the model group. In the administration group, after 1-8 American ginseng peptides intervene treatment on renal hypertension model rats, the AngII and ALD contents are both obviously reduced relative to the model group; in addition to peptide 1, the decrease in AngI content relative to the model group was also observed. Therefore, the American ginseng peptides 1-8 provided by the embodiment of the application can obviously reduce the content of AngI, angII and ALD in the serum of a rat with renal hypertension model.
In the modeling, the left renal artery of a rat is ligated to cause the blood flow of the renal artery to be reduced, and the renin, the angiotensin and the aldosterone system (RAAS) are sequentially activated to cause the RAAS to lose balance, then angiotensinogen generated by the liver is hydrolyzed into Ang I by the renin released into venous blood, and then is catalyzed by Angiotensin Converting Enzyme (ACE) in pulmonary circulation to be converted into Ang II; increase in Ang II content, contraction of arteriole smooth muscle, and increase in peripheral resistance. Meanwhile, the adrenal cortex bulbar band is stimulated, so that ALD secretion is increased, and therefore, the reabsorption of sodium in a collecting pipe at the far end of a renal tubule is enhanced, water and sodium in a body are retained, and blood pressure is increased. The content analysis of AngI, angII and ALD in rat serum shows that the American ginseng peptides 1-8 provided by the embodiment of the application can obviously reduce the content increase of AngI, angII and ALD caused by RAAS imbalance, and presumably can be realized by regulating the content of AngI and AngII through inhibiting ACE; the generation of ALD is mainly regulated by AngII, adrenocorticotropic hormone and sodium ions and potassium ions in body fluid, so the American ginseng peptides 1-8 provided by the embodiment of the application have the potential application prospects of improving RAAS imbalance and reducing blood pressure, and in the fields of improving secondary hypertension caused by renal hypertension, and improving the diastolic period with waist soreness and back pain.
In summary, the American ginseng is used for preparing the calluses of the American ginseng seedlings, the clone of the calluses is obtained by carrying out induction culture on the calluses, the high-yield cell strains are obtained by carrying out large-scale suspension culture on the calluses, and the steps of enzymolysis, purification and the like are carried out on the cell cultures of the calluses, so that 1-8 American ginseng peptides are obtained. In vitro experiments show that the American ginseng peptide 1-8 has obvious antioxidant performance. Finally, in vivo experiments prove that the American ginseng peptides 1-8 not only have the blood pressure reducing effect equivalent to that of captopril, but also specifically realize the blood pressure reducing effect by improving RAAS imbalance, and have potential application prospects in the fields of improving secondary hypertension caused by renal hypertension, developing diastole with waist soreness and back pain and the like.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Sequence listing
<110> Qing Feng Lianxi soda drink (Jilin) Co., ltd
<120> American ginseng polypeptide, composition thereof and application thereof in lowering blood pressure and improving immunity
<141> 2022-05-17
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> PRT
<213> Artificial Sequence
<400> 1
Leu Lys Tyr Met Ser Asp Leu Arg Asp Pro Ala Ile Phe Arg Phe Cys
1 5 10 15
Ala Ile Pro Gln Ile Met Ala Ile Gly Thr Leu Ala Leu Cys Tyr Asn
20 25 30
Asn Ile Gln Val Phe Arg Gly Val Val Lys Met Arg Arg Gly Leu Thr
35 40 45
Ala Lys
50
<210> 2
<211> 38
<212> PRT
<213> Artificial Sequence
<400> 2
Lys Glu Ala Ile Glu Asp Ile Thr Met Arg Met Gly Ala Gly Met Ala
1 5 10 15
Lys Phe Ile Cys Lys Glu Val Glu Thr Ile Asp Asp Tyr Asp Glu Tyr
20 25 30
Cys His Tyr Val Ala Gly
35
<210> 3
<211> 46
<212> PRT
<213> Artificial Sequence
<400> 3
Lys Met Arg Arg Gly Leu Thr Ala Lys Val Ile Asp Arg Thr Asn Thr
1 5 10 15
Met Ser Asp Val Tyr Gly Ala Phe Phe Asp Phe Ser Cys Met Leu Lys
20 25 30
Ser Lys Val Asp Asn Asn Asp Pro Asn Ala Thr Lys Thr Leu
35 40 45
<210> 4
<211> 42
<212> PRT
<213> Artificial Sequence
<400> 4
Lys Glu Ala Ile Glu Asp Ile Thr Met Arg Met Gly Ala Gly Met Ala
1 5 10 15
Lys Phe Ile Cys Lys Glu Val Glu Thr Ile Asp Asp Tyr Asp Glu Tyr
20 25 30
Cys His Tyr Val Ala Gly Leu Val Gly Leu
35 40
<210> 5
<211> 29
<212> PRT
<213> Artificial Sequence
<400> 5
Lys Val Pro Ile Val Met Ala Phe His Cys His Ile Tyr Asp Asn Asp
1 5 10 15
Trp His Phe Ser Cys Gly Thr Lys Glu Tyr Lys Val Leu
20 25
<210> 6
<211> 29
<212> PRT
<213> Artificial Sequence
<400> 6
Lys His Glu Tyr Ser Ser Tyr Gly Asn Gly Ser Pro Ser Met Phe His
1 5 10 15
Asn Pro Met His Gly Val His Gln Ser Val Glu Trp Lys
20 25
<210> 7
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 7
Leu Ser Cys Val Gly Thr Ala Val Leu Ala Val Cys Leu Arg Ile Gln
1 5 10 15
Val Asn Lys Glu Asn Lys
20
<210> 8
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 8
Lys Gly Ser Ala Glu Phe Gln Val Val Ser Phe Thr Asn Arg Ile Arg
1 5 10 15
Arg Leu
Claims (7)
1. An American ginseng polypeptide and a composition thereof, which comprises at least one of the polypeptides shown in SEQ ID No. 1-8.
2. A pharmaceutical composition comprising the american ginseng polypeptide of claim 1, or a composition thereof, and a pharmaceutically acceptable carrier or diluent.
3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable salt of the American ginseng polypeptide of claim 1, a buffer, a tonicity modifier, a pharmaceutically acceptable vehicle, and/or a stabilizer.
4. The pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable carrier or diluent is used to improve the tolerability of the composition and allows for better solubility and better bioavailability of the active ingredient.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable carrier or diluent comprises an emulsifier, a thickener, a redox component, a starch, an alcohol solution, a polyethylene glycol, or a lipid.
6. The method for preparing the American ginseng polypeptide and the composition thereof of claim 1, which comprises the following steps:
obtaining callus of American ginseng;
obtaining the clone of the American ginseng callus;
performing suspension culture in a liquid culture solution to obtain American ginseng cells with high protein yield;
collecting the American ginseng cells, and carrying out enzymolysis, gel chromatography purification and liquid chromatography purification on the American ginseng cells by adopting food-grade alkaline protease to obtain the American ginseng polypeptide and the composition thereof.
7. The use of the American ginseng polypeptide and composition thereof of claim 1, or the American ginseng polypeptide and composition thereof prepared by the preparation method of claim 6 in the preparation of medicaments related to blood pressure reduction.
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| KR20160001801A (en) * | 2014-06-26 | 2016-01-07 | 경희대학교 산학협력단 | PNA probe set for identifying ginseng cultivars and method for identifying ginseng cultivars using the same |
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| KR20160001801A (en) * | 2014-06-26 | 2016-01-07 | 경희대학교 산학협력단 | PNA probe set for identifying ginseng cultivars and method for identifying ginseng cultivars using the same |
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