A Chinese herbal medicine extracellular vesicle composition and its preparation method and application

Abstract

The present invention relates to a Chinese herbal extracellular vesicle composition, its preparation method, and application. The Chinese herbal extracellular vesicle composition comprises five or more of the following: extracellular vesicles from Rhizoma Corydalis, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonella ternatea, and Agrimoniae scabra. The Chinese herbal extracellular vesicle composition comprises Verbena officinalis and Agrimoniae scabra. The Chinese herbal extracellular vesicle composition provided by the present invention can safely and effectively relieve neck, shoulder, waist, leg, and joint pain.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a Chinese herbal medicine extracellular vesicle composition, a preparation method and an application thereof. Background Art

[0002] Pain is a common symptom, especially chronic pain, which seriously affects patients' quality of life. Traditional pain treatment mainly relies on chemical drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids, but these drugs have many side effects, such as gastrointestinal irritation and addiction.

[0003] In recent years, with the deepening of regenerative medicine and exosome research, exosomes have gradually attracted attention as a new biotherapy. Exosomes are natural nano-sized vesicles that have immune tolerance, biological stability, and natural targeting, and can effectively deliver therapeutic molecules.

[0004] Chinese herbal medicines are an integral part of traditional Chinese medicine and possess a wealth of medicinal value. Herbal vesicles (plant extracellular vesicles), derived from natural plants, are multi-component, multifunctional, and readily absorbed by organisms. They can serve as biotherapeutics and drug delivery vehicles, addressing the poor bioavailability of many active ingredients and enhancing biosafety. However, the development of plant extracellular vesicles for pain treatment remains insufficient, particularly for herbs with traditional analgesic properties (such as groundgrass, white schizonepeta, and verbena). Research on their use as pain treatment products is lacking.

[0005] In view of this, there is an urgent need for a composition based on extracellular vesicles of Chinese herbal medicine to safely and effectively relieve neck / shoulder / waist / leg / joint pain. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a Chinese herbal extracellular vesicle composition to safely and effectively relieve neck / shoulder / waist / leg / joint pain.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention provides a Chinese herbal extracellular vesicle composition, which comprises five or more of the following: extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbena officinalis, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Sophora flavescens, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonella ternate and extracellular vesicles of Agrimoniae scabra; wherein the Chinese herbal extracellular vesicle composition comprises extracellular vesicles of Verbena officinalis and extracellular vesicles of Agrimoniae scabra.

[0009] Preferably, the Chinese herbal extracellular vesicle composition includes: extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbena officinalis, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonum multiflorum and extracellular vesicles of Herba Agrimoniae.

[0010] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is (0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-10).

[0011] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is (0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5).

[0012] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae and extracellular vesicles of Herba Agrimoniae is 1:1:2:1:1:1:1:1:2.

[0013] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae and extracellular vesicles of Herba Agrimoniae is 1:1:1:1:1:1:1:1:1:1.

[0014] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is 1:1:1:1:1:1:1.5:1.5:1.

[0015] Preferably, the mass ratio of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is 1:1:2:2:1:2:1:2:2.

[0016] The present invention provides a preparation method of the Chinese herbal extracellular vesicle composition. The preparation method comprises: separately extracting extracellular vesicles of Oryza sativa, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum, and Agrimoniae scabra, and then compounding them according to proportion to obtain the Chinese herbal extracellular vesicle composition.

[0017] Preferably, the extraction of the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae comprises: taking Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae respectively, adding a solvent to break the cell walls, and collecting the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae by centrifugation.

[0018] Preferably, the solvent is PBS buffer.

[0019] Preferably, the specific steps of the centrifugation include:

[0020] (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant;

[0021] (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant;

[0022] (3) Centrifuge at 3000-5000g for 20-40 min and collect the supernatant;

[0023] (4) Centrifuge at 8000-12000g for 50-70min and collect the supernatant;

[0024] (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0025] The present invention also provides a composition for relieving pain, which comprises any of the above-mentioned Chinese herbal extracellular vesicle compositions or the Chinese herbal extracellular vesicle composition prepared by the above-mentioned method.

[0026] Preferably, the weight proportion of the Chinese herbal extracellular vesicle composition in the composition for relieving pain is 0.1 to 50 parts.

[0027] The present invention also provides the use of any of the above-mentioned extracellular vesicle compositions, or the above-mentioned extracellular vesicle compositions prepared by the above-mentioned method, or any of the above-mentioned compositions for relieving pain in the preparation of products for relieving pain.

[0028] Preferably, the dosage form of the product includes oil, emulsion, ointment, paste, film coating, gel, aerosol, spray, solution, and liniment.

[0029] Compared to existing technologies, the herbal extracellular vesicle composition provided by the present invention exhibits synergistic effects between its components. This allows the composition to significantly reduce the levels of inflammatory factors in macrophages, reduce the expression of cyclooxygenase COX2, and reduce the level of prostaglandin PGE2 at the cellular level, thereby effectively alleviating patients' neck, shoulder, waist, leg, and joint pain. The herbal extracellular vesicle composition provided by the present invention is safe and effective, suitable for long-term use, and well-compatible with most pharmaceutical or daily necessities. Its addition to pharmaceuticals or daily necessities is simple, making it suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 To test the infiltration of the Chinese herbal extracellular vesicle composition into the ex vivo skin of 8-week-old Bama miniature pigs in Example 1 (yellow arrows represent PKH26-labeled YHZT-001 Chinese herbal extracellular vesicles, and green arrows represent DAPI-stained cell nuclei);

[0031] Figure 2 To test the test results of the Chinese herbal extracellular vesicle composition pain-relieving gel patch on human body in Example 5;

[0032] Figure 3 This is the statistical result of the effectiveness of the Chinese herbal extracellular vesicle composition pain-relieving gel patch in the human body treatment pain test in Example 5. DETAILED DESCRIPTION

[0033] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0034] The present invention provides a Chinese herbal extracellular vesicle composition, which comprises five or more of the following: extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbena officinalis, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Sophora flavescens, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonella ternate and extracellular vesicles of Agrimoniae scabra; wherein the Chinese herbal extracellular vesicle composition comprises extracellular vesicles of Verbena officinalis and extracellular vesicles of Agrimoniae scabra.

[0035] In certain embodiments, the herbal extracellular vesicle composition comprises: extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbena officinalis, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae flavescentis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonum multiflorum and extracellular vesicles of Herba Agrimoniae scabra.

[0036] In certain embodiments, the mass ratio of the extracellular vesicles of Rhizoma Corydalis, Rhizoma Bletillae, Rhizoma Verbenae, Rhizoma Aconitum, Rhizoma Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae and Rhizoma Agrimoniae is (0.1-10): (0.1-10): (0.1-10): (0.1-10): (0.1-10): (0.1-10): (0.1-10): (0.1-10).1~10), for example, 1:1:1:1:1:1:1:1:1, 2:1:1:1:1:1:1:1:1, 3:1:1:1:1:1:1:1:1, 4:1:1:1:1:1:1:1:1, 5:1:1:1:1:1:1:1:1, 6:1:1:1:1:1:1:1:1, 7:1:1:1:1:1:1:1:1, 8:1:1:1:1:1:1:1:1, 9:1:1:1:1:1:1:1:1, 10:1:1:1:1:1:1:1:1, 1:2:1:1:1:1:1:1:1, 2:2:1:1:1:1:1:1, 3:2:1:1:1:1:1:1 :1, 4:2:1:1:1:1:1:1, 5:2:1:1:1:1:1:1, 6:2:1:1:1:1:1:1, 7:2:1:1:1:1:1:1, 8:2:1:1:1:1:1:1, 9:2:1:1:1:1:1:1, 10:2:1:1:1:1:1:1, 1:1:2:1:1:1:1:1, 2:1:2:1:1:1:1:1, 3:1:2:1:1:1:1:1, 4:1:2:1:1:1:1:1, 5:1:2:1:1:1:1:1, 6:1:2:1:1:1:1:1, 7:1 :2:1:1:1:1:1, 8:1:2:1:1:1:1:1, 9:1:2:1:1:1:1:1, 10:1:2:1:1:1:1:1, 1:1:1:2:1:1:1:1, 2:1:1:2:1:1:1:1, 3:1:1:2:1:1:1:1, 4:1:1:2:1:1:1:1, 5:1:1:2:1:1:1:1, 6:1:1:2:1:1:1:1, 7:1:1:2:1:1:1:1, 8:1:1:2:1:1:1:1, 9:1:1:2:1:1:1:1, 10:1:1:2 :1:1:1:1, 10:1:1:1:2:1:1:1, 10:1:1:1:1:1:2:1:1, 10:1:1:1:1:1:1:2:1, 10:1:1:1:1:1:1:1:2, 10:1:1:1:1:1:1:1:3, 10:1:1:1:1:1:1:4, 10:1:1:1:1:1:1:5, 10:1:1:1:1:1:1:6, 10:1:1:1:1:1:1:7, 10:1:1:1:1:1:1:8, 10:1:1:1:1:1:1:1:9, 10:1:1:1:1:1:1:1:10, etc. .

[0037] In certain embodiments, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae and extracellular vesicles of Herba Agrimoniae is (0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5):(0.1~5).

[0038] In certain embodiments, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae and extracellular vesicles of Herba Agrimoniae is 1:1:2:1:1:1:1:1:2.

[0039] In certain embodiments, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae, and extracellular vesicles of Herba Agrimoniae is 1:1:1:1:1:1:1:1:1:1.

[0040] In certain embodiments, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae Tonkinensis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Rhizoma Trigonellae, and extracellular vesicles of Herba Agrimoniae is 1:1:1:1:1:1:1.5:1.5:1.

[0041] In certain embodiments, the mass ratio of the extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbenaceae, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Sophora flavescens, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonum multiflorum and extracellular vesicles of Agrimoniae scabra is 1:1:2:2:1:2:1:2.

[0042] The present invention provides a preparation method of the Chinese herbal extracellular vesicle composition. The preparation method comprises: separately extracting extracellular vesicles of Oryza sativa, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum, and Agrimoniae scabra, and then compounding them according to proportion to obtain the Chinese herbal extracellular vesicle composition.

[0043] In certain embodiments, the extraction methods of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum, and Agrimoniae scabra include but are not limited to density gradient centrifugation, ultracentrifugation, tangential flow filtration, fractional filtration, precipitation, etc.

[0044] In certain embodiments, the extraction of the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconiti Kusnezoffii, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae comprises: taking Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconiti Kusnezoffii, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae respectively, adding a solvent to break the cell walls, and collecting the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconiti Kusnezoffii, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trillii, and Herba Agrimoniae by centrifugation.

[0045] In certain embodiments, the Herba Lycopodii, Bletilla striata, Verbena officinalis, Radix Aconiti Kusnezoffii, Radix Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae, and Herba Agrimoniae are soaked in a solvent before cell wall breaking.

[0046] In certain embodiments, the solvent is PBS buffer.

[0047] In certain embodiments, the Herba Lycopodii, Bletilla striata, Verbena officinalis, Radix Aconiti Kusnezoffii, Radix Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae, and Herba Agrimoniae are soaked in PBS buffer before cell wall breaking.

[0048] In certain embodiments, the cell wall is broken for 8 to 20 minutes until there are no obvious block fragments.

[0049] In certain embodiments, the centrifugation is density gradient centrifugation and / or ultracentrifugation.

[0050] In certain embodiments, the centrifugation is density gradient centrifugation and ultracentrifugation.

[0051] In certain embodiments, the centrifugation is density gradient centrifugation.

[0052] In certain embodiments, the centrifugation is ultracentrifugation.

[0053] In certain embodiments, the centrifugation step comprises:

[0054] (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant;

[0055] (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant;

[0056] (3) Centrifuge at 3000-5000g for 20-40 min and collect the supernatant;

[0057] (4) Centrifuge at 8000-12000g for 50-70min and collect the supernatant;

[0058] (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

[0059] In certain embodiments, the step (1) is centrifugation at 500 g for 10 min, and the supernatant is collected.

[0060] In certain embodiments, the step (2) is centrifugation at 2000 g for 20 min, and collecting the supernatant.

[0061] In certain embodiments, the step (3) is centrifugation at 4000 g for 30 min, and the supernatant is collected.

[0062] In certain embodiments, the step (4) is centrifugation at 10,000 g for 60 min, and the supernatant is collected.

[0063] In certain embodiments, the step (5) is centrifugation at 110,000 g for 70 min to collect the precipitate.

[0064] In certain embodiments, in step (1), the centrifugation uses a 4°C low-speed centrifuge.

[0065] In certain embodiments, in step (2), the centrifugation uses a 4°C low-speed centrifuge.

[0066] In certain embodiments, in step (3), the centrifugation uses a 4°C low-speed centrifuge.

[0067] In certain embodiments, in step (4), the centrifugation uses a 4°C high-speed centrifuge.

[0068] In certain embodiments, in step (5), the centrifugation uses a 4°C ultracentrifuge.

[0069] In certain embodiments, in step (5), the precipitate is resuspended using the solvent.

[0070] In certain embodiments, in step (5), the precipitate is resuspended using PBS buffer.

[0071] The present invention also provides a composition for relieving pain, which comprises the Chinese herbal extracellular vesicle composition described in any one of the above or the Chinese herbal extracellular vesicle composition prepared by the method described above.

[0072] In certain embodiments, the weight ratio of the Chinese herbal extracellular vesicle composition in the composition for relieving pain is 0.1 to 50 parts, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, etc.

[0073] In certain embodiments, the weight ratio of the Chinese herbal extracellular vesicle composition in the composition for relieving pain is 10 to 40 parts.

[0074] In certain embodiments, the weight ratio of the Chinese herbal extracellular vesicle composition in the composition for relieving pain is 10 to 30 parts.

[0075] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 10 parts of a skeleton material, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 1.1 part, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, 5.5 parts, 6 parts, 6.5 parts, 7 parts, 7.5 parts, 8 parts, 8.5 parts, 9 parts, 9.5 parts, 10 parts, etc.; 0.1 to 30 parts of a moisturizing agent, for example, 0 .1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, etc.; and 0.1 to 5 parts of a thickener, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 1.1 part, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc.;

[0076] The skeleton material is selected from one or more of polyacrylic acid, sodium polyacrylate, partially neutralized sodium polyacrylate and polyvinyl alcohol;

[0077] The moisturizing agent is selected from one or more of glycerin, propylene glycol, sodium PCA, 1,3-propylene glycol, butylene glycol, hexylene glycol, 1,2-pentanediol, caprylyl glycol, sorbitol, ethylhexylglycerin, trehalose, betaine, sodium hyaluronate, saccharide isomers, urea, allantoin and panthenol;

[0078] The thickener is selected from one or more of xanthan gum, tara gum, ammonium acryloyldimethyltaurate / VP copolymer, hydroxyethyl acrylate / sodium acryloyldimethyltaurate copolymer, carbomer, guar gum, locust bean gum, cellulose gum and hydroxyethyl cellulose.

[0079] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 2 to 8 parts of a skeleton material, 5 to 30 parts of a moisturizer, and 0.5 to 4 parts of a thickener.

[0080] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 3 to 6 parts of a skeleton material, 10 to 30 parts of a moisturizer, and 1 to 3 parts of a thickener.

[0081] In certain embodiments, the backbone material is selected from sodium polyacrylate.

[0082] In certain embodiments, the humectant is selected from glycerin.

[0083] In certain embodiments, the thickening agent is selected from cellulose gum.

[0084] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 5 parts of a cross-linking agent, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 1.1 part, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3 0.05 parts, 0.15 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1 part, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc.; and 0.1-5 parts of a cross-linking regulator, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc.;

[0085] The cross-linking agent is selected from one or more of aluminum glycolate, aluminum hydroxide and aluminum chloride;

[0086] The cross-linking regulator is selected from one or more of disodium edetate, ethylenediaminetetraacetic acid and tetrasodium edetate.

[0087] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 3 parts of a cross-linking agent and 0.1 to 3 parts of a cross-linking regulator.

[0088] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 2 parts of a cross-linking agent and 0.1 to 2 parts of a cross-linking regulator.

[0089] In certain embodiments, the cross-linking agent is selected from aluminum glyoxylate.

[0090] In certain embodiments, the cross-linking regulator is selected from edetate disodium.

[0091] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 5 parts of a pH regulator, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part, 1 part, 1.1 part, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc.;

[0092] The pH regulator is selected from one or more of tartaric acid, citric acid, phosphoric acid, malic acid and salts of their compounds.

[0093] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.1 to 3 parts of a pH regulator.

[0094] In certain embodiments, the composition for relieving pain further comprises the following components in parts by weight: 0.5 to 3 parts of a pH regulator.

[0095] In certain embodiments, the pH adjusting agent is selected from tartaric acid.

[0096] The present invention also provides the use of any of the above-mentioned extracellular vesicle compositions, or the above-mentioned extracellular vesicle compositions prepared by the above-mentioned method, or any of the above-mentioned compositions for relieving pain in the preparation of products for relieving pain.

[0097] In certain embodiments, the product is used to relieve neck / shoulder / lower back / leg / joint pain.

[0098] In certain embodiments, the dosage form of the product includes oil, emulsion, ointment, paste, paint, gel, aerosol, spray, solution, and liniment.

[0099] In certain embodiments, the use is use of the composition in preparing a product for inhibiting macrophage IL-6 levels.

[0100] In certain embodiments, the application is the application of the composition in the preparation of a product for inhibiting the expression level of macrophage cyclooxygenase COX2 mRNA.

[0101] In certain embodiments, the use is use of the composition in preparing a product for inhibiting macrophage prostaglandin E2 (PGE2) levels.

[0102] The present invention is further illustrated below by means of specific examples. Unless otherwise specified, "%" represents percentage by mass. The materials and reagents used in the following examples, unless otherwise noted, are commonly used in the art and are commercially available or synthesized by known methods. Experimental procedures in the following examples, where conditions are not specified, were generally performed according to conventional experimental conditions or those recommended by the manufacturers of the relevant reagents (kits). Example 1

[0103] Weigh 100 g of ground wormwood, white ginseng, verbena, raw aconite, sophora flavescens, zedoaria, trifoliate green, and agrimony respectively, wash them three times with ultrapure water to remove impurities, and place them in a clean beaker; add 500 mL of sterile PBS respectively, soak overnight at 4°C; discard the water after soaking, wash three times with sterile water, transfer to a wall breaking machine, add 500 mL of sterile PBS, cover tightly, and break the wall for 8-20 min to ensure that there are no obvious block fragments; transfer the squeezed juice to a 50 mL sterile centrifuge tube respectively, centrifuge at 500 × g at 4°C for 10 min, discard the precipitate, and collect the supernatant; centrifuge at 2000 × g at 4°C for 20 min, discard the precipitate, and collect the supernatant; centrifuge at 4000 × g at 4°C for 30 min, discard the precipitate, and collect the supernatant; centrifuge at 10000 × g at 4°C for 1 h, discard the precipitate and collect the supernatant; use a 4℃ ultracentrifuge at 110,000×g for 70 min, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain extracellular vesicle particles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Radix Aconiti Kusnezoffii, Radix Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae, and Herba Agrimoniae (labeled as BSU-Exo, BS-Exo, VA-Exo, KMR-Exo, VSR-Exo, CR-Exo, CBR-Exo, and HAB-Exo, respectively). Example 2

[0104] The extracellular vesicle particles from Example 1, including Herba Lycopodii, Bletilla striata, Verbena officinalis, Radix Aconiti Kusnezoffii, Radix Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae, and Herba Agrimoniae, were compounded in varying weight ratios to produce various herbal extracellular vesicle compositions for pain relief (as shown in Table 1). For short-term use, the compositions can be directly stored in a -80°C freezer. For long-term use and transportation, a lyoprotectant can be added for freeze-drying.

[0105] Table 1

[0106] Example 3

[0107] The Chinese herbal extracellular vesicle composition obtained from Example 2 was added to other raw material components in the formula to prepare 4 sets of gel patches. The specific formula is shown in Table 2 below.

[0108] Table 2

[0109]

[0110] Test Example 1: Skin penetration of Chinese herbal vesicle composition

[0111] Cut the skin of the back or abdomen of an 8-week-old Bama miniature pig into 1 cm x 1 cm sections. Take 1 mg of the Chinese herbal vesicle composition YHZT-001 prepared in Example 2 and adjust the sample volume to 1 mL using Diluent C from the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit MINI26-1KT). Add 6 μL of PKH26 dye from the kit to a test tube containing 1 mL of Diluent C. Mix gently by pipetting for 30 seconds. Let stand at room temperature for 5 minutes. Quench the sample by adding 2 mL of 10% BSA in PBS (Sigma-Aldrich, D8537). Adjust the volume to 30 mL using serum-free medium. Centrifuge at 110,000 g for 2 hours at 2-8°C. Resuspend the extracellular vesicle pellet in 1 mL of serum-free medium by gently pipetting and set aside.

[0112] A 1 cm x 1 cm section of 8-week-old Bama miniature pig skin was placed at 32°C. Then, 50 μL of 1 mg / mL PKH26-labeled herbal vesicle composition YHZT-001 was dripped onto the 1 cm x 1 cm section, evenly covering the entire epidermis. The skin was incubated for 2 hours. After incubation, cryosections were performed at a thickness of 10 μm. Cell nuclei were stained with DAPI, and the cells were observed and photographed using a fluorescence microscope.

[0113] The results show (such as Figure 1 ): The Chinese herbal vesicle composition of the present invention can quickly penetrate into the skin of 8-week-old Bama miniature pigs within 2 hours.

[0114] Test Example 2: The Chinese herbal vesicle composition can reduce the IL-6 level of macrophages (RAW264.7)

[0115] Macrophages were seeded into 96-well plates at a density of 20,000 cells / 200 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 200 μL of blank control reagent (BC: DMEM medium), 200 μL of negative control reagent (NC: DMEM medium), and 200 μL of experimental reagent (extracellular vesicle particles from different sources of YHZT-001 as described in Examples 1 and 2 (diluted in DMEM medium to a protein concentration of 20 μg / mL)) were added to each well. The cells were cultured for 24 hours. The cell culture supernatant was discarded, and 200 μL of blank control reagent (BC: DMEM medium), 200 μL of negative control reagent (NC: DMEM medium containing 1 μg / mL LPS), and 200 μL of experimental reagent (DMEM medium containing 1 μg / mL LPS) were added to each well. The cells were cultured for 6 hours. The supernatant was collected and the secretion of the pro-inflammatory cytokine interleukin-6 (IL-6) was measured using the Lianke Biotechnology inflammatory cytokine interleukin-6 (IL-6) ELISA kit.

[0116] The results showed (Table 3) that BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra) extracellular vesicle particles significantly inhibited macrophage IL-6 levels. The herbal vesicle composition YHZT-001 of the present invention also significantly inhibited macrophage IL-6 levels, and its effect was superior to that of each single BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra).

[0117] Table 3

[0118]

[0119] Test Example 3: The Chinese herbal vesicle composition can reduce the expression level of macrophage cyclooxygenase COX2 mRNA

[0120] Macrophages were seeded into 12-well plates at a density of 500,000 cells / 1000 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 1000 μL of blank control reagent (BC: DMEM medium), 1000 μL of negative control reagent (NC: DMEM medium), and 1000 μL of experimental reagent [extracellular vesicle particles from different sources of YHZT-001 as described in Examples 1 and 2 (diluted in DMEM medium to a protein concentration of 20 μg / mL)] were added to each well and cultured for 24 hours. The cell culture supernatant was discarded, and 1000 μL of blank control reagent (BC: DMEM medium), 1000 μL of negative control reagent (NC: DMEM medium containing 1 μg / mL LPS), and 1000 μL of experimental reagent (DMEM medium containing 1 μg / mL LPS) were added to each well and cultured for 6 hours. The supernatant was discarded, the cells were collected, the RNA of the cells was extracted, and the expression of cyclooxygenase-related genes (COX2) was detected. The results are shown in Table 4.

[0121] The results showed (Table 4) that BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra) extracellular vesicle particles significantly reduced the expression level of the cyclooxygenase COX2 in macrophages. The Chinese herbal vesicle composition YHZT-001 of the present invention also reduced the expression level of the cyclooxygenase COX2 in macrophages, and its effect was superior to that of each single BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra).

[0122] Table 4

[0123]

[0124] Test Example 4: The Chinese herbal vesicle composition can reduce the level of prostaglandin E2 (PGE2) in macrophages

[0125] Macrophages were seeded into 96-well plates at a density of 20,000 cells / 200 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 200 μL of blank control reagent (BC: DMEM medium), 200 μL of negative control reagent (NC: DMEM medium), and 200 μL of experimental reagent [extracellular vesicle particles from different sources of YHZT-001 as described in Examples 1 and 2 (diluted in DMEM medium to a protein concentration of 20 μg / mL)] were added to each well. The cells were cultured for 24 hours. The cell culture supernatant was discarded, and 200 μL of blank control reagent (BC: DMEM medium), 200 μL of negative control reagent (NC: DMEM medium containing 1 μg / mL LPS), and 200 μL of experimental reagent (DMEM medium containing 1 μg / mL LPS) were added to each well. The cells were cultured for 6 hours. The supernatant was collected and PGE2 secretion was measured using a Lianke Bio prostaglandin E2 ELISA kit.

[0126] The results showed (Table 5) that BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra) extracellular vesicle particles significantly inhibited PGE2 levels in macrophages. The Chinese herbal extracellular vesicle composition YHZT-001 of the present invention also significantly inhibited PGE2 levels in macrophages, and its effect was superior to that of each single BSU-Exo (Psoralea corylifolia), BS-Exo (Bletilla striata), VA-Exo (Verbena officinalis), KMR-Exo (Aconiti kusnezoffii), VSR-Exo (Sophora flavescens), CR-Exo (Zhuzhu), CBR-Exo (Sterilhaemoides lanceolata), and HAB-Exo (Agrimonia scabra).

[0127] Table 5

[0128]

[0129] Test Example 5: Test on the Treatment of Pain in Human Body Using Chinese Herbal Vesicle Composition

[0130] Fifty patients aged 30-75 years who suffered from pain or tenderness in the neck, shoulders, waist, legs, joints, etc., and those who had not used analgesics recently or had used analgesics but did not get relief were enrolled and randomly divided into 5 groups. The samples were prepared according to the gel patch of Example 3 of the present invention. They were divided into a blank control group (n=10) and 4 groups of the composition group of the present invention (n=10). According to the internationally commonly used single-dimensional pain intensity assessment scale VRS scoring method (verbal rating scale), the pain level was quantified into 6 levels: no pain, mild discomfort, discomfort, relatively painful / uncomfortable, very painful, and extremely painful. When the patient felt pain, the pain and tenderness level was recorded, and then the analgesic gel was applied to the affected area for 12 hours. After the application, the corresponding pain level was recorded. Each patient used it for 5 consecutive days and recorded his own situation for 5 consecutive days. The test results of the Chinese herbal extracellular vesicle composition for the treatment of pain in humans are shown in Figure 2 , the statistical results of the efficiency are shown in Figure 3 .

[0131] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A Chinese herbal extracellular vesicle composition for external use to relieve pain, characterized in that: The Chinese herbal extracellular vesicle composition comprises: extracellular vesicles of Polygonum multiflorum, extracellular vesicles of Bletilla striata, extracellular vesicles of Verbena officinalis, extracellular vesicles of Aconitum kusnezoffii, extracellular vesicles of Radix Sophorae flavescentis, extracellular vesicles of Curcuma zedoaria, extracellular vesicles of Trigonella ternate and extracellular vesicles of Herba Agrimoniae scabra. The mass ratio of the extracellular vesicles of Rhizoma Corydalis, Rhizoma Bletillae, Rhizoma Verbenae, Rhizoma Aconitum, Rhizoma Sophorae Tonkinensis, Rhizoma Curcumae, Rhizoma Trigonellae and Rhizoma Agrimoniae is 1:1:2:1:1:1:1:2; or, The mass ratio of the extracellular vesicles of Rhizoma Corydalis, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is 1:1:1:1:1:1:1:1:1; or, The mass ratio of the extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum and Agrimoniae scabra is 1:1:2:2:1:2:1:

2.

2. A method for preparing the external herbal extracellular vesicle composition for relieving pain according to claim 1, characterized in that: The preparation method comprises: extracting extracellular vesicles of Polygonum multiflorum, Bletilla striata, Verbena officinalis, Aconitum kusnezoffii, Sophora flavescens, Curcuma zedoaria, Trigonum multiflorum, and Agrimoniae scabra respectively, and then compounding them according to proportion to obtain the Chinese herbal medicine extracellular vesicle composition.

3. The preparation method according to claim 2, characterized in that The extraction of the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trigonellae, and Herba Agrimoniae comprises: taking Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trigonellae, or Herba Agrimoniae, adding a solvent to break the cell walls, and collecting the extracellular vesicles of Herba Lycopodii, Herba Bletillae, Herba Verbenae, Herba Aconitum, Herba Sophorae Tonkinensis, Herba Curcumae, Herba Trigonellae, and Herba Agrimoniae by centrifugation; and / or, The solvent is PBS buffer; and / or, The specific steps of the centrifugation include: (1) Centrifuge at 400-800 g for 5-20 min and collect the supernatant; (2) Centrifuge at 1000-3000 g for 10-30 min and collect the supernatant; (3) Centrifuge at 3000-5000g for 20-40 min and collect the supernatant; (4) Centrifuge at 8000-12000g for 50-70min and collect the supernatant; (5) Centrifuge at 100,000-120,000 g for 60-80 min and collect the precipitate.

4. A composition for external use to relieve pain, characterized in that: The composition is composed of the external herbal extracellular vesicle composition for relieving pain according to claim 1 or the external herbal extracellular vesicle composition for relieving pain prepared by the preparation method according to any one of claims 2 to 3.

5. Use of the external herbal extracellular vesicle composition for relieving pain according to claim 1, the external herbal extracellular vesicle composition for relieving pain prepared by the preparation method according to any one of claims 2 to 3, or the external herbal extracellular vesicle composition for relieving pain according to claim 4 in the preparation of an external pain-relieving product.

6. The use according to claim 5, characterized in that The dosage forms of the product include oil, emulsion, ointment, paste, film coating, gel, aerosol, spray and solution.

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