Medicated bath traditional Chinese medicine composition for treating rheumatoid arthritis and application thereof

Abstract

The invention relates to a medicated bath traditional Chinese medicine composition for treating rheumatoid arthritis and application thereof. The medicated bath traditional Chinese medicine composition is prepared by fermenting Tibetan medicine Wuwei mannan composed of Tibetan ephedra, juniperus formosana, artemisia sieversiana, rhododendron anthopogonoide and myricaria laxiflora with compound bacteria composed of lactobacillus plantarum and saccharomyces cerevisiae or compound bacteria composed of rhizopus oryzae and monascus. The fermented medicated bath composition has the remarkable treatment effects of reducing joint inflammation, improving the pain threshold value, reducing the swelling rate and the inflammation level and achieving rheumatoid arthritis.

Description

Technical Field

[0001] The invention relates to the field of Chinese medicine fermentation, and in particular to a Chinese medicine bath composition for treating rheumatoid arthritis and application thereof. Background Art

[0002] Rheumatoid arthritis is a chronic autoimmune disease with an increasing global incidence. Patients often experience synovial hyperplasia, cartilage destruction, and systemic inflammatory responses. Traditional treatments rely on nonsteroidal anti-inflammatory drugs and immunosuppressants, but long-term use can easily cause side effects such as gastrointestinal reactions and liver and kidney damage. Although biological agents have made certain progress in recent years, their high costs and potential infection risks limit their scope of application. Therefore, the development of new treatments with multi-target regulatory effects and low toxicity and side effects has become a key issue that needs to be urgently addressed in clinical practice.

[0003] Tibetan medicine bath therapy, as a treasure of ethnic medicine, has accumulated a lot of clinical experience in the treatment of rheumatic diseases. Among them, the medicinal bath prescriptions with Tibetan ephedra, juniper and other five-flavor nectar are the most widely used. However, the traditional preparation process mostly uses the water boiling extraction method, which has the defects of low dissolution rate of active ingredients and difficulty in transdermal absorption of large molecular substances. Studies have shown that the bioavailability of flavonoids and terpenes in unfermented medicinal materials is less than 40%, and high-temperature decoction is prone to degradation of heat-sensitive components, which directly affects the anti-inflammatory and analgesic effects. This has become the main technical bottleneck restricting the improvement of the efficacy of traditional medicinal baths.

[0004] The improvement of existing medicinal bath preparations is mostly focused on enzymatic assisted extraction or single strain fermentation process. However, such methods have the problems of single strain metabolic spectrum and low conversion efficiency of secondary metabolites, making it difficult to achieve multi-component synergistic enhancement. In particular, there is a lack of effective regulation on the directional conversion of phenylethanol glycosides and alkaloid components in the special matrix of Tibetan medicine, and the traditional fermentation process is prone to produce organic solvent residues, affecting the safety of the preparation. Therefore, the development of a new composite strain synergistic fermentation system, through the complementary effect of the metabolic network of multiple strains to increase the concentration of active ingredients and reduce toxic byproducts, and improve the therapeutic effect has become an important research direction to break through the existing technical barriers. Summary of the invention

[0005] In order to solve the above problems, the present invention first provides a medicinal bath Chinese medicine composition for treating rheumatoid arthritis, which is characterized by: the Tibetan medicine five-flavor nectar composed of Tibetan ephedra, juniper, Artemisia grandiflora, Rhododendron australis and water cypress branches is prepared by composite microbial fermentation; the composite microorganism is selected from a combination of Lactobacillus plantarum and Saccharomyces cerevisiae, or a combination of Rhizopus oryzae and Monascus, and the active ingredients of the medicinal materials are transformed through the synergistic metabolism of multiple strains, thereby significantly improving the anti-inflammatory and analgesic effects.

[0006] In certain embodiments, the composite microbial combination comprises any one of the following forms: (1) a mixed strain of Lactobacillus plantarum and Saccharomyces cerevisiae, with the seed liquid volume ratio of the two being 1:1, and the total inoculation amount being 5% of the volume of the extract; (2) a mixed strain of Rhizopus oryzae and Monascus purpurogenus, with the seed liquid volume ratio of the two being 1:1, and the fermentation temperature and dissolved oxygen content being controlled in stages; (3) a directional fermentation system of a composite bacterial community in koji.

[0007] The present invention also provides a preparation method, which comprises the following steps: (1) preparation of extract: mixing and grinding the five-flavor nectar medicinal materials of equal mass, passing through a No. 4 sieve, soaking with purified water at a ratio of 1:5 (mass to volume ratio) for 12 hours, sterilizing by steam sterilization at 100° C. for 40 minutes, and obtaining a sterile extract after cooling; (2) composite bacteria fermentation: inoculating composite microbial seed liquid into the extract, fermenting at 25-37° C. for 48-96 hours, optimizing the generation of metabolites by pH control (4.5-5.5) or staged temperature / dissolved oxygen control (30° C. standing + 28° C. stirring); (3) post-fermentation treatment: inactivating the fermentation liquid at 100° C. for 10 minutes, coarse filtering through a 200-mesh filter cloth and fine filtering through a 0.22 μm microporous filter membrane in sequence, low-temperature vacuum concentration to contain ≥30% solids, adding 1% natural menthol and 0.5% natural tea polyphenols, and obtaining a medicinal bath concentrate.

[0008] Finally, the present invention provides an application, which is: the application of the medicinal bath Chinese medicine composition in the preparation of an external preparation for treating rheumatoid arthritis, which reduces the levels of serum IL-1β and TNF-α inflammatory factors, increases the pain threshold, and inhibits the joint swelling rate, thereby achieving multi-target synergistic treatment.

[0009] In certain embodiments, the medicinal bath concentrate is diluted in 38°C warm water at a ratio of 1:10 (v / v) and immersed in water for 30 minutes daily for 28 consecutive days, which can significantly improve the pathological damage of joints in mice with collagen-induced arthritis.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] For the first time, a technical solution for the synergistic fermentation of Wuwei Ganlu and composite bacteria was proposed. This solution promotes the hydrolysis of glycosides through the joint action of Lactobacillus plantarum and yeast; the esterase and oxidase of Rhizopus oryzae and Monascus purpureus synergistically catalyze the conversion of flavonoid glycosides and terpenoid compounds in the medicinal materials into small molecular active substances, optimize the composition of Tibetan medicine Wuwei Ganlu, and improve its medicinal value and bioavailability. On the other hand, the use of microbial fermentation technology instead of traditional chemical extraction methods not only meets the requirements of green pharmaceutical manufacturing but also achieves the goal of being more environmentally friendly and safe. In addition, in the rheumatoid arthritis model, the therapeutic effect of the Tibetan medicine Wuwei Ganlu synergistically fermented by composite bacteria is significantly better than that of the traditional Tibetan medicine Wuwei Ganlu decoction, and the efficacy is equivalent to methotrexate but without liver and kidney toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Changes in arthritis index scores of mice in each group at different times after treatment.

[0013] Figure 2 Changes in tail-flick latency of mice in each group at different times before and after treatment.

[0014] Figure 3 Changes in paw swelling rate of mice in each group at different times before and after treatment.

[0015] Figure 4 Comparison of relative concentrations of inflammatory factors IL-1β and TNF-α in the serum of mice in each group. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below in conjunction with specific embodiments and drawings.

[0017] Example 1 Chinese medicinal composition of Tibetan medicine Wuwei Ganlu fermented by koji

[0018] Step 1: Weigh 200g of Tibetan ephedra, 200g of juniper, 200g of Artemisia grandis, 200g of Rhododendron serrata, and 200g of water cypress branches, grind them and pass through a No. 4 sieve to prepare Wuwei Ganlu powder. Mix the Wuwei Ganlu powder with pure water at a ratio of 1:5 (mass to volume ratio), soak for 12 hours, and allow the medicinal materials to fully absorb water and swell. Spread the soaked medicinal materials flat in a steamer, steam sterilize at 100°C for 40 minutes, and cool to room temperature to obtain Wuwei Ganlu extract.

[0019] Step 2: Grind the koji (purchased from Angel Yeast Co., Ltd.) into powder, add 30°C warm water (containing 2% glucose) at a ratio of 1:10, and let it stand for activation for 2 hours. The microbial concentration of the koji suspension after activation is ≥1×107CFU / mL. Inoculate the activated koji liquid into the five-flavor nectar extract at a 5% inoculation rate, let it stand for fermentation at 30°C for 96 hours, and after the fermentation is completed, inactivate it at 100°C for 10 minutes to obtain the fermentation liquid.

[0020] Step 3: coarsely filter the fermentation liquid through a 200-mesh filter cloth to remove the drug residue; further finely filter through a 0.22 μm microporous filter membrane to remove microbial cells and suspended particles to obtain a fine filtrate, and concentrate the fine filtrate under low-temperature vacuum at 40° C. and -0.08 MPa to 1 / 5 of the original volume to obtain a concentrated solution (containing ≥30% solids); add natural menthol (to improve skin permeability) at 1% of the volume of the concentrated solution, stir evenly, and finally add 0.5% natural tea polyphenols as a preservative to finally prepare a fermented medicinal bath composition I.

[0021] Example 2: Chinese medicinal composition of Tibetan medicine Wuwei Ganlu fermented by Lactobacillus plantarum and Saccharomyces cerevisiae

[0022] Step 1: Weigh 200g of Tibetan ephedra, 200g of juniper, 200g of Artemisia grandis, 200g of Rhododendron serrata, and 200g of water cypress branches, grind them and pass through a No. 4 sieve to prepare Wuwei Ganlu powder. Mix the Wuwei Ganlu powder with pure water at a ratio of 1:5 (mass to volume ratio), soak for 12 hours, and allow the medicinal materials to fully absorb water and swell. Spread the soaked medicinal materials flat in a steamer, steam sterilize at 100°C for 40 minutes, and cool to room temperature to obtain Wuwei Ganlu extract.

[0023] Step 2: Take Lactobacillus plantarum and inoculate it in MRS liquid medium, culture it anaerobically at 37°C for 24 hours, and adjust the concentration of the bacterial seed liquid to 1×108CFU / mL; take Saccharomyces cerevisiae and inoculate it in YPD liquid medium, culture it in a shaking incubator (150rpm) at 28°C for 18 hours, and adjust the concentration of the bacterial seed liquid to 1×107CFU / mL. Mix the two bacterial seed liquids in a volume ratio of 1:1, inoculate them into the five-flavor nectar extract at a 5% inoculation amount, and ferment them at 37°C for 96 hours, during which the pH is monitored at 4.5-5.5 every 12 hours. After the fermentation is completed, inactivate them at 100°C for 10 minutes to obtain the fermentation liquid.

[0024] Step 3: coarsely filter the fermentation liquid through a 200-mesh filter cloth to remove the medicinal residue; further finely filter through a 0.22 μm microporous filter membrane to remove microbial cells and suspended particles to obtain a fine filtrate, and concentrate the fine filtrate under low-temperature vacuum at 40° C. and -0.08 MPa to 1 / 5 of the original volume to obtain a concentrated solution (containing ≥30% solids); add natural menthol (to improve skin permeability) at 1% of the volume of the concentrated solution, stir evenly, and finally add 0.5% natural tea polyphenols as a preservative to finally prepare a fermented medicinal bath composition II.

[0025] Example 3: Chinese medicinal composition of Tibetan medicine Wuwei Ganlu fermented by Rhizopus oryzae and Monascus purpurogenus composite bacteria

[0026] Step 1: Weigh 200g of Tibetan ephedra, 200g of juniper, 200g of Artemisia grandis, 200g of Rhododendron serrata, and 200g of water cypress branches, grind them and pass through a No. 4 sieve to prepare Wuwei Ganlu powder. Mix the Wuwei Ganlu powder with pure water at a ratio of 1:5 (mass to volume ratio), soak for 12 hours, and allow the medicinal materials to fully absorb water and swell. Spread the soaked medicinal materials flat in a steamer, steam sterilize at 100°C for 40 minutes, and cool to room temperature to obtain Wuwei Ganlu extract.

[0027] Step 2: Take Rhizopus oryzae and inoculate it in PDA liquid medium, shake and culture it at 30℃ and 120rpm for 48 hours, adjust the spore concentration to 1×106 spores / mL, and prepare a Rhizopus oryzae spore suspension. Take Monascus purpureus in malt juice liquid medium, shake and culture it at 28℃ and 150rpm for 72 hours, the diameter of the mycelium ball is ≤0.5mm, and the biomass reaches 20g / L (wet weight), and prepare Monascus purpureus seed liquid. Mix the Rhizopus oryzae spore liquid and the Monascus purpureus seed liquid in a ratio of 1:1, and inoculate it into the five-flavor nectar extract at a 5% inoculation amount. Ferment at 30℃ for 48h; then continue to ferment at 28℃ for 48h, and maintain 30% saturation by adjusting the stirring rate of 250rpm. After the fermentation is completed, inactivate it at 100℃ for 10 minutes to obtain the fermentation liquid.

[0028] Step 3: coarsely filter the fermentation liquid through a 200-mesh filter cloth to remove the drug residue; further finely filter through a 0.22 μm microporous filter membrane to remove microbial cells and suspended particles to obtain a fine filtrate, and concentrate the fine filtrate under low-temperature vacuum at 40° C. and -0.08 MPa to 1 / 5 of the original volume to obtain a concentrated solution (containing ≥30% solids); add natural menthol (to improve skin permeability) at 1% of the volume of the concentrated solution, stir evenly, and finally add 0.5% natural tea polyphenols as a preservative to finally prepare a fermented medicinal bath composition III.

[0029] Example 4 Study on different fermented medicinal bath compositions for treating rheumatoid arthritis in mouse models

[0030] DBA / 1J male mice, 6-8 weeks old, weighing 18-22g (sensitive to type II collagen induction), were prepared in an SPF environment with a temperature of 22±2℃, a humidity of 50±10%, a 12-hour light-dark cycle, free access to food and water, and the experimental protocol was approved by the Animal Ethics Committee. After the mice adapted to the laboratory environment for 3 days, they were placed in a transparent plexiglass cabin (25×15×10cm) with their tails flat on the heat source area, which was generated by a thermal pain meter (UgoBasile, model 37360) with a heat source temperature set at 52±0.5℃. The latency from the start of the heat stimulus to the tail-flicking escape of the mouse was recorded, and each mouse was tested 3 times with an interval of 10 minutes. Mice with a latency of 5-10 seconds and a standard deviation of ≤1.5 seconds were selected for the experiment, and individuals with hypersensitive reactions (<5 seconds) or slow reactions (>10 seconds) were excluded. Among them, 10 mice were in the normal group, and the remaining 60 mice were used for modeling. Prepare bovine type II collagen (CII, Chondrex, catalog number 20022) dissolved in 0.1M acetic acid to a concentration of 2mg / mL; Freund's complete adjuvant (CFA, Sigma, catalog number F5881), containing 1mg / mL inactivated Mycobacterium tuberculosis. Mix CII solution and CFA in a volume ratio of 1:1, and push and mix 50 times with a double syringe (23G needles connected at both ends) under ice bath conditions until a viscous oil-in-water emulsion is formed. Anesthetize mice (2% isoflurane inhalation), and inject 100μL of emulsifier (containing 100μg CII) subcutaneously at the base of the tail. After injection, massage the local area for 30 seconds to promote the diffusion of the emulsion. On the 7th day of the first immunization, inject 50μL of emulsifier (containing 50μg CII) subcutaneously at the right ankle joint of the mouse. The normal group was anesthetized in the same way at the same time, and an equal amount of normal saline was injected at the same site. On the 21st day of the first immunization, joint inflammation was evaluated. The model was successfully established if the arthritis index AI ≥ 3. After successful modeling, the mice were randomly divided into model group, medicinal bath group I, medicinal bath group II, medicinal bath group III, control group I, and control group II, with 10 mice in each group. The treatments were as follows:

[0031] Normal group: No modeling, treated with physiological saline bath, mice were immersed in water for 30 minutes every day (water depth to the chest), and the intervention lasted for 28 days.

[0032] Model group: Model mice were treated with saline bath. The mice were immersed in water for 30 minutes every day (the water depth reached the chest) for 28 consecutive days.

[0033] Bathing group I: model mice were treated with the Jiuqu fermentation bathing composition I (Example 1) bathing treatment. The concentrate was diluted in 38°C warm water at a ratio of 1:10 (v / v). The mice were immersed in water for 30 minutes every day (water depth to the chest) for 28 consecutive days.

[0034] Bathing group II: model mice were treated with Lactobacillus plantarum + yeast fermentation bathing composition II (Example 2) bathing treatment. The concentrate was diluted in 38°C warm water at a ratio of 1:10 (v / v). The mice were immersed in water for 30 minutes daily (water depth to the chest) for 28 consecutive days.

[0035] Bathing group III: model mice were treated with bathing composition III (Example 3) fermented by Rhizopus oryzae and Monascus purpurogenus. The concentrate was diluted in 38°C warm water at a ratio of 1:10 (v / v). The mice were immersed in water for 30 minutes daily (water depth to the chest) for 28 consecutive days.

[0036] Control group I: model mice, methotrexate (MTX, 1 mg / kg, intraperitoneal injection, twice a week).

[0037] Control group II: Wuwei Ganlu Medicinal Bath Soup Powder provided by Tibet Shenhou Pharmaceutical Co., Ltd. (Tibetan medicine preparation number Z20210291000, batch number: 20231001) was used as a positive control. The mice were treated with a medicinal bath. The Wuwei Ganlu Medicinal Bath Soup Powder was diluted in 38°C warm water. The mice were soaked for 30 minutes every day (the water depth was to the chest) for 28 consecutive days.

[0038] Arthritis Index (AI): From the day of treatment to the 28th day, the arthritis index of each group of mice was scored every 7 days (the total score was the sum of the scores of the four feet). The standards for joint inflammation scoring and counting were: 0 points, no redness and swelling; 1 point, erythema and slight swelling limited to the ankle joint or tarsal bone; 2 points, erythema or slight swelling from the ankle joint to the tarsal joint; 3 points, erythema and moderate swelling from the ankle joint to the metatarsal bone; 4 points, swelling of the entire paw from the ankle joint to the toes. The cumulative score of the four limb joints is the AI ​​score of each mouse, with a maximum of 16 points, see Figure 1 .

[0039] Figure 1 The results showed that the scores of the medicated bath groups I-III and the control groups I-II decreased on the 7th day after the start of treatment (P<0.05); and after the end of treatment, that is, on the 28th day after the start of treatment, the scores of the medicated bath groups I-III and the control groups I-II were lower than those of the model group, and the medicated bath group II-III was significantly lower, and the differences were statistically significant compared with the control group I-II (P<0.01).

[0040] Pain threshold measurement: 1 day before modeling, 7 and 14 days after modeling, 7, 14, 21 and 28 days after treatment. The end 1 / 3 of the mouse tail was placed close to the heat source area, the temperature was 52±0.5℃, and the timing was started at the same time. The latency from the start of heat stimulation to the mouse's tail-flicking escape was recorded. The tail-flicking latency of each mouse was measured 3 times in total, with an interval of more than 20 minutes between each measurement. The average value was calculated. Figure 2 .

[0041] Figure 2 The results showed that from the time of modeling to the start of treatment on the 14th day after modeling, the pain threshold of each group decreased significantly except for the normal group, while that of the normal group increased slightly; after the start of treatment, the pain threshold of each treatment group began to increase, and the pain threshold of the normal group increased to about 8s and then stabilized; the pain threshold of the medicated bath I-III groups was significantly different from that of the model group from the 7th day of treatment (P<0.05), and from the 14th day of treatment until the end of treatment on the 28th day, the pain threshold of the medicated bath I group and the control I-II groups were lower than that of the medicated bath II-III groups, and the difference was statistically significant (P<0.01).

[0042] Mouse paw swelling rate detection: The ankle diameter of each group of mice was measured using an electronic digital caliper, and the swelling rate was calculated: swelling rate (%) = (diameter after intervention - basic diameter) / basic diameter × 100%, see Figure 3 .

[0043] Figure 3 The results showed that from the time of modeling until the start of treatment on the 14th day after modeling, the paw swelling rate of mice in each group, except the normal group, continued to increase, reflecting the progression of uncontrolled inflammation; after the start of treatment, the swelling rate of each group began to decrease, and the swelling rate of mice in the normal group tended to stabilize; the swelling rate of mice in the medicated bath groups I-III decreased significantly compared with the model group starting from the 7th day of treatment, and the rate of decline was significantly better than that of the control groups I-II, indicating the best treatment effect.

[0044] Inflammatory factor detection: After the treatment, blood was collected from the venous plexus of each group of mice, left to stand at room temperature for 30 minutes, centrifuged at 3000rpm for 15 minutes to separate the serum, and stored at -80℃ after packaging. Before testing, the serum samples were thawed on ice and operated according to the instructions of the kit (IL-1β: R&D Systems, catalog number RLB00; TNF-α: R&D Systems, catalog number RTA00). The absorbance (OD value) was measured at a wavelength of 450nm, and the concentrations of IL-1β and TNF-α (pg / mL) were calculated according to the standard curve. Each group of samples had 3 replicate wells, and the mean was taken for analysis. The data were expressed as Indicates, see Figure 4 .

[0045] Figure 4 The results showed that compared with the normal group, the levels of IL-1β and TNF-α in the serum of the model group were significantly increased; compared with the model group, the levels of IL-1β and TNF-α in the serum of the other groups were decreased, and the decrease of IL-1β and TNF-α in the serum of the medicinal bath II-III group was the most significant, which indicates that the fermented medicinal bath composition II-III can relieve joint pain by reducing the expression of inflammatory factors.

[0046] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicinal bath composition for treating rheumatoid arthritis, characterized in that: The medicinal bath Chinese medicine composition is prepared by fermenting Tibetan medicine five-flavor nectar consisting of Tibetan ephedra, juniper, Artemisia grandiflora, Rhododendron serrata and water cypress branches with composite microorganisms; the composite microorganisms are selected from a combination of Lactobacillus plantarum and Saccharomyces cerevisiae, or a combination of Rhizopus oryzae and Monascus purpurogenus.

2. The medicinal bath Chinese medicine composition according to claim 1, characterized in that: The fermentation comprises the following steps: sterilizing the five-flavor nectar extract and inoculating composite microbial seed liquid, fermenting at 25-37° C. for 48-96 hours, filtering and concentrating after inactivation, and adding 1% natural menthol and 0.5% natural tea polyphenols to prepare a medicinal bath concentrate.

3. The medicinal bath Chinese medicine composition according to claim 1, characterized in that: The composite microorganism is a mixed strain of plant lactobacillus and saccharomyces cerevisiae, the volume ratio of the seed liquid of the two is 1:1, the total inoculation amount is 5% of the volume of the extract, the fermentation conditions are standing at 37°C for 96 hours and the pH is controlled at 4.5-5.

5.

4. The medicinal bath Chinese medicine composition according to claim 1, characterized in that: The composite microorganism is a mixed strain of Rhizopus oryzae and Monascus purpureus, the volume ratio of the seed liquid of the two is 1:1, the fermentation process includes standing at 30°C for 48 hours and stirring and fermenting at 28°C for 48 hours, the stirring rate is 250rpm and the dissolved oxygen content is maintained at 30% saturation.

5. The medicinal bath Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The preparation method of the five-flavor nectar extract is as follows: Tibetan ephedra, juniper, Artemisia grandiflora, Rhododendron dasyphylla and water cypress branches are mixed and crushed in equal weight, passed through a No. 4 sieve, soaked in pure water at a mass-to-volume ratio of 1:5 for 12 hours, and sterilized by steam at 100° C. for 40 minutes to obtain the extract.

6. The medicinal bath Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The concentrated solution contains ≥30% solids and is diluted in 38°C warm water at a ratio of 1:10 (v / v) when used to form a medicinal bath solution for treating rheumatoid arthritis.

7. Use of the medicinal bath Chinese medicine composition according to any one of claims 1 to 6 in the preparation of an external preparation for treating rheumatoid arthritis, characterized in that: The application achieves the treatment of rheumatoid arthritis by reducing serum IL-1β and TNF-α levels, increasing pain threshold and inhibiting joint swelling rate.

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