Stem cell-traditional Chinese medicine compound and application thereof in acupoint injection

Abstract

The invention relates to a stem cell-traditional Chinese medicine compound and application of the stem cell-traditional Chinese medicine compound in acupoint injections. The stem cell-traditional Chinese medicine compound is prepared from the following raw materials: radix astragali seu hedysari extract, mesenchymal stem cells, human serum albumin, auxiliary materials and a solvent. As the mesenchymal stem cells can be homed to the damaged part after being injected into the body through the acupoints, the problem that the homing efficiency is not high due to the fact that the stem cells are intercepted by lung capillaries after being injected into the vein is solved. Under the influence of the local microenvironment of the acupuncture point, the exosome or various growth factors and cytokines can be secreted, the repair and regeneration of damaged tissues are promoted, and the structure and function of the tissues are recovered. The active ingredients of the radix astragali extract maintain the functions of cells and delay senescence by enhancing autophagy, resisting oxidation, resisting inflammation and the like and regulating related signal channels, so that the stem cells in the combined preparation can play a role more enduringly. The human serum albumin can maintain the osmotic pressure of the preparation and play a protection role.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a stem cell-traditional Chinese medicine complex and application thereof in acupuncture injections. Background Art

[0002] Acupoint injection therapy, based on traditional Chinese medicine meridian theory, involves injecting medicinal ingredients into relevant acupuncture points to treat diseases. Over the years, the use of acupoint injection has expanded from Western medicine injections to traditional Chinese medicine injections, and the injection sites have evolved from localized injections to acupoints. This technique aims to combine the efficacy of traditional Chinese medicine with the convenience of modern drug delivery methods, achieving rapid onset of action.

[0003] Currently, traditional Chinese medicine injections are primarily used for anti-inflammatory, analgesic, and microcirculatory improvement. For example, Shuxuening Injection is used to treat osteoarthritis, and Shuanghuanglian Injection is used for infection. Technological improvements in newer products focus on quality control in areas such as extraction, purification, and formulation stability, but there is a lack of research focused on enhancing efficacy and expanding therapeutic areas. Summary of the Invention

[0004] The purpose of the present invention is to disclose a stem cell-traditional Chinese medicine complex and its application in acupuncture injection, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] The first aspect of the present invention is to provide a stem cell-traditional Chinese medicine complex.

[0006] The second aspect of the present invention is to provide application directions of the stem cell-traditional Chinese medicine complex described in the first aspect of the present invention.

[0007] The raw materials of the stem cell-traditional Chinese medicine complex described in the first aspect of the present invention include mesenchymal stem cells, astragalus extract, human serum albumin, excipients and solvents. Since the mesenchymal stem cells can home to the damaged part after acupuncture injection into the body, the problem of low homing efficiency caused by the stem cells being intercepted by the pulmonary capillaries after intravenous injection is avoided. Under the influence of the local microenvironment of the acupuncture points, they can secrete exosomes or various growth factors and cytokines, promote the repair and regeneration of damaged tissues, and restore the structure and function of tissues. The active ingredients of the astragalus extract maintain cell function and delay aging by enhancing autophagy, anti-oxidation, anti-inflammatory and other effects and regulating related signal pathways, so that the stem cells in the combined preparation can play a more lasting role. The human serum albumin can maintain the osmotic pressure of the preparation and play a protective role.

[0008] In a further application embodiment, the method for preparing mesenchymal stem cells comprises the steps of: (1) Wash the umbilical cord tissue with a cleaning solution containing physiological saline, gentamicin sulfate, amphotericin, and red blood cell lysis solution; (2) The Wharton's gel in the cleaned umbilical cord tissue was separated and minced, and then digested with a digestion solution containing collagenase I, DNase, and Tryple Express enzyme. After digestion, the pellet was centrifuged and resuspended to obtain an umbilical cord mesenchymal stem cell suspension; (3) adding the umbilical cord mesenchymal stem cell suspension to a complete culture medium containing gentamicin, amphotericin B, and cytokines, culturing and passage; (4) The cells subcultured to the P1 generation are cultured in a hydrogel culture medium, wherein the hydrogel culture medium includes a matrix component and a cross-linking component, wherein the matrix component includes, by weight, 1-10% gelatin, 0.5-5% sodium alginate, 1-6% γ-polyglutamic acid, and 80-95% distilled water, and the cross-linking component is transglutaminase.

[0009] The above preparation method can place the stem cells in a three-dimensional culture environment during the culture process, and the obtained mesenchymal stem cells have higher stemness.

[0010] In a further application embodiment, the mesenchymal stem cells are stem cells cultured to passage P5.

[0011] In a further embodiment, the final concentration of the mesenchymal stem cells is 1×10 7 ~1×10 8 cells / mL.

[0012] In a further application embodiment, the preparation method of the astragalus extract comprises the steps of: 1) Wash and decoct Astragalus root three times, 1-2 hours each time. Combine the decoctions and filter. 2) Concentrate the filtrate to 50-100% of the original weight of the astragalus root, precipitate with ethanol, and refrigerate. 3) Recover the ethanol and concentrate it to 10% of the original weight of Astragalus; 4) Dilute with water to 100-150% of the original weight of the astragalus root and refrigerate. 5) Filter and concentrate to 15-20% of the original weight of Astragalus; 6) Adjust the pH to 7.5, heat to boiling, add activated carbon, boil for 5 minutes and filter; 7) Dilute with water to 50% of the original weight of the Astragalus root, filter, adjust the pH to 7.5, seal, and sterilize.

[0013] The astragalus extract contains active ingredients such as flavonoids, saponins, polysaccharides, and various amino acids, and has the effects of bidirectionally regulating immune responses, delaying cell aging, and providing anti-inflammatory, anti-oxidative, and tissue repair benefits. Existing astragalus injections are primarily suitable for viral myocarditis, heart failure, and hepatitis. The astragalus extract and the mesenchymal stem cells can act synergistically, allowing the stem cell-traditional Chinese medicine complex, when used as an injection, to nourish qi and calm the mind, achieving the effect of intervening in sub-health fatigue.

[0014] In a further application embodiment, the astragaloside concentration of the astragalus extract is ≥0.08 mg / mL.

[0015] In a further application embodiment, the final mass concentration of the human serum albumin is 5-10%.

[0016] In a further embodiment, the excipients include at least one of poloxamer, sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride, calcium chloride, L-malic acid, and sodium hydroxide. The poloxamer exhibits a thermosensitive liquid-gel transition, being liquid at lower temperatures and forming a soft gel at temperatures close to human body temperature. When the stem cell-traditional Chinese medicine complex is used as an injectable injection, the gelled poloxamer allows for slow drug release for sustained absorption by the human body. The remaining excipients can be directly replaced with Compound Electrolyte Injection Type I, Compound Electrolyte Injection Type II, Sodium Potassium Magnesium Calcium Glucose Injection, or Compound Sodium Lactate Glucose Injection.

[0017] In a further embodiment, the final volume concentration of the poloxamer is 10-20%. In a preferred embodiment, the poloxamer is composed of 85% poloxamer 407 and 15% poloxamer 188.

[0018] The second aspect of the present invention involves using the stem cell-traditional Chinese medicine complex to prepare acupuncture injections for human acupuncture. Acupuncture injections combine traditional Chinese medicine meridian theory with modern stem cell technology, providing a new therapeutic approach for traditional Chinese medicine. By stimulating specific acupuncture points, the body's physiological functions can be regulated, achieving the purpose of treating diseases. Traditional Chinese medicine emphasizes individualized treatment. Acupuncture injections can select appropriate acupuncture points based on the patient's specific symptoms and constitution, utilizing stem cells and appropriate traditional Chinese medicine for direct administration, thereby achieving a more personalized and precise treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a photograph of the P0 mesenchymal stem cells in Example 1; Figure 2 This is a photograph of the P5 mesenchymal stem cells in Example 1. DETAILED DESCRIPTION

[0020] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0022] Example 1: Preparation of the stem cell-traditional Chinese medicine complex 1) Preparation of mesenchymal stem cells: 1-1) Cut 15-18 cm of sterile umbilical cord tissue, rinse the surface with cleaning solution, cut into 2-3 cm lengths, and place in a 10 cm diameter culture dish. Add 75% alcohol to the culture dish and soak for 1-2 minutes. Then, remove the umbilical cord tissue and place it in a culture dish filled with clean cleaning solution. Squeeze the blood vessels in the umbilical cord tissue until the blood inside the vessels is cleaned. The cleaning solution is prepared from normal saline, gentamicin sulfate at a concentration of 25 μg / mL, amphotericin B at a concentration of 5 μg / mL, and red blood cell lysis solution at a volume percentage of 5%, with the remainder being normal saline.

[0023] 1-2) Remove the vein and artery of the umbilical cord, separate the Wharton's jelly, transfer the Wharton's jelly to a 50 mL centrifuge tube, and cut it into 1-2 mm pieces. 3 The tissue fragments were divided into 2 g, and an equal volume of digestion solution was added. The fragments were digested for 30 minutes, centrifuged at 4°C for 5 minutes at a speed of 1000 rpm, the supernatant was removed, the precipitate was retained, and culture medium was added to the precipitate for resuspending to obtain an umbilical cord mesenchymal stem cell suspension; the digestion solution contained 0.2 mg / mL collagenase I, 0.025 mg / mL DNase, and 40% Tryple Express enzyme.

[0024] 1-3) Add 20 mL of complete culture medium containing dual antibodies to the umbilical cord mesenchymal stem cell suspension, mix thoroughly, and aliquot into T75 culture medium, labeled as P0. Culture in an incubator. Change the culture medium every 5 days with complete culture medium containing dual antibodies. The incubator temperature is 37°C, saturated humidity, and a CO2 concentration of 5%. The complete culture medium contains 0.25 μg / mL gentamicin, 0.5 μg / mL amphotericin B, and 10 wt% cytokines. A photo of P0 mesenchymal stem cells during culture medium change is shown below. Figure 1 shown.

[0025] 1-4) Culture P1 stem cells on 3D culture matrix and prepare a density of 5×10 5cells / mL, and the final concentration of the cell suspension was 5×10 4 cells / mL, added 50 g / L mTG enzyme solution for cross-linking, and then cultured at 37°C until P5. Figure 2 shown.

[0026] 1-5) The mesenchymal stem cells at passage P5 were collected and resuspended in compound electrolyte injection type I, and 10% human serum albumin for injection was added to prepare a cell concentration of 5×10 7 cells / mL.

[0027] 2) Preparation of Astragalus Extract: 2-1) Wash 2000 g of Astragalus membranaceus, decoct in water three times for 1.5 hours each time, combine the decoctions, and filter.

[0028] 2-2) Concentrate the filtrate to 1000-2000 mL and precipitate it twice with ethanol, with the first solution containing 75% ethanol and the second solution containing 85% ethanol. Keep refrigerated each time.

[0029] 2-3) Recover the ethanol and concentrate to 200 mL.

[0030] 2-4) Dilute with water to 2000-3000 mL and refrigerate for 12 hours.

[0031] 2-5) Filter and concentrate to 300-400 mL, then allow to cool to room temperature.

[0032] 2-6) Adjust the pH to 7.5 with 20% sodium hydroxide solution, heat to boiling, add 0.375-0.5g activated carbon, boil for 5 minutes, and filter while hot.

[0033] 2-7) Add water for injection to a volume of 1000 mL, filter, adjust the pH to 7.5 with 20% sodium hydroxide solution, filter, seal, and sterilize.

[0034] 3) Preparation of the compound: 3-1) Weigh 2.5 g of pharmaceutical-grade poloxamer and dissolve it in 10 mL of Compound Electrolyte Injection Type I. Sterile filter the solution through a 0.22 μm filter and set aside.

[0035] 3-2) Take 20 mL of the Astragalus extract, add 8 mL of the poloxamer solution prepared in step 3-1), and mix thoroughly.

[0036] 3-3) Take 8 mL of the mesenchymal stem cell stock solution, add it to the mixed solution prepared in step 3-2), supplement it with 4 mL of compound electrolyte injection type I, and mix thoroughly to obtain the stem cell-traditional Chinese medicine complex.

[0037] Example 2: Optimizing the sustained-release effect of the stem cell-traditional Chinese medicine complex The poloxamer in the excipient is a temperature-sensitive material that can be converted into a hydrogel state at a specific temperature, thereby achieving the effect of sustained release of the active ingredient.

[0038] According to the preparation method provided in Example 1, based on a final concentration of 20% of the poloxamer, different ratios of poloxamer 407 and poloxamer 188 were mixed to form the poloxamer, and the phase transition critical temperature was measured.

[0039] Determination Method: 1 mL of each complex was plated on the bottom of a 10 mL Erlenmeyer flask. The flask was sealed and placed in a water bath with a digital thermometer. The water bath was set to a heating rate of 0.1°C / minute. The flask was tilted slightly manually. The desired phase transition temperature was recorded when the complex stopped flowing and formed a gel. The results are shown in Table 1.

[0040] Table 1 Phase transition temperatures of Poloxamer 407 and Poloxamer 188

[0041] The phase transition temperature of the combination of 17% poloxamer 407 and 3% poloxamer 188 is 32.1°C, which is closest to the human body temperature of 32°C under normal conditions. Therefore, the selection of 85% poloxamer 407 and 15% poloxamer 188 to form the poloxamer can achieve the best drug sustained-release effect.

[0042] Example 3: Control experiment of the stem cell-traditional Chinese medicine complex as acupuncture injection for human body 1) Grouping: Using the Fatigue Self-Assessment Scale (FSAS) developed by the Chinese Association of Traditional Chinese Medicine as an assessment tool, 80 volunteers with FSAS scores ≥ 30 for one month were recruited. They were randomly divided into groups of 20 into experimental, control 1, control 2, and control 3 groups.

[0043] 2) Experimental methods: Volunteers in the experimental group received the stem cell-TCM complex described in Example 1 as an injection, administered at the Zusanli acupoint. Volunteers in the control group 1 received an Astragalus injection (Shenwei Pharmaceutical Group Co., Ltd., National Medicine Approval No. Z13021000), administered at the Zusanli acupoint. Volunteers in the control group 2 received a stem cell injection, also administered at the Zusanli acupoint. The stem cell injection was prepared by replacing the Astragalus extract described in step 3-2) with Compound Electrolyte Injection Type I during the preparation of the stem cell-TCM complex described in Example 1. Volunteers in the control group 3 received the stem cell-TCM complex as an injection, administered intramuscularly into the gluteus maximus muscle.

[0044] The Zusanli acupoint location requirements were based on the "National Standard of the People's Republic of China: Acupoint Names and Locations," and the injection procedure was based on the "(GB / T21709.6-2008) Acupuncture Technical Operation Specifications Part 6: Acupoint Injection." Injections were administered bilaterally, once every other day, with 1 mL injected each time. The intervention period lasted three times per week for two weeks. FSAS assessments were performed before and one month after treatment. The one-month overall fatigue relief rate was used as the outcome measure. A score less than 0 on day 31 minus the overall fatigue score on day 0 was considered relief; otherwise, no relief was considered. The results are shown in Tables 2 and 3.

[0045] Table 2. Comparison of effective rates

[0046] Results: Using SPSS software analysis, the total effective rate of the experimental group was significantly higher than that of the control group 1 (X 2 =4.286, P=0.038<0.05); the total effective rate of the experimental group was significantly higher than that of the control group 2 and control group 3 (X 2 =5.584, P=0.018<0.05).

[0047] Table 3. Comparison of scores before and after the experiment

[0048] Results: SPSS software analysis showed that the scores of the experimental group were significantly different before and after treatment.

[0049] The Shenwei Astragalus Injection used in Control Group 1 achieves a synergistic effect of "tonifying Qi, strengthening the body's health, and unblocking the meridians" through multi-target regulation. It is a representative treatment for cardiovascular and cerebrovascular diseases, immune disorders, and organ dysfunction using integrated Chinese and Western medicine. However, its effectiveness in alleviating sub-health conditions when used alone was far lower than that of the experimental group. The effectiveness of Control Group 2 in alleviating sub-health conditions was also far lower than that of the experimental group. This suggests that the Astragalus extract and mesenchymal stem cells work synergistically, resulting in a longer-lasting efficacy and effective repair of damaged body parts.

[0050] The control group 3 received the same stem cell-TCM complex as the experimental group, but the injection site was the gluteus maximus, a common site for intramuscular injections. The control group's efficacy rate was also significantly lower than that of the experimental group. This demonstrates that acupuncture injections stimulating the Zusanli acupoint can enhance drug efficacy, consistent with the acupoint "reservoir" effect and the acupoint-drug effect of drug "amplification."

[0051] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A stem cell-traditional Chinese medicine complex, characterized in that: The raw materials include mesenchymal stem cells, astragalus extract, human serum albumin, excipients and solvents.

2. The stem cell-traditional Chinese medicine complex according to claim 1, characterized in that: The method for preparing mesenchymal stem cells comprises the steps of: (1) Wash the umbilical cord tissue with a cleaning solution containing physiological saline, gentamicin sulfate, amphotericin, and red blood cell lysis solution; (2) The Wharton's gel in the cleaned umbilical cord tissue was separated and minced, and then digested with a digestion solution containing collagenase I, DNase, and Tryple Express enzymes. After digestion, the pellet was centrifuged and resuspended to obtain an umbilical cord mesenchymal stem cell suspension; (3) adding the umbilical cord mesenchymal stem cell suspension to a complete culture medium containing gentamicin, amphotericin B, and cytokines, culturing and passage; (4) The cells subcultured to the P1 generation are cultured in a hydrogel culture medium, wherein the hydrogel culture medium includes a matrix component and a cross-linking component, wherein the matrix component includes, by weight, 1-10% gelatin, 0.5-5% sodium alginate, 1-6% γ-polyglutamic acid, and 80-95% distilled water, and the cross-linking component is transglutaminase.

3. The stem cell-traditional Chinese medicine complex according to claim 2, characterized in that: The mesenchymal stem cells are stem cells cultured and passaged to P5.

4. The stem cell-traditional Chinese medicine complex according to claim 3, characterized in that: The final concentration of the mesenchymal stem cells was 1×10 7 ~1×10 8 cells / mL.

5. The stem cell-traditional Chinese medicine complex according to claim 1, characterized in that: The preparation method of the astragalus extract comprises the steps of: 1) Wash and decoct Astragalus root three times, 1-2 hours each time. Combine the decoctions and filter. 2) Concentrate the filtrate to 50-100% of the original weight of the astragalus root, precipitate with ethanol, and refrigerate. 3) Recover the ethanol and concentrate it to 10% of the original weight of Astragalus; 4) Dilute with water to 100-150% of the original weight of the astragalus root and refrigerate. 5) Filter and concentrate to 15-20% of the original weight of Astragalus; 6) Adjust the pH to 7.5, heat to boiling, add activated carbon, boil for 5 minutes and filter; 7) Dilute with water to 50% of the original weight of the Astragalus root, filter, adjust the pH to 7.5, seal, and sterilize.

6. The stem cell-traditional Chinese medicine complex according to claim 5, characterized in that: The astragaloside concentration of the astragalus extract is ≥0.08 mg / mL.

7. The stem cell-traditional Chinese medicine complex according to claim 1, characterized in that: The final concentration of the human serum albumin is 5-10%.

8. The stem cell-traditional Chinese medicine complex according to claim 1, characterized in that: The auxiliary material comprises at least one of poloxamer, sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride, calcium chloride, L-malic acid and sodium hydroxide.

9. The stem cell-traditional Chinese medicine complex according to claim 8, characterized in that: The final volume concentration of the poloxamer is 10-20%. Preferably, the poloxamer is composed of 85% poloxamer 407 and 15% poloxamer 188.

10. Use of the stem cell-traditional Chinese medicine complex according to any one of claims 1 to 9 in the preparation of acupuncture injections for human acupoints.

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