Tripterygium wilfordii outer vesicle as well as extraction method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a tripterygium wilfordii outer vesicle as well as an extraction method and application thereof. The extraction method comprises the following steps: taking tripterygium wilfordii raw juice, carrying out stepped centrifugation on the tripterygium wilfordii raw juice by virtue of 6000-7000 g and 8000-12000 g to remove impurities, extracting by virtue of a plant tissue external vesicle extraction kit, carrying out centrifugation on the tripterygium wilfordii raw juice by virtue of 10000-14000 g and 12000-15000 g in combination with low-temperature standing to separate and precipitate, carrying out resuspension on the precipitate, and taking supernate again, so as to obtain the tripterygium wilfordii external vesicles. Animal experiment results show that the kidney coefficient, urea nitrogen, urine protein and cholesterol levels of mice with chronic nephritis can be remarkably reduced by intragastric administration of 0.1 mg of tripterygium wilfordii external vesicles per day, the pathological injury improvement effect is superior to that of traditional tripterygium wilfordii polyglycoside tablets, and a new way for effectively relieving chronic nephritis is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a Tripterygium wilfordii exovesicle, its extraction method, and its application. Background Technology

[0002] Tripterygium wilfordii has the effects of dispelling wind and dampness, promoting blood circulation and reducing swelling, and relieving pain by clearing the meridians. Its diterpenoids and triterpenoids contain active substances with anti-inflammatory, oxidative stress-improving, and immune-regulating effects. Extracellular vesicles are released from cells and contain nanovesicles that deliver proteins, nucleic acids, and other bioactive substances. Studies have shown that the structure of extracellular vesicles from various plants is similar to that of mammalian extracellular vesicles, and they can regulate intercellular communication between mammalian cells across species. Chronic nephritis is a disease characterized by immune-mediated glomerular inflammation, clinically manifested as proteinuria, hematuria, edema, hypertension, and renal function impairment. Due to its insidious onset and prolonged course, chronic nephritis can easily progress to end-stage renal disease and lead to death if not diagnosed and treated early. Therefore, it is necessary to explore a new and effective way to alleviate chronic nephritis. Summary of the Invention

[0003] To explore a new approach to alleviate chronic nephritis, this invention provides Tripterygium wilfordii exovesicles, their extraction method, and applications. Oral administration of 0.1 mg / mouse / day of the Tripterygium wilfordii exovesicles provided by this invention significantly reduces kidney coefficient, blood urea nitrogen, urinary protein, and cholesterol levels in mice with chronic nephritis, and its effect on improving pathological damage is superior to that of traditional Tripterygium wilfordii polyglycoside tablets.

[0004] This invention provides an application of Tripterygium wilfordii exovesicles for the preparation of drugs for treating chronic nephritis.

[0005] The Tripterygium wilfordii vesicles provided by this invention, administered by gavage at a dose of 0.1 mg / mouse / day, can significantly reduce the renal coefficient, blood urea nitrogen, urinary protein, and cholesterol levels in mice with chronic nephritis, and its effect on improving pathological damage is superior to that of traditional Tripterygium wilfordii polyglycoside tablets.

[0006] Furthermore, the drug is used to reduce kidney coefficient, blood urea nitrogen, urinary protein, or cholesterol levels.

[0007] Furthermore, the drug also includes a pharmaceutically acceptable excipient, wherein the excipient is physiological saline.

[0008] Furthermore, the drug was used to treat mice with chronic nephritis.

[0009] Furthermore, the Tripterygium wilfordii exovesicles are obtained by extracting the juice from Tripterygium wilfordii through cutting it into pieces, centrifuging to collect the supernatant, and then using a plant tissue exovesicle extraction kit.

[0010] This invention also provides a method for extracting extravesicles from Tripterygium wilfordii, specifically including the following steps: Take fresh Tripterygium wilfordii, wash it, cut it into pieces and extract the juice to obtain Tripterygium wilfordii juice; Centrifuge the Tripterygium wilfordii juice at 4℃~10℃ and 6000 g~7000 g for 5 min~15 min, and take the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 4℃~10℃ and 8000 g~12000 g for 15 min~25 min, and take the supernatant to obtain the second supernatant; The second supernatant was extracted using a plant tissue extravesicular vesicle extraction kit. The extract was then centrifuged at 12000 g to 15000 g for 25 min to 35 min at 4℃ to 10℃, and the precipitate was collected. Resuspend the precipitate in sterile physiological saline, centrifuge at 1500 g to 2500 g for 5 min to 15 min, and collect the supernatant to obtain the Tripterygium wilfordii vesicles.

[0011] Furthermore, the plant tissue extravesicular vesicle extraction kit includes extraction reagent A and extraction reagent B. The extraction steps of the kit are as follows: the second supernatant is mixed with extraction reagent A at a volume ratio of 1.5~2.5:1 and allowed to stand. Then, it is centrifuged at 4℃~10℃ and 10000 g~14000 g for 5 min~15 min, and the supernatant is collected to obtain the third supernatant. The third supernatant is mixed with extraction reagent B at a volume ratio of 2~4:1 and allowed to stand to obtain the extract.

[0012] The present invention also provides exovesicles of Tripterygium wilfordii, extracted by any of the extraction methods described herein, with an average particle size of 175.5 nm to 210.9 nm and a concentration of 2.11 × 10⁻⁶. 10 particle / mL ~2.61×10 10 particle / mL.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, the Tripterygium wilfordii external vesicle group further reduced urea nitrogen, urinary protein, cholesterol, and renal index compared to the Tripterygium wilfordii polyglycoside tablet group (*p<0.05), approaching the levels of the blank group; HE staining showed that the Tripterygium wilfordii external vesicle group exhibited reduced glomerular sclerosis, decreased inflammatory cell infiltration, and cell morphology closer to normal. These results indicate that, at the same dosage, the Tripterygium wilfordii external vesicles provided by this invention have a significantly better effect on alleviating chronic nephritis than traditional Tripterygium wilfordii polyglycoside tablets, providing a new and effective approach to relieving chronic nephritis.

[0014] The embodiments of this invention use the reagent kit method (Isolation Reagent A / B) combined with stepwise centrifugation to extract Tripterygium wilfordii exovesicles. The extraction purity is comparable to that of the ultra-high speed centrifugation method of 120,000 g × 120 min in the comparative example, but the operation is simpler, the extraction time is reduced from >2 h to <1 h, and no ultra-high speed centrifugation equipment is required, which significantly reduces the operating cost and threshold. This shows that the reagent kit method extraction scheme provided by this invention can replace the traditional ultra-high speed centrifugation method. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Electron microscopy and particle size analysis of Tripterygium wilfordii exoves extracted by two different extraction methods.

[0017] Figure 2 The images show the morphological changes of the kidneys in mice with nephritis in each group, with four samples per row representing parallel samples.

[0018] Figure 3 The kidney pathological changes of mice in each group with nephritis are shown.

[0019] Figure 4 The renal function indicators of each group of mice with nephritis; In the figure, A represents the cholesterol levels of each group of mice with nephritis; B represents the urinary protein index of each group of nephritis mice; C represents the blood urea nitrogen level in each group of mice with nephritis; D represents the kidney coefficient index of each group of mice with nephritis. Detailed Implementation

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0021] Example 1: A method for extracting exovesicles from Tripterygium wilfordii Take 1 kg of fresh Tripterygium wilfordii, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water. Cut the washed Tripterygium wilfordii into pieces and extract the juice to obtain Tripterygium wilfordii juice.

[0022] Centrifuge the Tripterygium wilfordii juice at 10℃ and 6500 g for 10 min to remove residual plant tissue and cells, and obtain supernatant A.

[0023] Centrifuge supernatant A at 10℃ and 10000 g for 20 min to remove plant cell debris and obtain supernatant B.

[0024] Add extraction reagent A at a volume ratio of 2:1 (supernatant B: extraction reagent A), mix well, and let stand at 4℃ for 10 min. After standing, centrifuge at 10℃ and 12000 g for 10 min to obtain supernatant C. Add extraction reagent B at a volume ratio of 3:1 (supernatant C: extraction reagent B), mix well, and let stand at 4℃ for 1 h. After standing, centrifuge at 10℃ and 13500 g for 30 min, discard the supernatant, and collect the precipitate (extraction reagent A and extraction reagent B are Isolation Reagent A and Isolation Reagent B from the Plant Tissue Vesicle Extraction Kit sold by Shenzhen Shifangjie Technology Co., Ltd.).

[0025] The precipitate was resuspended in sterile saline and centrifuged at 2000 g for 10 min to obtain supernatant D, which is the Tripterygium wilfordii vesicle.

[0026] Example 2: A method for extracting exovesicles from Tripterygium wilfordii Take 1 kg of fresh Tripterygium wilfordii, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water. Cut the washed Tripterygium wilfordii into pieces and extract the juice to obtain Tripterygium wilfordii juice.

[0027] Centrifuge the Tripterygium wilfordii juice at 4℃ and 6000 g for 15 min to remove residual plant tissue and cells, and obtain supernatant A.

[0028] Centrifuge supernatant A at 4℃ and 8000 g for 25 min to remove plant cell debris and obtain supernatant B.

[0029] Add extraction reagent A at a volume ratio of 1.5:1 (supernatant B: extraction reagent A), mix well, and let stand at 4℃ for 10 min. After standing, centrifuge at 10000 g for 15 min at 4℃ to obtain supernatant C. Add extraction reagent B at a volume ratio of 2:1 (supernatant C: extraction reagent B), mix well, and let stand at 4℃ for 1 h. After standing, centrifuge at 12000 g for 35 min at 4℃, discard the supernatant, and collect the precipitate (extraction reagent A and extraction reagent B are Isolation Reagent A and Isolation Reagent B from the Plant Tissue Vesicle Extraction Kit sold by Shenzhen Shifangjie Technology Co., Ltd.).

[0030] The precipitate was resuspended in sterile saline and centrifuged at 1500 g for 15 min to obtain supernatant D, which is the Tripterygium wilfordii vesicle.

[0031] Example 3: A method for extracting exovesicles from Tripterygium wilfordii Take 1 kg of fresh Tripterygium wilfordii, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water. Cut the washed Tripterygium wilfordii into pieces and extract the juice to obtain Tripterygium wilfordii juice.

[0032] Centrifuge the Tripterygium wilfordii juice at 4℃ and 7000 g for 5 min to remove residual plant tissue and cells, and obtain supernatant A.

[0033] Centrifuge supernatant A at 4℃ and 12000 g for 15 min to remove plant cell debris and obtain supernatant B.

[0034] Add extraction reagent A at a volume ratio of 2.5:1 (supernatant B: extraction reagent A), mix well, and let stand at 4℃ for 10 min. After standing, centrifuge at 14000 g for 5 min at 4℃ to obtain supernatant C. Add extraction reagent B at a volume ratio of 4:1 (supernatant C: extraction reagent B), mix well, and let stand at 4℃ for 1 h. After standing, centrifuge at 15000 g for 25 min at 4℃, discard the supernatant, and collect the precipitate (extraction reagent A and extraction reagent B are Isolation Reagent A and Isolation Reagent B from the Plant Tissue Extravesicular Vesicle Extraction Kit sold by Shenzhen Shifangjie Technology Co., Ltd.).

[0035] The precipitate was resuspended in sterile saline and centrifuged at 2500 g for 5 min to obtain supernatant D, which is the Tripterygium wilfordii vesicle.

[0036] Comparative Example 1: A method for extracting exovesicles from Tripterygium wilfordii Take 1 kg of fresh Tripterygium wilfordii, put it in a water tank and rinse it with drinking water, then rinse it with ultrapure water. Cut the washed Tripterygium wilfordii into pieces and extract the juice to obtain Tripterygium wilfordii juice.

[0037] Centrifuge the Tripterygium wilfordii juice at 10℃ and 6500 g for 10 min to remove residual plant tissue and cells, and obtain supernatant A.

[0038] Centrifuge supernatant A at 10℃ and 10000 g for 20 min to remove plant cell debris and obtain supernatant B.

[0039] Supernatant B was filtered using a 0.22 μm microporous membrane to remove suspended low-density impurities. The supernatant was then centrifuged at 120,000 g for 120 min, and the supernatant was discarded to collect the precipitate.

[0040] The precipitate was resuspended in sterile saline and centrifuged at 2000 g for 10 min to obtain supernatant D, which is the Tripterygium wilfordii vesicle.

[0041] The results of the Tripterygium wilfordii vesicles obtained by the extraction methods of Examples 1 to 3 of this invention are similar. This invention takes Example 1 as an example to identify and compare the Tripterygium wilfordii vesicles extracted with those obtained by Comparative Example 1, and conducts an application study on the preparation of drugs for treating chronic nephritis.

[0042] I. Identification of Tripterygium wilfordii exovesicles Example 1 used a kit method, while Comparative Example 1 used ultracentrifugation. The particle size distribution and concentration of *Tripterygium wilfordii* exovesicles extracted by both methods were determined using a nanoparticle size analyzer (NTA). Purity was initially assessed based on the distribution of particle size peaks; fewer peaks indicated higher purity. The yield of *Tripterygium wilfordii* exovesicles was determined by the number of exovesicles per milliliter; a higher number of exovesicles indicated a higher yield. Results are as follows: Figure 1 As shown in Example 1, the NTA results using the kit method showed that the average particle size of the isolated Tripterygium wilfordii exovesicles was 175.5 nm, and the concentration was 2.11 × 10⁻⁶. 10 particle / mL. Comparative Example 1, NTA results from ultracentrifugation showed that the average particle size of the separated Tripterygium wilfordii exovesicles was 210.9 nm, and the concentration was 2.61 × 10⁻⁶. 10 particle / mL. The Tripterygium wilfordii vesicles extracted by both methods conformed to the particle size range of vesicles, and had high purity and high yield. There was no significant difference between the two methods. However, the kit method does not require the use of an ultracentrifuge, making it simpler and faster. Therefore, the Tripterygium wilfordii vesicles extracted by the kit method in Example 1 were selected for application research in the preparation of drugs for treating chronic nephritis.

[0043] II. Application of Tripterygium wilfordii exovesicles in the preparation of drugs for treating chronic nephritis 1. Test materials Twelve six-week-old male C57 mice were selected.

[0044] 2. Test instruments and reagents The instruments and reagents used in the experiment are shown in Table 1.

[0045] Table 1. Test Instruments and Reagents 3. Test methods Twelve mice were randomly divided into four groups: blank group, model group, Tripterygium wilfordii polyglycoside tablet group, and Tripterygium wilfordii external vesicle group.

[0046] Mice in the model group, Tripterygium wilfordii polyglycoside tablet group, and Tripterygium wilfordii exovesicle group were administered 80 mg / kg / mouse of adenine suspension (97.5 mg adenine dissolved in 21 mL of physiological saline by ultrasound) by gavage for 7 consecutive days to establish a chronic nephritis model. Simultaneously, mice in the blank control group were administered an equal volume of physiological saline by gavage as a control. On day 8, mice in the Tripterygium wilfordii polyglycoside tablet group were administered 0.1 mg / mouse of Tripterygium wilfordii polyglycoside tablets by gavage, mice in the Tripterygium wilfordii exovesicle group were administered 0.1 mg / mouse of Tripterygium wilfordii exovesicles by gavage, and mice in the blank control and model groups were administered an equal volume of physiological saline by gavage. This gavage treatment continued for 7 days.

[0047] On day 15, the body weight (g) of mice in each group was measured. After anesthesia, both kidneys were removed and weighed (g). Photos were taken and the kidney coefficient (g / g) of each group was calculated as: bilateral kidney weight (g) / mouse body weight (g). Blood was collected from the eyeballs to measure plasma cholesterol, urinary protein, and blood urea nitrogen. After weighing bilateral kidney tissue, each kidney was evenly cut in half along the midline, fixed, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

[0048] 4. Test Results The kidney morphology of each group of mice is as follows Figure 2 As shown, the kidneys of mice in the control group were bright red, while the kidneys of mice in the model group were swollen and whitish, indicating that the kidneys of mice in the model group were abnormal. The size and color of the kidneys of mice in the Tripterygium wilfordii polyglycoside tablet group and the Tripterygium wilfordii exovesicle group were between those of the control group and the model group, but the kidneys of the Tripterygium wilfordii exovesicle group were redder and slightly larger than those of the Tripterygium wilfordii polyglycoside tablet group.

[0049] HE staining results are as follows Figure 3 As shown in the figure. Compared with the control group, the model group mice showed glomerular sclerosis, increased mesangial matrix and mesangial cells, Bowman's capsule wall adhesion, and increased inflammatory cell infiltration. Compared with the model group, the pathological changes in the kidneys of mice in the Tripterygium wilfordii polyglycoside tablet group and the Tripterygium wilfordii external vesicle group were alleviated, with the degree of alleviation being slightly stronger in the Tripterygium wilfordii polyglycoside tablet group. The cells in the Tripterygium wilfordii external vesicle group were more relaxed than those in the Tripterygium wilfordii polyglycoside tablet group.

[0050] Results of cholesterol, urine protein, blood urea nitrogen, and kidney coefficient ( Figure 4 The results (A-D) showed that, compared with the control group, the mice in the model group had significantly higher levels of urea nitrogen, urinary protein, cholesterol, and kidney coefficient, indicating that the chronic nephritis mouse model was successfully established. The urea nitrogen, urinary protein, cholesterol, and kidney coefficient in the Tripterygium wilfordii polyglycosides group and the Tripterygium wilfordii exovesicle group decreased compared with the model group, indicating that the nephritis in these groups was alleviated to some extent. The Tripterygium wilfordii exovesicle group significantly downregulated these indicators on the basis of the model, but did not return them to normal levels.

[0051] The above experimental results demonstrate that both Tripterygium wilfordii polyglycoside tablets and Tripterygium wilfordii exovesicles can alleviate chronic nephritis caused by adenine, but compared with Tripterygium wilfordii polyglycoside tablets, Tripterygium wilfordii exovesicles are more effective in alleviating chronic nephritis caused by adenine.

[0052] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0053] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An application of Tripterygium wilfordii exovesicles, characterized in that, The application is for preparing drugs to treat chronic nephritis.

2. The application of the Tripterygium wilfordii exovesicles according to claim 1, characterized in that, The drug is used to reduce kidney coefficient, blood urea nitrogen, urinary protein, or cholesterol levels.

3. The application of the Tripterygium wilfordii exovesicles according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients, wherein the excipients are physiological saline.

4. The application of the Tripterygium wilfordii exovesicles according to claim 1, characterized in that, The drug was used to treat mice with chronic nephritis.

5. The application of the Tripterygium wilfordii exovesicles according to claim 1, characterized in that, The Tripterygium wilfordii exovesicles are obtained by extracting the juice from Tripterygium wilfordii through cutting it into pieces, centrifuging to collect the supernatant, and then using a plant tissue exovesicle extraction kit.

6. A method for extracting exovesicles from Tripterygium wilfordii, characterized in that, The Tripterygium wilfordii exovesicles are those described in claim 1; The extraction method of the extravesicles of Tripterygium wilfordii specifically includes the following steps: Take fresh Tripterygium wilfordii, wash it, cut it into pieces and extract the juice to obtain Tripterygium wilfordii juice; Centrifuge the Tripterygium wilfordii juice at 4℃~10℃ and 6000 g~7000 g for 5 min~15 min, and take the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 4℃~10℃ and 8000 g~12000 g for 15 min~25 min, and take the supernatant to obtain the second supernatant; The second supernatant was extracted using a plant tissue extravesicular vesicle extraction kit. The extract was then centrifuged at 12000 g to 15000 g for 25 min to 35 min at 4℃ to 10℃, and the precipitate was collected. Resuspend the precipitate in sterile physiological saline, centrifuge at 1500 g to 2500 g for 5 min to 15 min, and collect the supernatant to obtain the Tripterygium wilfordii vesicles.

7. The method for extracting Tripterygium wilfordii exovesicles according to claim 6, characterized in that, The plant tissue extravesicular vesicle extraction kit includes extraction reagent A and extraction reagent B. The extraction steps of the kit are as follows: the second supernatant is mixed with extraction reagent A at a volume ratio of 1.5~2.5:1 and allowed to stand. Then, it is centrifuged at 4℃~10℃ and 10000 g~14000 g for 5 min~15 min, and the supernatant is collected to obtain the third supernatant. The third supernatant is mixed with extraction reagent B at a volume ratio of 2~4:1 and allowed to stand to obtain the extract.

8. A type of Tripterygium wilfordii exovesicle, characterized in that, Extracted by the method described in any one of claims 6 to 7, the average particle size is 175.5 nm to 210.9 nm, and the concentration is 2.11 × 10⁻⁶. 10 particle / mL ~2.61×10 10 particle / mL.

Images