Use of black garlic extract in the preparation of a product for treating rheumatoid arthritis

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of black garlic extract in the preparation of products for treating rheumatoid arthritis. This invention is the first to demonstrate that black garlic extract can exert its anti-arthritis effect by regulating intestinal flora and its metabolites as direct effector molecules, thereby modulating immune balance. Based on black garlic extract, which is naturally derived, convenient to take orally, and has a clearly defined mechanism, this invention provides a safe, economical, and convenient alternative for the treatment of rheumatoid arthritis, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of black garlic extract in the preparation of products for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by erosive, symmetrical polyarthritis, with its pathogenesis remaining unclear. Current clinical treatment primarily relies on nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and biologics; however, long-term use often leads to increased risk of infection, liver and kidney damage, and high costs. Therefore, further novel interventions are needed.

[0003] Black garlic is a product made by heat-processing the whole bulb of fresh garlic at high temperatures with controlled humidity. Its biological activity is enhanced due to the physicochemical changes caused by enzymatic hydrolysis and non-enzymatic browning reactions such as Maillard reaction, caramelization, and chemical oxidation of polyphenols during heat processing. Currently, there are no studies on the use of black garlic in the treatment of rheumatoid arthritis (RA). Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of black garlic extract in the preparation of products for treating rheumatoid arthritis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of black garlic extract in the preparation of products for treating rheumatoid arthritis.

[0006] A second aspect of the present invention provides a pharmaceutical composition for treating rheumatoid arthritis, said pharmaceutical composition comprising black garlic extract.

[0007] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention is the first to demonstrate that black garlic extract exerts its anti-arthritis effect by regulating the intestinal flora and its metabolites as direct effector molecules, thereby modulating immune balance. Based on black garlic extract, which is naturally derived, conveniently taken orally, and has a clearly defined mechanism, this invention provides a safe, economical, and convenient alternative for the treatment of rheumatoid arthritis, with broad application prospects. Attached Figure Description

[0008] Figure 1 A schematic diagram illustrating the improvement of RA symptoms in CIA mice by BGE; where A represents the degree of swelling in the forepaws and hindaws of mice in different treatment groups; B represents the arthritis score of mice, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0009] Figure 2 BGE inhibited inflammatory cytokines in the serum of CIA mice; where A is the concentration of IL-1β; B is the concentration of IL-6; C is the concentration of TNF-α; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0010] Figure 3 BGE altered the abundance of gut microbiota in mice; where A and B represent α-diversity analysis; C represents β-diversity analysis; *P<0.05.

[0011] Figure 4 BGE altered the abundance of gut microbiota in mice; where A represents changes in phylum-level species abundance (top 10); and B represents changes in genus-level species abundance (top 10).

[0012] Figure 5 The T-test was used to analyze differences in species abundance at the phylum level. Detailed Implementation

[0013] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] Terminology Explanation Black Garlic Extract: Black garlic extract is extracted from the underground bulbs of black garlic, and sulfur-containing compounds are the main bioactive substances.

[0015] Rheumatoid arthritis (RA) is an autoimmune disease with synovitis as its pathological basis, which may eventually lead to joint deformities.

[0016] Collagen-induced arthritis (CIA) best reflects the clinical and pathological features of human rheumatoid arthritis (RA). As an autoimmune disease mediated by endogenous autoantigens, CIA is currently the gold standard model for RA research. Collagen is a component of the extracellular matrix and is classified into type I, type II, and type III, with type II collagen being abundant in articular cartilage. When animals are immunized with heterologous type II collagen (such as bovine CII or chicken CII), an autoimmune response against type II collagen in articular cartilage can be induced.

[0017] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0018] In a typical embodiment of the present invention, the use of black garlic extract in the preparation of products for treating rheumatoid arthritis is provided.

[0019] The active ingredient in the black garlic extract of this invention is S-allyl cysteine ​​(SAC). This invention does not impose special requirements on the content of S-allyl cysteine, nor on the extraction method of the black garlic extract. The content of the extract varies depending on the extraction method, and the black garlic extract generally contains 0.05% to 1% S-allyl cysteine.

[0020] Furthermore, the product has any one or more of the following functions: (1) Reduce joint circumference; (2) Relieve joint swelling; (3) Reduce the level of inflammatory factors; (4) Reduce Firmicutes abundance.

[0021] Furthermore, the inflammatory factors include one or more of IL1β, TNFα, IL6, IL26, IL22, IL20, IL10, IL38, IL33, and IL18.

[0022] Furthermore, the product is a medicine.

[0023] In another typical embodiment of the present invention, a pharmaceutical composition for treating rheumatoid arthritis is provided, the pharmaceutical composition comprising black garlic extract.

[0024] In some embodiments, the pharmaceutical composition also includes other medications for treating rheumatoid arthritis.

[0025] In some embodiments, other medications for treating rheumatoid arthritis include, but are not limited to, celecoxib capsules, ibuprofen, loxoprofen sodium tablets, meloxicam, etoricoxib tablets, diclofenac sodium extended-release tablets, indomethacin tablets, diclofenac sodium extended-release tablets, leflunomide tablets, methotrexate, ellamod, sulfasalazine, tripterygium wilfordii glycoside tablets, glucosamine, chondroitin sulfate, hydroxychloroquine sulfate, etanercept, adalimumab, tocilizumab, secukinumab, prednisolone acetate, methylprednisolone, calcium carbonate D3 tablets, calcitriol, and alendronate sodium.

[0026] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0027] In some embodiments, a pharmaceutically acceptable carrier is used to refer to a material that is compatible with the recipient, preferably a mammal, more preferably a human, and is suitable for delivering the active agent to the target site without terminating the activity of the agent. The toxicity or side effects (if present) associated with the pharmaceutically acceptable carrier are preferably proportionate to a reasonable risk / benefit ratio for the intended use of the active agent.

[0028] In one embodiment, pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. These pharmaceutically acceptable carriers are used as needed to aid in the stability of the formulation or to contribute to its activity or bioavailability, or to produce an acceptable taste or odor when taken orally. Formulations that can be used in such pharmaceutical compositions may be in the form of the original compound itself or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical compositions thus formulated can be administered as needed by any suitable method known to those skilled in the art.

[0029] The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water.

[0030] Adhesives include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose.

[0031] Surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, and glyceryl monostearate.

[0032] Humectants include, but are not limited to, glycerin.

[0033] Adsorption carriers include, but are not limited to, bentonite, silica gel, kaolin and soap clay.

[0034] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0035] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythritol, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0036] Disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0037] In some embodiments, the dosage form of the pharmaceutical composition includes solutions, tablets, powders, suspensions, emulsions, granules, capsules, pills, inhalers, gels, suppositories, creams, gels, and sprays.

[0038] In some embodiments, the route of administration of the pharmaceutical composition includes intravenous administration, oral administration, mucosal administration, nasal administration, rectal administration, subcutaneous administration, intramuscular administration, transdermal administration, or inhalation administration.

[0039] In some embodiments, the medicament of this application can be administered orally. For oral administration, for example, the medicament can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

[0040] In some embodiments, the pharmaceutical composition can be manufactured using methods well-known in the art, such as conventional granulation, mixing, dissolving, encapsulation, lyophilization, or emulsification. The pharmaceutical composition can be formulated in various forms, including but not limited to solutions, tablets, powders, suspensions, emulsions, granules, capsules, pills, inhalers, gels, suppositories, creams, gels, and sprays.

[0041] In some embodiments, solid dosage forms for oral administration may contain binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants, and / or time-delay agents acceptable in human and veterinary pharmaceutical practice.

[0042] Adhesives include gum arabic, gelatin, corn starch, sodium alginate, carboxymethyl cellulose, or polyethylene glycol.

[0043] Sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginate, or agar.

[0044] Diluents include lactose, sorbitol, mannitol, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate.

[0045] Flavorings include peppermint oil, wintergreen oil, cherry, citrus, or raspberry flavorings.

[0046] Coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, corn gluten, shellac, or gluten.

[0047] Preservatives include sodium benzoate, vitamin E, and alpha-carotene. Tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite.

[0048] Lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable delay agents include glyceryl monostearate or glyceryl distearate.

[0049] In some embodiments, the liquid dosage form for oral administration may, in addition to the aforementioned drugs, also include a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides, or mixtures thereof.

[0050] In some embodiments, the suspension for oral administration may further include a dispersant and / or a suspending agent.

[0051] Suspension agents include sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, sodium alginate, or acetylglucosamine.

[0052] Dispersants include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, and polyoxyethylene sorbitol monophosphate. Or two Oleate, stearate or laurate, polyoxyethylene sorbitan mono- or di-oleate, stearate or Laurate esters and analogues.

[0053] In some embodiments, the pharmaceutical composition will vary depending on the desired effect. Therefore, the optimal content of the pharmaceutical composition of this application can be readily determined by those skilled in the art and can be adjusted based on various factors including: the type and severity of the disease, the content of other components in the pharmaceutical composition, the type of formulation, the patient's age, weight, general health condition, sex, and diet, the time of administration, the route of administration, the rate of drug secretion, the duration of treatment, and concurrent medications. The amount of the pharmaceutical composition should be determined taking these factors into account, and it may be administered once daily or in several divided doses. However, it should be understood that the actual dose of the active ingredient should be determined taking into account various relevant factors, including the disease being treated, the severity of the disease, the route of administration, and the patient's weight, age, and sex. Therefore, dosage does not limit the scope of the invention in any way.

[0054] In some implementations, treatment refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and may be performed for prevention or in the course of clinicopathology. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] The black garlic extract used in the examples was purchased from Fufeng Ciyuan Biotechnology Co., Ltd. Its active ingredient is S-allyl cysteine ​​(SAC), with a purity of 0.1%, CAS NO: 21593-77-1, and molecular formula: C6H. 11 NO2S.

[0057] In this example, the black garlic extract is referred to as BGE.

[0058] Example 1: Black garlic extract can improve the inflammatory response and immune regulation of RA. 1. Experimental animals: DBA1 / J mice, purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0059] 2. Experimental grouping, modeling, and drug administration procedures Experimental groups: Blank control group (Ctrl; n=8), no modeling, only the same dose of physiological saline was injected into the feet and tail; CIA model group (CIA+Water; n=7), after successful modeling, the same dose of water was administered by gavage daily as a control; Experimental group (CIA+BGE; n=6), CIA modeling, 200 mg / kg of black garlic extract was administered by gavage once daily.

[0060] Modeling and drug administration procedures: For the first immunization, bovine type II collagen and complete Freund's adjuvant (1:1) were emulsified, and then 100 μL was injected subcutaneously into the base of the tail of mice. Twenty-one days later, a second immunization was administered using bovine type II collagen and incomplete Freund's adjuvant (1:1). The degree of joint swelling in mice was observed and recorded daily. Three days before the second immunization, mice were administered BGE and water by gavage for two consecutive weeks.

[0061] 3. Arthritis score Scoring criteria for each paw: 0 points for normal paws without erythema or swelling; 1 point for erythema and mild swelling at the ankle joint; 2 points for erythema and mild swelling at the ankle to metatarsophalangeal joint and forefoot joint; 3 points for moderate swelling; and 4 points for severe swelling. The scores for each mouse's four limbs are added together to form the mouse arthritis index, with a maximum score of 16 points.

[0062] The results are as follows Figure 1 As shown, compared with the control group, gavage administration of BGE alleviated RA symptoms in CIA mice, including the degree of paw swelling ( Figure 1 A) Arthritis score ( Figure 1 B).

[0063] 4. Expression of inflammatory cytokines Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease. To investigate whether BGE can alleviate the inflammatory response in CIA mice, mouse serum was collected, and the expression of inflammatory cytokines in the serum was detected using an ELISA kit (Linke Biotech: IL6, EK206; TNFα, EK282; IL1β, EK201B). The procedure is as follows: Add 50 μL of serum and standard solution to each well of the ELISA plate coated with the corresponding antibody. Incubate at 37°C and 200 rpm on a shaker for 1 h. Discard the liquid, wash 5 times with PBST, pat dry, add HRP-labeled secondary antibody, incubate at 37°C and 200 rpm on a shaker for 30 min. Discard the liquid, wash 5 times with PBST, pat dry, add 100 μL of chromogenic solution, incubate at 37°C for 5 min, observe the color change, add 50 μL of stop solution, and immediately perform detection under an OD450nm microplate reader.

[0064] The results showed that, compared with the control group, serum IL-1β ( Figure 2 A), IL-6 ( Figure 2 B), TNF-α ( Figure 2 C) The expression of inflammatory cytokines in serum decreased significantly. The above results indicate that BGE can significantly inhibit the expression of inflammatory cytokines in serum and improve the RA symptoms in CIA mice.

[0065] Example 2: Black garlic extract alleviates RA symptoms based on gut microbiota To investigate whether BGE works by regulating the gut microbiota, we performed 16S RNA sequencing on mouse feces (primer sequences F: GTGCCAGCMGCCGCGGTAA (SEQ ID NO:1), R: GGACTACHVGGGTWTCTAAT (SEQ ID NO:2)). Genomic DNA was extracted from mouse fecal samples. After the DNA samples passed amplification and testing, the PCR products were mixed and purified. The library preparation process, including end repair, A-tailing, sequencing adapter addition, and purification, was then performed before sequencing. The raw sequencing data contained a certain proportion of interference data. To ensure more accurate and reliable information analysis, the raw data was first spliced ​​and filtered to obtain valid data. Then, noise reduction was performed using DADA2 on the valid data to obtain the final ASVs. For the obtained ASVs, species annotation was performed on the representative sequence of each ASV to obtain the corresponding species information and abundance distribution based on the species. Simultaneously, abundance and alpha diversity calculations were performed on ASVs to obtain information on species richness and evenness within the samples, as well as shared and unique ASVs among different samples or groups. On the other hand, multiple sequence alignment of ASVs and construction of phylogenetic trees were conducted. Dimensionality reduction analyses such as PCoA and PCA, along with sample clustering tree visualization, could be used to explore differences in community structure among different samples or groups. To further explore the differences in community structure among grouped samples, the T-test statistical analysis method was used to test the significance of differences in species composition and community structure among grouped samples. The study found that, compared with the control group, the α-diversity and β-diversity of the gut microbiota in mice after gavage with BGE were significantly altered (…). Figure 3 A, B, C).

[0066] Further analysis of gut microbiota using species abundance clustering heatmaps revealed that at the apical level after BGE gavage, the abundance of Bacteroidota, Deferribacterota, Proteobacteria, and Cyanobacteria was significantly increased, while the abundance of Firmicutes, Desulfobacterota, and Patescibacteria was significantly decreased. Figure 4 A). At the genus level, compared with the control group, significantly elevated levels were found in Lachnospiraceae_NK4A136_group, Bacteroides, and Odoribacter. Figure 4 B). The above results indicate that BGE can alter the abundance of gut microbiota.

[0067] T-tests revealed that BGE gavage increased Bacteroidota abundance while decreasing Firmicutes abundance. Figure 5 ).

[0068] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of black garlic extract in the preparation of products for the treatment of rheumatoid arthritis.

2. The application as described in claim 1, characterized in that, The black garlic extract contains 0.05% to 1% S-allyl cysteine.

3. The application as described in claim 1, characterized in that, The product has one or more of the following functions: (1) Reduce joint circumference; (2) Relieve joint swelling; (3) Reduce the level of inflammatory factors; (4) Reduce Firmicutes abundance.

4. The application as described in claim 3, characterized in that, The inflammatory factors include one or more of IL1β, TNFα, IL6, IL26, IL22, IL20, IL10, IL38, IL33, and IL18.

5. The application as described in claim 1, characterized in that, The product is a medicine.

6. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that, The pharmaceutical composition includes black garlic extract.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also includes other medications for treating rheumatoid arthritis.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable... The carrier of the received.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage forms of the pharmaceutical composition include solutions, tablets, powders, suspensions, emulsions, granules, capsules, pills, inhalers, gels, suppositories, creams, gels, and sprays.

10. The pharmaceutical composition according to claim 6, characterized in that, The drug composition can be administered via intravenous, oral, mucosal, nasal, or rectal routes.

Images