Preparation method and application of vesicle-like nanoparticles derived from fresh medicine perfoliote knotweed herb

Abstract

The invention provides a preparation method and application of vesicle-like nanoparticles derived from fresh medicine perfoliote knotweed herb. The vesicle-like nanoparticles are especially used for improving psoriasis-like inflammation. According to the method, firstly, differential centrifugation is adopted, supernate is collected, and impurity protein is effectively removed; and then ultracentrifugation is carried out, precipitates are collected, water-soluble impurities are effectively removed, and the concentration of the vesicle-like nanoparticles is improved. Compared with a traditional fresh medicine perfoliote knotweed herb active ingredient extract, the vesicle-like nano-particles derived from the fresh medicine perfoliote knotweed herb extracted by the method disclosed by the invention have anti-inflammatory biological activity, naturally carry active molecules derived from plants, have lower toxicity, higher bioavailability and better biological compatibility, and can be used for preparing the anti-inflammatory drug-loaded vesicle-like nano-particles for preparing the anti-inflammatory drug-loaded vesicle-like nano-particles for preparing the anti-inflammatory drug-loaded vesicle-like nano-particles. The psoriasis-like skin inflammation can be effectively relieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, specifically to a preparation method and application of vesicle-like nanoparticles derived from fresh medicinal plants of Polygonum perfoliatum L., especially for the improvement of psoriasiform inflammation. Background Art

[0002] Psoriasis is an immune-mediated inflammatory skin disease, and its occurrence and development are affected by various factors. Currently, more than 60 million people worldwide are suffering from this disease, and its clinical symptoms are diverse, not only in the skin, but more importantly, in the psychological aspect. At present, the main treatments for it are topical therapy, systemic therapy, and biological agents, but there are some disadvantages for traditional preparations, which seriously endanger human health. Therefore, there is an urgent need to develop new products for improving psoriasiform inflammation.

[0003] Polygonum perfoliatum L. is one of the traditional Chinese medicines in China. It is the dried above-ground part of the plant Polygonum perfoliatum L. of the Polygonaceae family, and has the effects of clearing heat and detoxifying, promoting blood circulation to remove stasis, softening hardness and dissipating swelling, and removing dampness and astringing. It is included in the first part of the Chinese Pharmacopoeia. Its clinical application is extensive and its pharmaceutical value is quite high. Polygonum perfoliatum L. grows in the wild, and due to its low price and rich resources, it has now been reported that it can be externally used to treat a variety of skin diseases.

[0004] Vesicle-like nanoparticles contain various proteins, lipids, and nucleic acids, and can play important physiological functions by mediating cell-to-cell communication. Plant vesicle-like nanoparticles usually show a vesicular structure and are micronano-scale biological particles wrapped by a phospholipid bilayer. Some studies have found that plant vesicle-like nanoparticles have cross-species regulatory functions, which can not only regulate the physiological functions of mammals, but also intervene in and prevent the disease process, and play a therapeutic role in diseases. These research conclusions prove that plant vesicle-like nanoparticles, as a new type of natural product, may become a good candidate source for new drug development.

[0005] Fresh medicinal plants of Polygonum perfoliatum L. are a traditional Chinese medicinal material, and its main active ingredients are flavonoids and anthraquinones, which have significant anti-inflammatory activity, and its active ingredients can improve psoriasiform skin inflammation. However, due to the high difficulty in extracting its active ingredients and the difficulty in large-scale application, etc., it hinders its clinical application. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum and its application in psoriasiform inflammation. The vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum prepared by the method of the present invention have good stability, are easy to absorb, and have a certain effect on improving psoriasiform skin inflammation, and to a certain extent solve the technical problems of the transdermal drug delivery preparation of Polygonum perfoliatum for external use.

[0007] To achieve the above object, the present invention provides the following technical solutions: In the first aspect, the present invention provides a method for preparing vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum, and the method comprises the following steps: Step 1: Wash, chop and juice the fresh herb Polygonum perfoliatum to obtain the original tissue solution of the fresh herb Polygonum perfoliatum; Step 2: Subject the obtained original tissue solution of Polygonum perfoliatum to differential centrifugation at 4°C to obtain the supernatant of Polygonum perfoliatum; Step 3: Subject the supernatant of Polygonum perfoliatum to ultracentrifugation at 4°C to obtain a precipitate, and resuspend it to obtain vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum.

[0008] Preferably, in Step 2, the differential centrifugation conditions are: 4°C, 200 g, 20 min; 4°C, 2000 g, 20 min; 4°C, 10000 g, 30 min.

[0009] Preferably, in Step 3, the ultracentrifugation conditions are: 4°C, 100000 g, 60 min.

[0010] Preferably, after resuspension in Step 3, it further comprises filtering with a 0.22 µm filter membrane to obtain vesicle-like nanoparticles.

[0011] In the second aspect, the present invention provides the use of vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum for preparing a drug for improving psoriasiform inflammation, and the vesicle-like nanoparticles are prepared by the method as described above.

[0012] In the third aspect, the present invention provides a drug for improving psoriasiform inflammation, and the drug comprises vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum prepared by the method as described above.

[0013] Preferably, the drug for improving psoriasiform inflammation is an external preparation. More preferably, the external preparation is a solution, a lotion, a liniment, a gel, an ointment, a plaster, a paste or a patch.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the prior art, the present invention has the advantages of simple preparation method, low cost, large production, etc. The vesicle-like nanoparticles derived from fresh herbs themselves have a variety of active ingredients, and have advantages such as high solubility, low immunogenicity, easy metabolism by the human body, and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the preparation process of vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum L.; Figure 2 It is a particle size distribution diagram of the vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum L. of the present invention detected by a Malvern laser particle size analyzer; Figure 3 It is a Zeta potential distribution diagram of the plant nanovesicles derived from the fresh herb Polygonum perfoliatum L. of the present invention detected by a nanoparticle size and potential analyzer; Figure 4 It is a morphological photograph of the plant nanovesicles derived from the fresh herb Polygonum perfoliatum L. prepared according to the method of the present invention observed by a transmission electron microscope; Figure 5 It is a photograph of the improvement effect of the plant nanovesicles derived from the fresh herb Polygonum perfoliatum L. prepared according to the method of the present invention on imiquimod-induced psoriasis-like skin inflammation in mice; Figure 6 It is a curve graph of the body weight change of each group of mice during the experiment; Figure 7 It is a curve graph of the change of PASI score of each group of mice during the experiment; Figure 8 It is a graph of the H&E staining result of the back skin of psoriasis-like mice 7 days after administration (20x). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention aims to solve, to a certain extent, one of the technical problems of the transdermal drug delivery preparation of Polygonum perfoliatum L. For the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method of vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum L. The vesicle-like nanoparticles derived from fresh herbs prepared by the method of the present invention have good stability, are easy to absorb, and have a certain effect of improving psoriasis-like skin inflammation.

[0017] In the first exemplary embodiment, the present invention provides a preparation method of vesicle-like nanoparticles derived from the fresh herb Polygonum perfoliatum L., which comprises the following steps: Step 1, wash the fresh herb Polygonum perfoliatum L., cut it into pieces, and squeeze the juice to obtain the original tissue solution of the fresh herb Polygonum perfoliatum L.

[0018] Step 2, perform differential centrifugation on the original tissue solution of the fresh herb Polygonum perfoliatum L. at 4°C to obtain the supernatant of Polygonum perfoliatum L.

[0019] Step 3: Ultracentrifuge the supernatant of Polygonum perfoliatum L. at 4°C to obtain a precipitate, and resuspend and disperse the precipitate to obtain vesicle-like nanoparticles derived from fresh Polygonum perfoliatum L.

[0020] According to the preparation method of the vesicle-like nanoparticles derived from fresh Polygonum perfoliatum L. provided by the embodiments of the present invention, differential centrifugation is first used to collect the supernatant, effectively removing impurity proteins; secondly, ultracentrifugation is used to collect the precipitate, effectively removing water-soluble impurities and increasing the concentration of the vesicle-like nanoparticles.

[0021] Compared with the traditional extract of the active ingredients of fresh Polygonum perfoliatum L., the vesicle-like nanoparticles derived from fresh Polygonum perfoliatum L. extracted by this method have significant anti-inflammatory biological activities, and naturally carry the active molecules of the source plant, with lower toxicity, higher bioavailability and better biocompatibility, and can effectively alleviate psoriasis-like skin inflammation.

[0022] In addition, the following preferred operating process parameters are further provided for the method for preparing nano-vesicles from fresh Polygonum perfoliatum L. proposed in the above embodiments of the present invention: In Step 1, when using a juicer to crush fresh Polygonum perfoliatum L., the rotation speed is 1000 - 15000 rpm / min, and the crushing time is 1 min. The time should not be too long to avoid over-crushing.

[0023] In Step 2, the differential centrifugation is carried out at 4°C, 200 g for 20 min; 4°C, 2000 g for 20 min; 4°C, 10000 g for 30 min.

[0024] In Step 3, the ultracentrifugation is carried out at 4°C, 100000 g for 60 min. This can increase the yield rate compared to a shorter time and ensure the safety of the experimenters.

[0025] In addition, in Step 3, when using PBS buffer to resuspend and disperse the ultracentrifuged precipitate, try to disperse it evenly. In this embodiment, a 0.22 µm filter membrane is used for filtration.

[0026] It should be noted that the centrifugation operations in Step 2 and Step 3 need to be carried out at 4°C to avoid the fragmentation of the nano-vesicles caused by the high temperature generated during centrifugation.

[0027] It should also be noted that in Step 3, when resuspending and dispersing the precipitate, it needs to be carried out on an ice surface.

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the specific embodiments and the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", etc. is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0030] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0031] The following uses specific examples to illustrate the experimental technical solution of the present invention. It should be noted that the method steps used in this example do not exclude experimental solutions that can produce the same results. This experimental technical solution provides an extraction solution for vesicle-like nanoparticles from fresh Herba Polygoni Perfoliati. Other experimental steps can also be inserted in the experimental steps. It should also be noted that the technical steps used in this example are not only applicable to this example, but also to similar plant categories. At the same time, the method steps of this example are only used for distinction and do not limit the experimental steps.

[0032] The materials used in the embodiments of the present invention are all commercially available ordinary products and can be purchased in the market. The technical solution of the present invention will be described in detail below through specific embodiments to facilitate the understanding of those of ordinary skill in the art. Example 1

[0033] Prepare vesicle-like nanoparticles from fresh Herba Polygoni Perfoliati as follows.

[0034] Step 1: Take an appropriate amount of fresh Herba Polygoni Perfoliati, wash it with ultrapure water and then cut it into pieces. Add ultrapure water according to the weight ratio of the medicinal material 1:1, place it in a juicer and crush it to obtain the original liquid of the plant tissue of fresh Herba Polygoni Perfoliati.

[0035] Step 2: Perform differential centrifugation on the original liquid of the Herba Polygoni Perfoliati tissue obtained in Step 1 (4°C, 200g, 20 min; 4°C, 2000g, 20 min; 4°C, 10000g, 30 min) to obtain the supernatant of fresh Herba Polygoni Perfoliati.

[0036] Step 3: Ultracentrifuge the supernatant of the fresh Polygonum perfoliatum tissue obtained in Step 2 (at 4°C, 100,000 g, for 1 h), retain the supernatant, and take the precipitate.

[0037] Step 4: Resuspend and disperse the precipitate obtained in Step 3, and filter through a 0.22-μm filter membrane to obtain the fresh Polygonum perfoliatum nanovesicles. The extraction process is as Figure 1 shown. Example 2

[0038] Characterize and detect the nanovesicle particles obtained according to Example 1 as follows.

[0039] 1. Use a Malvern laser particle size analyzer to detect the particle size distribution of the fresh Polygonum perfoliatum nanovesicles obtained in Example 1. The results are as Figure 2 shown.

[0040] 2. Use a nanoparticle size potentiometer to detect the Zeta potential of the fresh Polygonum perfoliatum nanovesicles obtained in Example 1. The results are as Figure 3 shown.

[0041] 3. Use a transmission electron microscope to observe the morphology of the fresh Polygonum perfoliatum nanovesicles obtained in Example 1. The results are as shown in the Figure 4 photographs. Example 3

[0042] In this example, healthy female C57BL / 6 mice with a body weight between (20 ± 2) g were used and raised in a constant temperature environment. They were randomly divided into a blank control group, a model group, a low-dose group (3 mg / mL), and a high-dose group (6 mg / mL).

[0043] The fresh Polygonum perfoliatum nanovesicles obtained in Example 1 were administered to the depilated areas on the backs of mice with psoriasis-like skin inflammation induced by 5% imiquimod using the method of back application. Take pictures of the psoriasis-like skin inflammation, and record the body weight and PASI score every day.

[0044] As Figure 5 shown, compared with the back skin of mice in the normal group, model group, and treatment group, the skin of the normal group was smooth and without wrinkles; the back skin of mice in the model group showed redness, keratinous thickening, and obvious scaly symptoms; the back skin of mice in the treatment group also showed redness, keratinous thickening, and scaly symptoms, but compared with the model group, the symptoms were alleviated.

[0045] Figure 6 Shows the comparison curve of the body weight changes of mice in the normal group, model group, and treatment group. As Figure 6 shown, the body weight of mice in the normal group did not change and remained stable within a certain range; the body weight of mice in the treatment group and model group showed a phenomenon of first decreasing and then increasing within the overall range.

[0046] Figure 7 Shows the comparison chart of the changes in PASI scores of the dorsal skin of mice in the normal group, model group and treatment group. As Figure 7 shown, the PASI scores of the dorsal skin of mice in the normal group were all 0, indicating no lesions in the dorsal skin; the PASI scores of the dorsal skin of mice in the model group increased sharply from the second day, decreased slightly on the sixth day, and then increased, indicating that the psoriatic skin inflammation of the dorsal skin of mice in the model group was very severe; the PASI of the dorsal skin of mice in the treatment group increased slowly, but compared with the model group, the increase was very slow, and its PASI score was significantly lower than that of the model group. Therefore, it can be considered that the polygonum perfoliatum nanovesicles extracted in Example 1 have the effect of improving the symptoms of imiquimod-induced psoriatic skin inflammation in mice.

[0047] Figure 8 Shows the HE staining results of the dorsal skin of mice in the normal group, model group and treatment group. As Figure 8 shown, the cell morphology of the skin tissue of mice in the normal group was normal, the epidermis was thinner, and there was no inflammatory infiltration. The model group was the opposite; compared with the model group, the psoriatic mice intervened in the treatment group had significant improvement in the lesion thickness and inflammatory infiltration. This shows that polygonum perfoliatum nanovesicles can inhibit the inflammatory cell infiltration at the lesion site of psoriatic mice and reduce the severity of the disease.

[0048] In the description of this specification, some embodiments show the use of specific technical methods in combination with specific embodiments to better elaborate the specific features, structures and applications of the present invention. And it should be noted that the technical methods used in the embodiments of this specification are not only applicable to this embodiment, but also can be applied to other embodiments.

[0049] Where the present invention is not elaborated, it is all well-known techniques to those skilled in the art.

[0050] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified and equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of vesicle-like nanoparticles derived from fresh Chinese medicine Polygonum perfoliatum, characterized in that, The method includes the following steps: Step 1: Wash fresh Polygonum perfoliatum L., cut it into pieces, and squeeze juice to obtain the original tissue solution of fresh Polygonum perfoliatum L. Step 2: Subject the obtained original tissue solution of Polygonum perfoliatum L. to differential centrifugation at 4°C to obtain the supernatant of Polygonum perfoliatum L. Step 3: Subject the supernatant of Polygonum perfoliatum L. to ultracentrifugation at 4°C to obtain a precipitate, and resuspend it to obtain vesicle-like nanoparticles derived from fresh Polygonum perfoliatum L.

2. The method according to claim 1, characterized in that, In Step 2, the conditions for differential centrifugation are: 4°C, 200 g, 20 min; 4°C, 2000 g, 20 min; 4°C, 10000 g, 30 min.

3. The method according to claim 1, wherein In Step 3, the conditions for ultracentrifugation are: 4°C, 100000 g, 60 min.

4. The method according to claim 1, characterized in that, After resuspension in Step 3, it further includes filtering with a 0.22 µm filter membrane to obtain vesicle-like nanoparticles.

5. Use of vesicle-like nanoparticles derived from the fresh medicine Polygonum perfoliatum L. for preparing a medicament for improving psoriasiform inflammation, characterized in that, The vesicle-like nanoparticles are prepared by the method described in any one of Claims 1 to 4.

6. A drug for improving psoriasiform inflammation, characterized in that, The drug contains vesicle-like nanoparticles derived from fresh Polygonum perfoliatum L. prepared by the method described in any one of Claims 1 to 4.

7. The drug according to claim 6, characterized in that, The drug for improving psoriasiform inflammation is in an external preparation form.

8. The drug according to claim 7, characterized in that, The external preparation form is a solution, lotion, liniment, gel, ointment, plaster, paste or patch.

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