CN115466709B

CN115466709B - Trigonella exosome extraction method and application

Info

Publication number: CN115466709B
Application number: CN202211084583.1A
Authority: CN
Inventors: 刘琳娜; 王蕾; 程鹏; 李燕林; 王宏; 徐娟; 陈冠; 黄清秀; 李昕; 安海文; 袁玲; 杨文钦; 江茜; 康利; 李锦山; 林芬娜; 甘沛敏; 阮绍伟; 潘钰榕
Original assignee: Tianjin Institute Of Pharmaceutical Sciences Tianjin Pharmaceutical And Health Research Center; Zhongshan Hospital of TCM
Current assignee: Tianjin Institute Of Pharmaceutical Sciences Tianjin Pharmaceutical And Health Research Center; Zhongshan Hospital of TCM
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2023-10-20
Anticipated expiration: 2042-09-06
Also published as: CN115466709A

Abstract

The invention discloses a method for extracting the exosome of Trigonella Foenum-Graecum and application thereof, which utilizes a density gradient centrifugation method to extract the active ingredients of the exosome of Trigonella Foenum-Graecum, and has higher safety in clinical application because the exosome does not have a cell nucleus structure and can not be amplified in a host body, can be used for treating wound surface repair and preparing medicines for treating wound surface repair, and has shuttle characteristics and rich miRNA components, thereby being capable of regulating other organisms from plant bodies in a 'crossing' way, being possible to replace skin transplantation for wound healing in the future and having extremely broad application prospect.

Description

Trigonella exosome extraction method and application

Technical Field

The invention relates to a plant extraction method and application, in particular to a method for extracting Trigonella and application.

Background

Trigonella is a plant of Liliaceae, and has effects of clearing heat, detoxicating, diminishing inflammation, and relieving pain, and is commonly used for treating venomous snake bite and traumatic injury swelling pain in Guangdong region (reference documents: mei Quanxi, zhong Xiwen, zhang Xiaojun, etc.. Trigonella is analgesic, anti-inflammatory action research [ J ] Chinese medicinal material, 2000, 23 (10): 632. Mei Quanxi, zhong Xiwen, huifei, etc.. The influence of micro-ring of Rabbit ear when traumatic injury is analgesic [ J ] Chinese medicinal material, 2002, 25 (7): 493). The compound triangle grass tablet developed by taking the triangle grass as the main medicine has definite research evidence on detumescence, analgesia and snake venom resolving effect (reference documents: mei Quanxi, zhong Xiwen, lin Hui, etc.. The experimental study on the snake venom resisting effect of the compound triangle grass tablet is: chinese medicine industry. 2005, 14 (1): 24-25) and clinical curative effect (reference documents: miao Yingnian, mei Quanxi, chen Maochao, etc.. The compound triangle grass preparation is used for treating snake venom bite curative effect observation is: chinese traditional Chinese medicine emergency, 2005, 14 (5): 429-431). The team in the middle mountain market learns from clinical research data of 80 cases of patients suffering from the snake bite caused by the blood circulation type through the conventional treatment of the compound triangle grass preparation and western medicine, and the medicine has good curative effect on various snake bites caused by the blood circulation type. It is worth mentioning that the wound periphery is easy to cause extensive tissue necrosis after venomous snake bite, and difficult to heal after difficult to cure. The compound Trigonella Foenum preparation has no wound ulcer (reference: in year Mei Quanxi, chen Maochao, etc. the compound Trigonella Foenum preparation has therapeutic effect on snake venom bite, chinese traditional medicine emergency, 2005, 14 (5): 429-431). In addition, it is well documented that fresh Trigonella Foundation is mashed and applied externally in Guangdong Zhongshan (reference: guangdong Chinese Committee, guangdong journal of Chinese medicine (volume 2) [ M ] Guangzhou: guangdong scientific and technical Press, 1996, 10-11). This inspires the inventor team to develop the external preparation of the Trigonella Foenum-graecum, and has great popularization space for repairing the wound surface of the damaged skin.

In recent years, research on exosomes of medicinal plants has been increasingly paid attention to in the academy. Because the exosome does not have a cell nucleus structure, the exosome can not be amplified in a host, has higher safety in clinical application, can replace skin transplantation in the future for wound healing, and has extremely broad application prospect. Cells in the skin communicate with each other using a variety of mechanisms, one of which is the exosome. As a key loop in cell secretion, exosomes can reach distant injury sites through most capillaries.

Exosomes are a type of microvesicles with diameters of 40-100nm, and can transfer biomolecules such as lipids, carbohydrates, proteins, mRNA, miRNA, DNA and the like from one cell to another, so as to exchange genetic information and reprogram Cheng Suzhu cells for intercellular communication. Regent (ref: rege second nte M, corti-Monz n G, maldonado AM,et al. Vesicular fractions of sunflower apoplastic fluidsare associated with potential exosome marker proteins FEBSLett,2009, 583: 3363-3366) exosome-like vesicles were first found in 2009 in plants. The biological characteristics of the plant cell exosomes are the same as those of animal exosomes, and DNA, specific proteins, complex RNA and the like are contained in the plant cell exosomes, so that substances such as nucleic acid, bioactive lipid, protein and the like can be transferred among cells. miRNA contained in plant exosomes can be detected and stably exist in human blood, and plays a specific pharmacological role.

Recent studies have been carried out to suggest that mirnas can regulate gene expression across species, and that cross-species regulation of mirnas has been reported in a number of papers, the first of which reported that, 2012, a study paper published in the journal of Cell Research by the university of south Beijing Zhang Chenyu laboratory (ref: linzhang, dongxia Hou, xi Chen,et alexogenous plant MIR168a specifically targets mammalian LDLRAP: evidence of cross-kingdom regulation by microRNA, cell Research,2012, jan;22 (1): 107-26), it was confirmed that plant mirnas can be directly introduced into the body without being digested by animals, and can also regulate the expression of mammalian target genes. Abundant mirnas not only perform biological functions in the original system, but also move between plants and interacting organisms, achieving "cross-gene" expression regulation (reference: huang F, du J, liang Z,et allarge-scale analysis of smallRNAs derived from traditional chinese herbs in human tissues, sci China Life Sci,2019, 62 (3): 321-332), plays an important role (reference: de Oca, A.M.; madue ñ o, j.a.; martinez-Moreno, J.M.et alHigh-phosphate-induced calcification is related to SM22 a promoter methylation in vascular smooth muscle cells, J.Bone Miner.Res,2010, 25, 1996-2005). Thus, mirnas from medicinal plant exosomes are likely to interact as new bioactive ingredients with biological animal systems.

The biological activity of the "cross-over" regulation of the exosomes of medicinal plants successively accumulates a large number of basic research data (ref: alexander Tsukunnidas. Plant-Derived extracelluar vesicles as therapeutic nanoca)rriers, international Journal of Melecular Sciences,2022, 23, 191). Ginger exosomes regulate the intestinal flora (reference: teng Y, ren Y, safe M,et alplant-derived exosomal micrornas shape the gut microbiota, cell Host Microbe,2018, 24 (5): 637-652), inhibits colonic inflammation (reference: zhang M, viennois E, prasad M,et almedible ginger-derived nanoparticles: a novel therapeutic approach for the prevention and treatment ofinflammatory bowel disease and colitis-associated cancer Biomaterials,2016, 101: 321-340), the anti-inflammatory and antiviral effects of honeysuckle are exerted by miRNA2911 contained in the water decoction exosomes (reference: zhou Z, li X, liu J,et alhoneysuckle-encoded atypical microRNA2911 directly targets influenza a viruses [ J ]. Cell Research,2014, 25 (1): 39-49), rhodiola exosomes inhibit pulmonary fibrosis (reference: du J, liang Z, xu J,et alplant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibroticHJT-sRNA-m7, sci China Life Sci,2019, 62 (3): 309-320), astragalus water decoction nano-sample particles can regulate blood sugar by regulating the ratio of firmicutes/bacteroides in intestinal flora (reference: gao Wen, hou Min, chen Xiaoxiao, et al Astragalus membranaceus vesicle-like nanoparticles were analyzed for their mechanism of action by modulating intestinal flora to reduce blood glucose in db/db diabetic mice, journal of Chinese experimental prescription, 2021, 27 (14): 111-118), ginseng root exosome myocardial protection (reference: liu Tianjia, zhidong, ye, et al. Extraction of ginseng root exosomes, characterization and mechanism of action for protecting against doxorubicin-induced myocardial injury, chinese herbal medicine, 2021, 52 (12): 3514-3522), the cortex Mori exosome inhibits colitis (reference: mukesh K Sriwastva, deng Zhong-Bin, wang Bo-mei,et alexosome-like nanoparticles from Mulberry bark prevent Dss-induced colitis via the AhR/COPS8path, EMBO,2022,1 (7): 1243-1246) and may also promote wound healing (ref: lu Shuyan and Yang Song, any Li Mei, etc. Ginseng exosomes promote proliferation of HaCaT cells and wound healing, chinese lifeJournal of biochemistry and molecular biology, 2021, 37 (11): 1510-1519).

At present, no report on a method for extracting the Trigonella Foenum-graecum exosome is available, and no report on the application of the Trigonella Foenum-graecum exosome extract is available, and the invention is based on the premise of research and development of the applicant and is used for continuously researching and developing the Trigonella Foenum-graecum exosome.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for extracting the trigonella foenum-graecum exosome by using a density gradient centrifugation method and application thereof.

The technical scheme adopted for solving the technical problems is as follows:

a method for extracting an exosome of Trigonella Foenum-graecum comprises the following steps:

(1) The whole herb of the Trigonella Foenum-Graecum with 1.5. 1.5 kg root system removed is washed and crushed by a processor to obtain a sample A.

(2) Sample a was transferred to a centrifuge tube and centrifuged at 1000×g for 10min.

(3) The rotation speed was adjusted to 3000 Xg and centrifuged for 20min.

(4) The temperature was set at 4℃and 10000 Xg was centrifuged for 30min, and the supernatant was collected to obtain sample B.

(5) Sample B was subjected to a 37℃water bath for 1-2 hours to give sample C.

(6) Sample C was cooled to 4℃and centrifuged at 2000 Xg for 30min, and the supernatant was taken to give sample D.

(7) Sample D was transferred to a new centrifuge tube, centrifuged at 10000 Xg for 45 min at 4℃and the supernatant was taken to give sample E.

(8) Sample E was transferred to a new centrifuge tube, centrifuged at 100000 Xg for 120 min at 4℃and the supernatant was taken to give sample F.

(9) Sample F was transferred to a new centrifuge tube, resuspended in 20 mL pre-chilled (4 ℃) 1 XPBS, then kept at 4 ℃, centrifuged at 2000 Xg for 30min, and the supernatant was taken to give sample G.

(10) Sample G was transferred to a fresh centrifuge tube, centrifuged at 2000 Xg for 30min at 4℃and the supernatant was taken to give sample H.

(11) Sample H was transferred to a fresh centrifuge tube, centrifuged at 2000 Xg for 30min at 4℃and the supernatant was taken to give sample I.

(12) Sample I was transferred to a fresh centrifuge tube, centrifuged at 100000 Xg for 120 min at 4℃and the supernatant removed to give sample J.

(13) 1 XPBS pre-cooled (4 ℃) at 1 mL was added to sample J to re-suspend the pellet to give sample K, which was stored at 4℃for further use.

(14) Along the tube wall, 40% iodixanol, 20% iodixanol, 10% iodixanol and 5% iodixanol were added to the super-separation tube, followed by 1 mL sample K at the uppermost layer, and then ultracentrifugation was performed at 4 ℃,100000×g, for 120 min.

(15) After the centrifugation, the liquid in the super-separation tube is separated into 12 layers, the liquid in the middle 6-9 layers is taken out, and the super-separation tube is subjected to the super-centrifugation again at 4 ℃ and 100000 Xg for 120 min.

(16) The supernatant was removed and resuspended in 200. Mu.L of pre-chilled (4 ℃) 1 XPBS to give a Trigonella foenum-graecum exosome-like nanoparticle suspension.

The preservation condition of the triangular grass exosome-like nanoparticle suspension is-80 ℃ for sealing preservation.

The external application of the triangular grass exosome-like nanoparticle suspension can be applied to the application of wound repair treatment and the application of preparing a wound repair treatment medicine.

And drying the triangular grass exosome-like nanoparticle suspension to obtain triangular grass exosome-like nanoparticles, wherein the triangular grass exosome-like nanoparticles can be applied to the application of wound repair treatment and the application of preparing a wound repair treatment medicine in an external application manner.

The beneficial effects of the invention are as follows: the invention extracts the active ingredients of the Trigonella Foenum-Graecum exosome by using a density gradient centrifugation method, and has higher safety in clinical application because the exosome does not have a cell nucleus structure and can not be amplified in a host, and can be used for treating wound surface repair and preparing medicines for treating wound surface repair.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 is a distribution sequencing result of all sequences in different groups;

FIG. 2 is a statistical table of the classification of the number of small RNA deduplication sequences (Unique);

FIG. 3 is a table of Total number of Total de-duplication sequences of small RNA in the class statistics (Total);

FIG. 4 is a statistical table of miRNA expression levels;

fig. 5 is a photograph of the drug administration side and control side wounds on day 1;

fig. 6 is a photograph of the drug administration side and control side wounds on day 15;

fig. 7 is a photograph of the drug administration side and control side wounds on day 22.

Detailed Description

(1) The whole herb of the Trigonella Foenum-Graecum with 1.5. 1.5 kg root system removed is washed and crushed by a processor to obtain a sample A. The current household food processor can meet the crushing requirement. Before crushing, the whole herb of Trigonella Foenum-graecum can be soaked for 2 hours.

(3) The rotation speed was adjusted to 3000 Xg and centrifuged for 20min.

(5) Sample B was subjected to a water bath at 37℃for 2 hours to give sample C.

(14) Along the tube wall, sequentially adding the prepared 40% iodixanol, 20% iodixanol, 10% iodixanol and 5% iodixanol 3.6, mL respectively into a super-separation tube, adding 1 mL sample K at the uppermost layer along the tube wall, and then performing ultracentrifugation at 4 ℃ and 100000 Xg for 120 min.

(15) After centrifugation, the liquid in the ultracentrifugation tube was separated into 12 layers, the middle 6-9 layers of liquid were removed (separation layers from color or transparency aspects), and ultracentrifugation was performed again at 4℃for 120 min at 100000 Xg.

(16) The supernatant was removed and resuspended in 200. Mu.L of pre-chilled (4 ℃) 1 XPBS to give a Trigonella foenum-graecum exosome-like nanoparticle suspension which was stored under sealed conditions at-80 ℃.

After the triangular grass exosome-like nanoparticle suspension is dried (distilled and volatilized solvent is dried), triangular grass exosome-like nanoparticles are obtained, and the triangular grass exosome-like nanoparticles can be applied to the application of wound repair treatment and the application of wound repair treatment medicaments.

Three samples grass1, grass2, grass3 were prepared according to the method described above, 20. Mu.L each was subjected to SmallRNA sequencing, and the sequencing results are shown in FIG. 1: distribution of all sequences in different groups sequencing results, in fig. 1, seq is the sequence measured, and grass1, grass2, grass3 is the number of sequences measured for three samples.

Small RNA sequences are short and generally easily matched to different fragments, and thus there are a variety of annotation results. For each small RNA with Unique annotation, the existing annotation results were ranked according to the priority of the known miRNA > rRNA > tRNA > snRNA > level miRNA, and the small RNA deduplication sequence number classification statistics (Unique) are shown in fig. 2: the Total number of small RNA deduplication sequences classification statistics (Total) is shown in FIG. 3. In FIG. 2, the first column is the sample number, and columns 2 to 8 are the number of deduplication sequences annotated to each RNA, respectively. In FIG. 3, the first column is the sample number, and columns 2 to 8 are the total number of deduplication sequences annotated to each RNA, respectively.

According to the sequence number of the conservative miRNA, the Reads Count value of the miRNA is counted, and the result is shown in the figure 4: in the miRNA expression amount statistics table, in fig. 4, the first column is known mirnas of the present species, named "species short-miRNA family-distinguishing sequence number", and the 2 nd column to the last column are the expression amount Reads Count values of the mirnas in each sample.

The wound repair experiment is carried out on the sample manufactured by the invention, and the steps are as follows:

1. wistar rats were shaved on both sides of the back spine with an electric shaver to expose 6cm by 6cm of skin. The left skin is the administration side and the right skin is the control side.

2. The gauze is dipped in boiling water at 100 ℃ and is immediately applied to the skin to prepare the skin scald model.

3. Diluting the triangular grass exosome-like nanoparticle suspension extracted by the invention with PBS (phosphate buffered saline) solution, wherein the volume ratio of the triangular grass exosome-like nanoparticle suspension to the PBS solution is 1:9, and preparing Ch-ELNs application liquid;

4. the administration side was coated with the Ch-ELNs application solution and the control side was coated with the PBS solution.

5. The smeared part is wrapped by sterile medical gauze.

6. The dressing was changed 1 time on days 1, 5, 10, 15, 20, respectively.

7. Wound healing was observed daily. According to observation, from the 5 th day of the application, the wound healing of the skin at the application side is obviously improved compared with that at the control side, from the 10 th day, the effect is gradually obvious, the 1 st day of the wound is shown in fig. 5, the 15 th day of the wound is shown in fig. 6, the 22 nd day of the wound is shown in fig. 7, and from the wound healing diagrams at the application side and the control side, the application side has good promotion effect on the healing of the wound surface of the skin scald of the rat.

The embodiment adopts the triangular grass exosome-like nanoparticle suspension as an example for experiments, and the prepared triangular grass exosome-like nanoparticle is diluted by PBS (phosphate buffered saline) solution, so that the triangular grass exosome-like nanoparticle has a good promoting effect on healing of scalded wounds.

The triangular grass exosome-like nanoparticle suspension and the triangular grass exosome-like nanoparticle prepared by the invention can be combined with other medicines to prepare an external application medicine containing the triangular grass exosome-like nanoparticle suspension or the triangular grass exosome-like nanoparticle.

The invention utilizes density gradient centrifugation to extract the active ingredients of the Trigonella foenum-graecum exosome, and the exosome has no cell nucleus structure, can not be amplified in a host body, has higher safety in clinical application, can be used for treating wound surface repair and preparing medicaments for treating wound surface repair, and has shuttle characteristics and is rich in miRNA components, other organisms can be regulated and controlled from plant bodies in a crossing way, and can be predicted according to the existing experimental results, and the exosome can possibly replace skin transplantation for wound healing in the future, and has extremely broad application prospect.

The above embodiments do not limit the protection scope of the invention, and those skilled in the art can make equivalent modifications and variations without departing from the whole inventive concept, and they still fall within the scope of the invention.

Claims

1. A method for extracting an exosome of Trigonella Foenum-graecum is characterized by comprising the following steps:

(1) Cleaning whole grass of Trigonella Foenum-Graecum with root system removed 1.5. 1.5 kg, and crushing with a processor to obtain sample A;

(2) Sample A was transferred to a centrifuge tube and centrifuged at 1000 Xg for 10min;

(3) Regulating the rotating speed, and centrifuging for 20min at 3000 Xg;

(4) Setting the temperature to 4 ℃, centrifuging for 30min at 10000 Xg, and taking the supernatant to obtain a sample B;

(5) Carrying out water bath on the sample B at 37 ℃ for 1-2 hours to obtain a sample C;

(6) Cooling the sample C to 4 ℃, centrifuging for 30min at 2000 Xg, and taking the supernatant to obtain a sample D;

(7) Transferring the sample D into a new centrifuge tube, centrifuging at 4 ℃ and 10000 Xg for 45 min, and taking a supernatant to obtain a sample E;

(8) Transferring the sample E into a new centrifuge tube, centrifuging at 4 ℃ and 100000 Xg for 120 min, and taking the supernatant to obtain a sample F;

(9) Transferring the sample F into a new centrifuge tube, adding 20 mL precooled 1 XPBS for resuspension, then keeping the temperature at 4 ℃, centrifuging at 2000 Xg for 30min, and taking the supernatant to obtain a sample G;

(10) Transferring the sample G into a new centrifuge tube, centrifuging at 4 ℃ and 2000 Xg for 30min, and taking the supernatant to obtain a sample H;

(11) Transferring the sample H into a new centrifuge tube, centrifuging at 4 ℃ and 2000 Xg for 30min, and taking the supernatant to obtain a sample I;

(12) Transferring the sample I into a new centrifuge tube, centrifuging at 4 ℃ and 100000 Xg for 120 min, and removing the supernatant to obtain a sample J;

(13) Adding 1X PBS (phosphate buffer solution) precooled by 1 mL into the sample J for re-suspending and precipitating to obtain a sample K, and preserving the sample K at 4 ℃ for later use;

(14) Sequentially adding 40% iodixanol, 20% iodixanol, 10% iodixanol and 5% iodixanol 3.6, mL into a super-separation tube along the tube wall, adding 1 mL sample K at the uppermost layer, and then performing ultracentrifugation at 4 ℃ for 120 min at 100000 Xg;

(15) After the centrifugation is completed, the liquid in the super-separation tube is divided into 12 layers, the liquid in the middle 6-9 layers is taken out, and the temperature is 4 ℃ again, and the super-separation tube is subjected to 100000 Xg super-centrifugation for 120 min;

(16) The supernatant was removed and resuspended with 200 μl of pre-chilled 1×pbs to give a deltoid exosome-like nanoparticle suspension that was used to prepare a therapeutic wound repair drug.

2. The method for extracting Trigonella Foenum-graecum exosome as claimed in claim 1, wherein the preservation condition of the Trigonella Foenum-graecum exosome-like nanoparticle suspension in the step (16) is-80 ℃ sealed preservation.

3. The method for extracting Trigonella foenum-graecum exosome according to claim 1, wherein the Trigonella foenum-graecum exosome-like nanoparticle suspension extracted in step (16) is dried to obtain Trigonella foenum-graecum exosome-like nanoparticles, and the Trigonella foenum-graecum exosome-like nanoparticles are used for preparing a medicament for treating wound repair.