CN113559175A

CN113559175A - Extract for enhancing immunity and delaying senescence and preparation method thereof

Info

Publication number: CN113559175A
Application number: CN202110801428.6A
Authority: CN
Inventors: 黎攀; 陈宏著; 杜冰; 徐雅囡; 黄达荣; 邓新宇
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2021-10-29

Abstract

The invention discloses an extract for enhancing immunity and delaying senescence, which belongs to the technical field of functional foods and comprises the following raw material components: antler, tortoise shell, purified water, cinnamon, protease, American ginseng, medlar and resistant dextrin. The American ginseng and the cinnamon are selected, the mouth feel is sweet and pleasant, yin is nourished, kidney yang is nourished warmly, the tortoise shell glue and the antler glue have the effect of prolonging the life in the traditional Chinese pharmacology, the tortoise shell has the function of improving the immunity, the service life of the caenorhabditis elegans can be effectively prolonged, the ROS and MDA levels in the body are reduced, the activity of the antioxidase is improved, the good anti-aging effect is achieved, and the development prospect is wide.

Description

Extract for enhancing immunity and delaying senescence and preparation method thereof

Technical Field

The invention belongs to the technical field of functional foods, and relates to an extract for enhancing immunity and delaying senescence and a preparation method thereof.

Background

The tortoise shell has the effects of nourishing yin, suppressing yang, tonifying kidney, strengthening bone, nourishing blood, tonifying heart, consolidating channels, relieving metrorrhagia, reducing thyroid and adrenal cortex function of hyperthyroidism type rat, improving cellular immunity and blood immunity, and delaying aging; the antler has the effects of warming and recuperating kidney yang, generating essence, benefiting blood, strengthening tendons and bones and has an anti-inflammatory effect; the American ginseng has the effects of tonifying qi and yin, clearing away fire and promoting the production of body fluid, and can calm and calm the nerves, regulate blood pressure, improve immunity and resist aging; fructus Lycii has effects of nourishing liver and kidney, calming liver wind, replenishing vital essence, improving eyesight, nourishing yin, invigorating qi, invigorating tendons and bones, and relieving asthenia, and can tonify kidney, strengthen body constitution, regulate immunity, nourish liver, and improve eyesight; cinnamon has the effects of tonifying fire and supporting yang, guiding fire to the origin, dispelling cold and relieving pain, activating blood and dredging channels, warming and nourishing spleen and stomach, warming channels and dredging collaterals, controlling blood sugar, eliminating phlegm and relieving cough, and helping digestion and guiding qi. The resistant dextrin is a functional dietary fiber, has the functions of improving the composition of human intestinal flora, absorbing harmful substances in intestinal tracts, helping to prevent chronic diseases such as hypertension, diabetes and the like, and can improve the flavor of extract.

At present, most of the anti-aging drugs on the market are mainly simple stacking of multiple antioxidants, and lack scientificity and cooperativity. Most of health-care foods with the functions of resisting oxidation and delaying senility realize health-care effects by western medicine analysis, reduction and countermeasures, such as free radical removal and oxidation resistance, and the effect of fundamentally delaying senility is difficult to realize. Therefore, the health food which is reasonable and harmonious, has both principal and secondary aspects and can be taken for a long time, enhance the immunity and delay the aging needs to be designed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an extract which is beneficial to enhancing immunity, delaying senility and being easy to be absorbed by human bodies and a preparation method thereof. The invention applies a model organism caenorhabditis elegans to evaluate the specific effect of the caenorhabditis elegans in delaying senility. The invention can effectively prolong the service life of caenorhabditis elegans, reduce the levels of ROS and MDA in vivo, improve the activity of antioxidase, have good anti-aging effect and have great development prospect.

An extract for enhancing immunity and delaying senility comprises the following raw material components: antler, tortoise shell, purified water, cinnamon, protease, American ginseng, medlar and resistant dextrin.

A preparation method of an extract for enhancing immunity and delaying senescence comprises the following steps:

s1, cleaning and soaking: cleaning cornu Cervi and carapax et Plastrum Testudinis with ultrasonic wave, soaking cornu Cervi for 20-40 days, cleaning with ultrasonic wave every day, and changing water once every day;

s2, primary boiling: decocting the soaked cornu Cervi with purified water at a ratio of 1:10-30, boiling for 10-30min, draining water, adding water to submerge the cornu Cervi, adding cortex Cinnamomi, decocting at constant temperature, concentrating the extractive solution, and collecting;

s3, decocting again: continuously adding purified water into cornu Cervi, decocting, adding cortex Cinnamomi, decocting at constant temperature, concentrating the extractive solution, collecting, and changing water;

s4, boiling for the third time: adding carapax et Plastrum Testudinis and cornu Cervi, decocting with purified water, adding cortex Cinnamomi, decocting at constant temperature, concentrating the extractive solution, collecting, and changing water;

s5, enzymolysis: adjusting pH to 8.5, adding protease, reacting at 50-60 deg.C for 3-5 hr, boiling, and sterilizing to obtain enzymatic hydrolysate;

s6, fishy smell removal: adding cortex Cinnamomi, radix Panacis Quinquefolii and fructus Lycii into the enzymolysis solution, decocting for 1-6 hr, maintaining the temperature at 90-100 deg.C, and decocting at constant temperature;

s7, blending: adding resistant dextrin into the concentrated solution, and filling to obtain the extract.

Preferably, in S1, the antler is Cervus Nippon Temminck, Cervus elaphus Linnaeus or antler base, and the carapax et Plastrum Testudinis is dorsal and abdominal shell of Testudinis.

Preferably, the cinnamomum japonicum is added into the S2, the temperature is kept between 90 and 100 ℃ and the boiling time is 36 to 60 hours.

Preferably, the cinnamomum japonicum is added into the S3, the temperature is kept between 90 and 100 ℃ and the boiling time is 36 to 60 hours.

Preferably, the cinnamomum japonicum is added into the S4, the temperature is kept between 90 and 100 ℃ and the boiling time is 36 to 60 hours.

Preferably, the protease contains alkaline protease 0.50-1.00% and neutral protease 0.20-0.30%.

Preferably, the concentrated solution is added with resistant dextrin according to the solid content of 1: 1.

The invention has the beneficial effects that:

compared with the traditional Guilu Erxian gum, the extract has sweet and pleasant taste, no fishy smell and coffee aroma, wherein the American ginseng is sweet and slightly bitter in taste compared with the ginseng, is slightly warm, mainly tonifies yang, is sweet, slightly bitter and slightly cool, mainly nourishes yin, has the effect of nourishing kidney, has synergistic effect with other medicinal materials, is matched with each other, nourishes yin, warms and tonifies kidney yang, and the cinnamon is pungent and sweet in taste, can be used for treating impotence, cold pain in waist and knees, asthma due to kidney deficiency, floating of deficient yang and the like, dizziness, conjunctival congestion, cold pain in heart and abdomen and the like;

before the boiling process, the antler and the tortoise plastron are subjected to ultrasonic cleaning and soaking, so that the effective components can be efficiently protected, the soaking process can be shortened, and the antler is subjected to ultrasonic cleaning in the soaking process, so that the soaking time can be shortened; cinnamon is added in batches as a seasoning in the boiling process, so that the fragrance can be improved, and the fishy smell can be removed; after decocting, carrying out enzymolysis to improve the peptide content, wherein the dry weight of the peptide content accounts for 35.21%, the amino acid composition of the animal peptide is more abundant and easier to absorb, and the tortoise shell glue and the antler glue have the effect of prolonging the life in the traditional Chinese medicine, and the tortoise shell has the function of improving the immunity;

after being boiled, the resistant dextrin is added to improve digestion and absorption, and has the effects of reducing blood sugar, regulating blood fat, adjusting intestinal environment, controlling weight and preventing obesity; the specific effect of delaying senescence of the caenorhabditis elegans is evaluated by applying a model organism caenorhabditis elegans, and the evaluation result of a caenorhabditis elegans model shows that the caenorhabditis elegans anti-aging biological agent has the effects of prolonging the health life of the caenorhabditis elegans, enhancing the motor ability, reducing the levels of ROS and MDA in vivo and improving the activity of antioxidase (SOD, CAT and GSH-Px); modern medical research finds that the compatibility of the selected traditional Chinese medicines can increase the activity of T lymphocytes and reduce the apoptosis of the T lymphocytes, thereby playing a role in enhancing immunity;

compared with the traditional method, the invention adopts a double enzymolysis process to ensure that the enzymolysis rate of the protein reaches more than 20 percent, the peptide content is 19.2g/100g and is more than 8.9g/100g of the traditional method, the protein content is 70.5g/100g, the protein content is 38.4g/100g and is much more than 12.1g/100g of the traditional method, and the protein can be better absorbed. Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a flow chart of the production process of the present invention;

FIG. 2 shows the effect of the extracts of examples and comparative examples of the present invention on the longevity of C.elegans (n-5);

FIG. 3 shows the effect of the extracts of examples and comparative examples of the present invention on the motility of C.elegans (n-5);

FIG. 4 is a graph showing the effect of extracts of examples and comparative examples of the present invention on the amount of ROS accumulated in C.elegans (n-5);

FIG. 5 shows the effect of the extracts of the examples and the comparative examples of the present invention on the activity of SOD enzyme in C.elegans (n-5);

FIG. 6 shows the effect of the extracts of examples and comparative examples of the present invention on CAT enzyme activity in C.elegans (n-5);

FIG. 7 is a graph showing the effect of extracts of examples and comparative examples of the present invention on the amount of MDA accumulated in C.elegans (n-5);

FIG. 8 shows the effect of the extracts of examples and comparative examples of the present invention on the activity of GSH-Px enzyme in C.elegans (n-5).

Detailed Description

In order to make the technical means, objectives and functions of the present invention easy to understand, the present invention will be further described with reference to specific examples. The embodiments described below with reference to the accompanying drawings are illustrative only and are not intended to be limiting. The experimental procedures used in the following examples are conventional ones unless otherwise specified, and materials, reagents and the like used therein are commercially available.

1. An extract for enhancing immunity and delaying senility:

comprises the following raw material components: antler, tortoise shell, purified water, cinnamon, protease, American ginseng, medlar and resistant dextrin.

A preparation method of an extract for delaying senescence comprises the following steps:

s1, cleaning and soaking: cleaning 500 parts of cornu Cervi (Cervus Nippon Temminck, Cervus Elaphus L. or Cervus elaphus Linnaeus) and 250 parts of carapax et Plastrum Testudinis (carapax et Plastrum Testudinis and abdomen carapax et Plastrum Testudinis of Testudinidae) with ultrasonic wave, completely soaking cornu Cervi for 30 days, ultrasonic cleaning every day and changing water once every day;

s2, primary boiling: adding purified water into the soaked antler according to the proportion of 1:20, boiling, draining after boiling for 10-30min for the first time, adding water to submerge the antler, keeping the temperature at 90-100 ℃, adding 10 parts of cinnamon, boiling at constant temperature for 48 hours, and collecting the extracting solution after concentration;

s3, decocting again: continuously adding purified water into the deer horn to boil, keeping the temperature at 100 ℃, adding 10 parts of cinnamon, boiling at constant temperature for 48 hours, concentrating the extracting solution, collecting, and then changing water;

s4, boiling for the third time: adding carapax et Plastrum Testudinis and cornu Cervi, decocting with purified water, maintaining the temperature at 100 deg.C, adding 10 parts of cortex Cinnamomi, decocting at constant temperature for 48 hr, concentrating the extractive solution, collecting, and changing water;

s5, enzymolysis: adjusting pH to 8.5, adding protease, wherein the alkaline protease is 0.75%, the neutral protease is 0.25%, maintaining the temperature at 55 deg.C, reacting for 4 hr, boiling, and sterilizing to obtain enzymolysis solution;

s6, fishy smell removal: decocting 10 parts of cinnamon, 45 parts of American ginseng and 90 parts of medlar in the enzymolysis liquid for 1 hour, keeping the temperature at 100 ℃, and decocting at constant temperature;

s7, blending: adding resistant dextrin (solid content is 1:1) into the concentrated solution, and filling to obtain an extract.

Examples

10mL of extract is obtained by adding resistant dextrin into the enzymolysis liquid.

Comparative example

The traditional formula is that 10mL of Guilu Erxian glue is prepared by decocting antler, tortoise shell, ginseng and medlar. Experimental example 1:

TABLE 1 sensory evaluation tables of examples and comparative examples

In conclusion, the method described in the examples gives the best sensory evaluation of the extract (score 95).

2. Evaluation of senescence-delaying Activity

2.1 test specimens

Uracil-deficient E.coli (op50), examples 1-3 and positive control (64uM astaxanthin standard):

after the extract is dissolved in water, the extract is respectively mixed with op50 to prepare samples of 0.5, 2.5 and 5.0(mg/mL) for intervention; blank group of subjects contained only op 50; the positive control group astaxanthin sample is dissolved by dimethyl sulfoxide (DMSO) and then is mixed with op50 to prepare a 64uM sample for intervention.

2.2 synchronization of caenorhabditis elegans

The synchronization of caenorhabditis elegans was performed by bleaching. Selecting most NGM culture dishes of nematodes in the egg-laying period, flushing the NGM culture dishes into an EP (European patent document) tube by using an M9 buffer solution, adding a lysis solution prepared from sodium hypochlorite and sodium hydroxide to obtain synchronized eggs, transferring the eggs to a blank NGM culture dish for about 12 hours, incubating the eggs into larvae to obtain synchronized L1-stage larvae, then transferring the synchronized L1-stage larvae to an NGM culture plate containing op50, and culturing the eggs in an incubator at 20 ℃ for 48 hours to obtain L4-stage nematodes.

2.3 experimental grouping:

group of	Specific test sample
		Blank control group	op50
Example 1	Op50 containing 0.5mg/mL extract
		Example 2	Op50 containing 2.5mg/mL extract
Example 3	Op50 containing 5.0mg/mL extract
		Positive control group	Op50 containing 64uM astaxanthin

2.4 Life and running force test:

caenorhabditis elegans life test: preparing a culture plate containing op50 and a culture plate containing op50 and different test samples, transferring 20 synchronized L4 nematodes in different groups of NGM culture plates containing 12.5ug/ml of 5-Fu, culturing 80-120 nematodes in an incubator at 20 ℃, and recording the number of live, dead and unexpectedly dead nematodes and nematode states every day until the last nematode dies.

Judging the death standard that the caenorhabditis elegans does not react to strong light or knocking a flat plate, no movement of muscles at the hypopharynx part of a high power mirror, finally knocking the head of the caenorhabditis elegans by using a pest picking needle, and judging the caenorhabditis elegans as death if the caenorhabditis elegans still do not react.

After the life test was started, and simultaneously with the life test, the motor behavior ability of the surviving caenorhabditis elegans was rated every four days, with the behavior being divided into three grades, A, B, C, with the following criteria: the nematode can crawl autonomously is defined as A, the nematode only starts moving after the pick-up needle touches the head is defined as C, and the state between the two is defined as B.

Statistical analysis of lifetime was performed using Kaplan-Meier. As can be seen from the figures (FIGS. 2 and 3), the extract of the example significantly prolongs the healthy life of C.elegans and improves the exercise ability. Example 1 and the positive control astaxanthin standard work best extended caenorhabditis elegans longevity and increased locomotor ability.

2.5 measurement of Reactive Oxygen Species (ROS) accumulation in C.elegans

Synchronization of caenorhabditis elegans: the same as 2.2.

Experiment of ROS accumulation of caenorhabditis elegans: the synchronized nematodes were cultured on NGM plates without 5-FU, and C.elegans was taken on day 4 after continuous administration, transferred to NGM plates to remove E.coli, and after three transfers 80 nematodes were transferred to 96-well plates with 50. mu. L M9 buffer solution. At the same time, 50. mu.L of freshly prepared 100. mu. M H2-DCF-DA solution was added to a 96-well plate (final concentration of 50. mu.M). H2-DCF-DA solution containing no nematodes was used as a control. The fluorescence intensity was measured immediately by placing the 96-well plate in a microplate reader (Bio-Tek) controlled by KC4 software. The reaction temperature is 25 ℃, the emission wavelength is 528nm, and the excitation wavelength is 485 nm. The reaction was carried out for 2 hours at intervals of 10 minutes.

As can be seen from the figure (FIG. 4), the extract of the example significantly reduces the relative amount of ROS in C.elegans.

2.6 in vivo superoxide dismutase (SOD) level assay for caenorhabditis elegans

Synchronization of caenorhabditis elegans: the same as 2.2.

Experiment of SOD activity of caenorhabditis elegans: culturing the synchronized nematodes on NGM culture plate without 5-FU, collecting caenorhabditis elegans continuously administered on day 4, homogenizing with bead mill, centrifuging at 10000r for 5min, collecting supernatant, and determining SOD with reference to SOD kit built from Nanjing.

As can be seen from the figure (fig. 5), compared with the blank control group, the extractum of the example significantly improves the activity of SOD in caenorhabditis elegans, and the activity of SOD of the example 3 is the highest.

2.7 in vivo Catalase (CAT) level assay for caenorhabditis elegans

Synchronization of caenorhabditis elegans: the same as 2.2.

CAT activity assay of caenorhabditis elegans: culturing the synchronized nematodes on NGM culture plate without 5-FU, collecting caenorhabditis elegans continuously administered on day 4, homogenizing with bead mill, centrifuging at 10000r for 5min, collecting supernatant, and determining CAT with reference to CAT kit established by Nanjing.

As can be seen from the figure (FIG. 6), the extracts of the examples all significantly improved CAT activity in C.elegans, wherein the CAT activity of example 3 was the highest.

2.8 caenorhabditis elegans in vivo Malondialdehyde (MDA) accumulation test

Synchronization of caenorhabditis elegans: the same as 2.2.

Experiment of the level of MDA of caenorhabditis elegans: culturing the synchronized nematodes on NGM culture plate without 5-FU, collecting caenorhabditis elegans continuously administered on day 4, homogenizing with bead mill, centrifuging at 10000r for 5min, collecting supernatant, and measuring MDA with reference to Nanjing MDA kit.

As can be seen from the figure (FIG. 7), the extract of the example significantly reduces the relative content of MDA in C.elegans.

2.9 assay of glutathione peroxidase (GSH-Px) levels in C.elegans

Synchronization of caenorhabditis elegans: the same as 2.2.

GSH-Px activity assay for C.elegans: culturing the synchronized nematodes on NGM culture plate without 5-FU, collecting caenorhabditis elegans continuously administered on day 4, homogenizing with bead mill, centrifuging at 10000r for 5min, collecting supernatant, and determining GSH-Px by constructing GSH-Px kit with reference to Nanjing.

As can be seen from the figure (figure 8), the extracts of the examples all significantly improve the activity of GSH-Px in caenorhabditis elegans, wherein the activity of GSH-Px of the example 3 is the highest.

TABLE 2 analysis of peptide content data of extract

TABLE 3 analysis of the extract and the content data of polypeptide and protein of traditional Guilu Erxian Gum

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. An extract for enhancing immunity and delaying senility is characterized by comprising the following raw material components: antler, tortoise shell, purified water, cinnamon, protease, American ginseng, medlar and resistant dextrin.

2. A preparation method of an extract for enhancing immunity and delaying senescence is characterized by comprising the following steps:

3. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: s1 the cornu Cervi is Cervus Nippon Temminck, Cervus elaphus Linnaeus or cornu Cervi Degelatinatum, and the carapax et Plastrum Testudinis is the dorsal and ventral concha of Testudinis.

4. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: s2, adding the cinnamon into the raw materials, decocting at constant temperature of 90-100 ℃ for 36-60 hours.

5. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: s3, adding the cinnamon into the raw materials, decocting at constant temperature of 90-100 ℃ for 36-60 hours.

6. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: s4, adding the cinnamon into the raw materials, decocting at constant temperature of 90-100 ℃ for 36-60 hours.

7. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: adding alkaline protease 0.50-1.00% and neutral protease 0.20-0.30%.

8. The preparation method of the extract for enhancing immunity and delaying aging according to claim 2, which is characterized in that: adding resistant dextrin into the concentrated solution according to the solid content of 1: 1.