CN117899132A - Quinoa exosome and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a quinoa exosome (QDNPs) and a preparation method and application thereof. The preparation method comprises the following steps: pulverizing quinoa, and obtaining QDNPs extract by adopting a method of combining differential centrifugation and sucrose gradient centrifugation. QDNPs prepared by the invention can improve metabolic syndrome by reducing indexes such as lipid accumulation, insulin resistance and the like in a high-fat diet mouse obesity model; meanwhile, indexes such as glutamic pyruvic transaminase, glutamic oxaloacetic transaminase activity, NAFLD Activity Score (NAS) and the like can be obviously reduced to relieve nonalcoholic fatty liver diseases. The invention provides experimental basis for edible plant derived nano particles as relieving metabolic syndrome and nonalcoholic fatty liver disease, and provides a new thought for further research and development of the edible plant derived nano particles into related functional foods, foods for special diet, health-care products and/or medicines, and has wide application prospect.

Description

Quinoa exosome and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a quinoa exosome (QDNPs) and a preparation method thereof, and application of QDNPs in preparing functional food, special diet food, health-care food and/or medicine for improving non-alcoholic fatty liver and metabolic syndrome.

Background

Metabolic syndrome (Metabolic Syndrome, MS), which refers to a pathological state of metabolic disorder of substances such as carbohydrates, fats, proteins, etc., of an organism, is a complex group of metabolic disorder syndrome, wherein insulin resistance, central obesity, dyslipidemia, abnormal glucose metabolism, and hypertension are main indicators for evaluating MS. With the development of people's high pressure, fast paced life status and unhealthy eating habits, MS has become a worldwide public health problem severely threatening human health, bringing a heavy burden to society and home. Epidemiological investigation shows that: the current global incidence of MS is 20% -45%, which is expected to increase to 53% by 2035. Nonalcoholic fatty liver disease (Nonalcoholic FATTY LIVER DISEASE, NAFLD) is an early warning of metabolic syndrome as a liver manifestation of metabolic syndrome. NAFLD is often a progressive disease, closely related to major complications of cirrhosis, liver cancer, cardiovascular disease, etc., and mortality from liver related diseases. At present, the treatment means for MS and NAFLD are mainly improved by controlling diet, enhancing exercise and cultivating healthy life style, or are treated aiming at the characteristics of each metabolic disease, and no effective medicine exists clinically. Therefore, there is an urgent need to develop a novel drug for preventing and treating MS and NAFLD.

The exosomes are extracellular vesicles with the size of about 30-200nm, are rich in various bioactive substances, can be found in sheep reticulocytes at the earliest, can wrap and carry various signal molecules such as RNA, DNA, protein, lipid and the like, play a role in transmitting the signal molecules among cells, and have wide application prospects in aspects of beauty treatment, anti-aging, drug treatment, delivery vectors, in-vitro diagnosis and scientific research reagents. Such as: the bone marrow mesenchymal stem cell derived exosome drug ExoFlo ^TM can be used to treat novel coronavirus infection and acute respiratory distress syndrome at stage iii clinical stage; the exosomes exoSTING ^TM produced unmodified or engineered can be transported to the tumor tissue treatment solid tumor by mounting targeting ligands or therapeutic molecules onto the exosome proteins. Exosomes are not only present in animals, but also widely present in plants and microorganisms, wherein exosomes of edible plant origin can exert not only biological activity in plant bodies but also cross-border regulatory activity. Isolated nanovesicles, e.g., grapefruit, can alleviate UC mouse colitis; the lipid nanovesicles purified from ginger can effectively alleviate alcoholic liver disease in mice; the nano particles derived from the honeysuckle decoction are found to contain miRNA2911 which exists stably, and can target and act on various universal influenza A viruses and the like. Research shows that compared with the existing drug delivery system, the exosome-like nano particles of edible plant sources have better biocompatibility, low toxicity and other effects, and are excellent candidates for drug delivery carriers. Therefore, the active exosomes with medicinal efficacy in the mined plants can be used as a new strategy for drug development.

Quinoa (Chenopodium quinoa willd.) is an annual herb of the genus quinoa of the family amaranthaceae, which has high nutritional value and is honored by ancient printing as a "master of food". In particular, quinoa contains rich functional components such as protein, flavonoid, unsaturated fatty acid, saponin and the like, and has the medical effects of resisting oxidation, reducing blood fat, reducing cholesterol, enhancing immunity and the like. However, the biological efficacy of quinoa exosome cross-border regulation has not been reported at present. The exosome nano-particles QDNPs for relieving the metabolic syndrome and the nonalcoholic fatty liver disease are successfully prepared from quinoa for the first time, so that a new thought is provided for further research and development of related functional foods, special meal foods, health-care products and/or medicines for relieving the diseases, and the exosome nano-particles have wide clinical application prospects.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide a quinoa exosome and a preparation method thereof, and application of QDNPs in preparing functional foods, foods for special diet, health-care foods and/or medicines for improving non-alcoholic fatty liver and metabolic syndrome.

In order to achieve the above purpose, the invention discloses a preparation method of QDNPs, which comprises the following steps:

The first step: pulverizing clean quinoa, adding appropriate amount of PBS solution, and soaking for 2-2.5 hr to obtain coarse extract of quinoa exosome.

And a second step of: and (3) sequentially centrifuging 3000g of the crude extract for 30min, 10000g of the crude extract for 1h, and collecting supernatant.

And a third step of: the supernatant was centrifuged at 150000g for 1.5-2h, the pellet resuspended in an appropriate amount of PBS and transferred to a prepared sucrose gradient (8%/30%/45%/60%) solution and centrifuged at 150000g for 2-3h. Collecting 8% -30% of strips between layers, namely: quinoa exosomes QDNPs are stored at-80 ℃.

The result of the activity of the quinoa exosome QDNPs prepared by the invention for relieving the metabolic syndrome and the nonalcoholic fatty liver disease in vivo shows that the QDNPs prepared by the invention can improve the metabolic syndrome by reducing the indexes such as lipid accumulation, insulin resistance and the like in a mouse obesity model induced by high-fat diet; meanwhile, the indexes such as the activity of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase and NAFLD activity score can be obviously reduced to relieve the nonalcoholic fatty liver disease. The following is indicated: QDNPs has potential to develop into related pharmaceutical products for preventing and treating metabolic diseases.

Further, QDNPs nanoparticles obtained by extraction may be used alone or in the form of pharmaceutical compositions; the pharmaceutical composition comprises QDNPs components as active components and a pharmaceutically acceptable carrier.

Further, the QDNPs nano-particles can be prepared into the following dosage forms for preparing medicines for preventing and treating metabolic diseases: granules, capsules, soft capsules, oral liquid preparations, injections, transdermal drug delivery preparations and the like.

Compared with the prior art, the invention has the beneficial effects that:

The results of the in vivo activity experimental study for relieving the metabolic syndrome and the nonalcoholic fatty liver disease show that: the QDNPs prepared by the invention can improve the metabolic syndrome by reducing the indexes such as lipid accumulation, insulin resistance and the like in a high-fat diet induced mouse obesity model; meanwhile, the indexes such as the activity of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase and NAFLD activity score can be obviously reduced to relieve the nonalcoholic fatty liver disease. QDNPs has the potential to develop related medical products for preventing and treating syndrome and nonalcoholic fatty liver disease.

Drawings

Fig. 1: extraction results of quinoa exosome-like nanoparticles.

Fig. 2: characterization results of quinoa exosome-like nanoparticles, wherein a: QDNPs transmission electron microscope images; b: QDNPs particle size diagram; c: QDNPs Zeta potential.

Fig. 3: effect of quinoa exosome-like nanoparticles on the metabolic syndrome in mice, wherein a: representative epididymal white adipose tissue photographs of each group of mice at week 14; b: white fat index of each group of mice; C-F: TC, TG, LDL and HDL levels in mouse serum; g: fasting blood glucose values of mice; h: insulin in mouse serum; j: mouse insulin resistance index.

Fig. 4: remission of nonalcoholic fatty liver following oral quinoa exosome-like nanoparticle in mice, wherein a: representative photographs of mice and mouse livers; b: a mouse liver weight statistical graph; c, scoring NAFLD activity of the liver tissue of the mouse; ALT and AST levels in mouse liver.

Detailed Description

The following examples are illustrative of the invention but do not limit the scope of the invention. Modifications and substitutions of the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.

Example 1: extraction method of quinoa exosome QDNPs

The invention, when embodied, comprises the following steps:

(1) Treatment of quinoa

60G of quinoa seeds are weighed, crushed by a crusher and the powder is collected according to the following weight ratio of 1:2 in a proportion of Phosphate Buffer Solution (PBS) and soaking for 2h.

(2) QDNPs preparation of crude extract: sequentially centrifuging the soaking solution with 3000g for 30min to obtain supernatant; centrifuging 10000g for 1h to obtain supernatant; the supernatant was removed by centrifugation at 150000g for 2h and the pellet was resuspended in PBS. A QDNPs crude extract was obtained, the centrifugation temperature in this step was 4 ℃.

(3) QDNPs purification: preparing 8%, 30%, 45% and 60% sucrose solution by using ultrapure water, sequentially injecting 2mL of sucrose solution from the bottom of a centrifuge tube according to the concentration from low to high to prepare discontinuous sucrose density gradient solution, re-suspending the QDNPs crude extract obtained in the step 2) in 2mL of PBS, transferring the re-suspended QDNPs crude extract into the sucrose density gradient solution, centrifuging for 2h at 150000g, and layering the solution at the centrifugation temperature of 4 ℃; collecting 8% -30% of the strips, namely quinoa exosome (named QDNPs), and storing in a refrigerator at-80deg.C for use. The yield was 0.919g and 1.53%.

Example 2: QDNPs characterization analysis

(1) Transmission electron microscope analysis

Morphology of QDNPs was observed by transmission electron microscopy, QDNPs particle pellet was fixed and examined by transmission electron microscopy using conventional procedures, and QDNPs was found to be a spherical morphology resembling exosome-like nanoparticles as shown in fig. 2A.

(2) Particle size and Zeta potential analysis

The particle size and Zeta potential of QDNPs were measured using a Zeta View particle size analyzer, and the results showed that QDNPs particle size range was between 85.6 and 267.0nm, the peak particle size was 152.8nm, and Zeta potential was about-4.7 mV, as shown in FIGS. 2B-2C.

The results show that: QDNPs extracted in this study had a dished or hemispherical concave side structure resembling exosome-like nanoparticles, demonstrating the feasibility of the extraction procedure used in this study.

Example 3: QDNPs Effect on the metabolic syndrome in mice

1. Grouping animals

(1) 40 Male C57BL/6J mice, weighing 13-15g, were kept and tested in SPF class rodent laboratory (China radiation protection institute). Its ambient temperature: 25+/-1 ℃; humidity: 60+/-5%; illumination conditions: and (5) circulating for 12 hours.

(2) After one week of normal diet adaptive feeding, experiments were started, 40 mice were weighed and randomly grouped according to body weight into 10 mice per group, control (Control), high-fat Model (Model), QDNPs low-dose (QDNPs-L) and QDNPs high-dose (QDNPs-H) groups, respectively. Wherein the control group is given normal free drinking water diet, the high-fat model group starts to feed 60% of high-fat feed in the second week, QDNPs-L groups are fed with high-fat feed in the second week, each mouse is filled with stomach 2.5mg QDNPs,QDNPs-H groups every day, and each mouse is filled with stomach 5-mg QDNPs every day until the 14 th week;

(3) All mice were euthanized by isoflurane inhalation prior to dissection.

2. White adipose tissue picture of epididymis of mouse and weight

Mice after the end of 14 weeks of feeding were dissected, the epididymal white adipose tissue (eWAT) of the mice was removed, representative eWAT of each group of mice was picked up for photographing, and their weights were weighed as shown in FIGS. 3A-3B.

As a result, it was found that white adipocyte volume was significantly increased in high fat diet mice (DIO) compared to the control group, while the volume and weight of white adipocyte of QDNPs dosing treated DIO mice was significantly reduced compared to HFD group, and dose dependency was exhibited, improving obesity phenomenon in DIO mice.

3. Blood fat four index detection

(1) Mice were fasted for 14h before sacrifice. Isoflurane is used for anaesthetizing animals, the left beard is trimmed, blood is taken from the inner canthus vein, and the operation is performed to avoid the occurrence of hemolysis caused by the hair staining. Collecting blood sample into 1.5mL EP tube, standing on ice for 30min, centrifuging at 3000rpm and 4deg.C for 15min, carefully collecting upper yellowish clear liquid, which is serum, packaging, and storing at-80deg.C;

(2) Referring to the specifications of six detection kits for blood lipid in Nanjing built serum, the operation and analysis are strictly performed according to the specifications, and the results are shown in figures 3C-3F.

Conventional blood lipid index results showed that QDNPs treatment induced a decrease in TG, T-CHO and LDL levels and an increase in HDL levels in the serum of mice.

4. Measurement of fasting blood glucose

(1) One week before mice were sacrificed, tail-end blood was taken 14h in advance, and fasting blood glucose was measured using a rogowski glucometer.

(2) The experimental mice were subjected to glucose tolerance experiments after feeding to week 14: after all mice were starved for 14h, 2.5G/kg glucose solution was infused into each mouse, tail vein blood collection was performed at 0, 30, 60, 140min, and blood glucose concentration was measured by a rogowski glucometer, as shown in fig. 3G.

As a result, it was found that the fasting blood glucose of mice in the model group was significantly increased compared to the control group, indicating that DIO mice may have type two diabetes; and the fasting blood glucose of the DIO mice is obviously reduced after QDNPs treatment.

5. Insulin and insulin resistance assay

The concentration of insulin in the serum of each group of mice was determined by an insulin detection kit, as shown in fig. 3H. The insulin resistance index calculation formula is: fasting blood glucose values x fasting insulin values/22.5. Insulin resistance index in this study was calculated and counted using the formula as shown in fig. 3J.

As a result, the serum insulin content of mice in the model group is far higher than that of the normal group, and insulin in the serum of the mice in the model group is obviously reduced by QDNPs gastric lavage treatment, and dose dependency is presented, and meanwhile, consistent results of insulin resistance are presented. These results indicate that QDNPs is able to improve insulin levels in serum of obese mice and reduce the insulin resistance index.

Example 4: remission of mouse oral quinoa exosome QDNPs on non-alcoholic fatty liver

1. Mouse and liver representative picture

After isoflurane anesthesia, mice were euthanized, organs were dissected, the livers of the mice were removed with a scalpel, and after residual blood stains and tissues were washed out in pre-chilled PBS buffer, the mice and livers were photographed as shown in fig. 4A.

The mice gross anatomical data show that QDNPs treatment significantly reduced liver steatosis in model mice.

2. Mouse liver weight and NAFLD Activity score (NAFLD ACTIVITY score, NAS)

The dissected mouse livers were weighed and each experimental mouse was graded for liver injury severity according to NAFLD Activity Scoring (NAS) criteria as follows. The results are shown in FIGS. 4B-4C. (1) hepatocyte adiposity: score 0 (< 5%); 1min (5% -33%); 2 minutes (34% -66%); 3 minutes (> 66%). (2) intralobular inflammation (20-fold mirror count necrotic foci): 0 minutes, none; score 1 (< 2); 2 (2-4); 3 minutes (> 4). (3) hepatocyte balloon-like changes: 0 minutes, none; 1 minute, rare; 2 minutes, see more.

After statistics of body weight and liver weight data for each group of mice, significantly increased liver weight and NAS scores in the model group were found to decrease significantly after QDNPs intervention.

3. Liver AST and ALT index detection

(1) Mice were fasted for 12h before sacrifice. Animals were anesthetized with isoflurane and their livers were dissected after euthanasia. Accurately weighing the weight of the liver according to the weight (g): volume (mL) =1: 9, adding 9 times of physiological saline, mechanically homogenizing in ice water bath at 2500rpm, centrifuging for 10min, and taking supernatant as a test.

(2) Reference is made to the instructions of the Nanjing's as-built detection kit, which is strictly followed by the instructions and the results are shown in FIGS. 4D-4E.

The liver index results of mice show that the QDNPs treatment significantly induces the reduction of serum ALT and AST levels of mice. These data indicate that QDNPs can significantly alleviate liver steatosis in NAFLD model mice, suggesting that QDNPs has a good protective effect on liver steatosis.

According to the invention, QDNPs nano particles are extracted through the above embodiment, and pathological experiments of a high-fat diet induced metabolic syndrome and non-alcoholic fatty liver disease mouse model are carried out on the nano particles, so that the metabolic syndrome effect and the non-alcoholic fatty liver disease of the mouse can be relieved by improving lipid metabolism disorder, and the effect of the nano particles is found to be free from toxic and side effects.

Therefore, QDNPs nano-particles obtained by extraction according to the invention can be used as ideal choice for preparing medicines for treating metabolic syndrome and nonalcoholic fatty liver disease. The embodiments described above and features of the embodiments herein may be combined with each other without conflict. The present invention is not limited to the details and embodiments described herein, and further advantages and modifications may readily be achieved by those skilled in the art, so that the present invention is not limited to the specific details, representative solutions and illustrative examples shown and described herein, without departing from the spirit and scope of the general concepts defined by the claims and the equivalents.

Claims (5)

1. A method for preparing quinoa exosomes QDNPs, comprising the steps of:

The first step: pulverizing clean quinoa, adding appropriate amount of PBS solution, and soaking for 2-2.5 hr to obtain coarse extract of quinoa exosome.

And a second step of: and (3) sequentially centrifuging 3000g of the crude extract for 30min, 10000g of the crude extract for 1h, and collecting supernatant.

And a third step of: the supernatant was centrifuged at 150000g for 1.5-2h, the pellet resuspended in an appropriate amount of PBS and transferred to a prepared sucrose gradient of 8%/30%/45%/60% solution and centrifuged at 150000g for 2-3h. Collecting 8% -30% of strips between layers, namely: quinoa exosomes QDNPs are stored at-80 ℃.

2. The method of making QDNPs according to claim 1, wherein: QDNPs is nanometer vesicle, the grain diameter is 85.6-267.0nm, and the peak value is 152.8nm.

3. QDNPs prepared according to the process of any one of claims 1-2.

4. Use of QDNPs according to claim 3 for the preparation of functional foods, foods for special diets, health-care products and/or pharmaceuticals for the prevention and treatment of metabolic syndrome.

5. Use of QDNPs according to claim 3 for the preparation of a functional food, a special meal food, a health product and/or a pharmaceutical product for the prevention and treatment of non-alcoholic fatty liver disease.