Neutralizing agent composition for microbial limit test of traditional Chinese medicine and application thereof

Abstract

The application discloses a neutralizer composition for microbial limit examination of Chinese patent medicines, a dilution composition containing the neutralizer composition, and application of the dilution composition. Specifically, the neutralizer composition can effectively neutralize bacteriostatic components in Chinese patent medicines such as Dabaidu capsules, and improve the accuracy of microbial detection of Chinese patent medicines. The dilution composition can be used in combination with culture medium dilution to simply and accurately perform microbial limit examination.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical microbiology detection technology, specifically to a neutralizing agent composition for microbial limit testing of traditional Chinese medicine preparations and its application. Background Technology

[0002] Traditional Chinese medicine (TCM) preparations contain a complex and diverse array of ingredients. Many of these heat-clearing and detoxifying TCM preparations contain antibacterial components, such as Dabaidu Capsules, Compound Berberine Tablets, and Pudilan Anti-inflammatory Tablets. Previous in vitro drug sensitivity tests, cell structure studies, and clinical relevance analyses have shown that heat-clearing and detoxifying TCM preparations can achieve broad-spectrum antibacterial activity through multiple antibacterial mechanisms from various herbs. For example, Dabaidu Capsules have antibacterial properties covering bacteria, fungi, viruses, and drug-resistant bacteria, making it a model TCM compound for combating complex infections. Many other similar TCM preparations exist, such as Huanglian Shangqing Pills and Pudilan Anti-inflammatory Tablets.

[0003] The Chinese Pharmacopoeia lists three methods for microbial counting: plate count, membrane filtration, and maximum probable number (MPN) count. The Chinese Pharmacopoeia prioritizes the plate count and dilution method for removing antibacterial activity, followed by the addition of a suitable neutralizing or inactivating agent. The United States Pharmacopoeia (USP) 42-NF37 2S General Chapter <1227> also favors convenient and rapid chemical neutralizing agents to remove interference from the antibacterial activity of pharmaceuticals.

[0004] In routine use, traditional Chinese medicine preparations for clearing heat and detoxifying are mostly tested using the plate test or membrane filtration method. With the plate test, a neutralizing agent is often added to the diluent during sample preparation; with the membrane filtration method, the rinsing solution also contains neutralizing components. The choice between the plate test and membrane filtration method, the addition of neutralizing agents to the diluent and rinsing solution, and the type and amount of neutralizing agent added are all determined by the manufacturer through validation experiments. This results in different manufacturers choosing different validation methods for the same drug, making it impossible to compare the accuracy of the tests.

[0005] In summary, there is an urgent need in this field to develop a simple, safe, and effective method for detecting microbial limits in order to eliminate the antibacterial interference of antibacterial traditional Chinese medicine, thereby improving the detection rate of microbial limits and effectively avoiding false negative results. Summary of the Invention

[0006] The purpose of this invention is to provide a neutralizing agent composition for microbial limit testing of antibacterial traditional Chinese medicine and its application.

[0007] In a first aspect, the present invention provides a neutralizing agent composition for the microbial limit detection of traditional Chinese medicine preparations, comprising: (a) Tween-80, (b) lecithin, (c) glutathione, (d) cyclodextrin and (e) optionally sodium pyruvate, wherein the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin and sodium pyruvate is about 3-10:0.5-4:1-6:40-140:0-5.

[0008] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 3-10:0.5-4:1-6:40-140:1-5.

[0009] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 5-9:1-3:2-4:60-100:1-3.

[0010] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 7:1.5:3:8:2.

[0011] In another preferred embodiment, the neutralizing agent composition comprises: 3-10 g / L Tween-80, 0.5-4 g / L lecithin, 50-200 mM glutathione, 40-140 g / L cyclodextrin, and 1-5 g / L sodium pyruvate.

[0012] In another preferred embodiment, the neutralizing agent composition comprises: 5-9 g / L Tween-80, 1-3 g / L lecithin, 80-150 mM glutathione, 60-100 g / L cyclodextrin and 1-3 g / L sodium pyruvate.

[0013] In another preferred embodiment, the neutralizing agent composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin and 2 g / L sodium pyruvate.

[0014] In another preferred embodiment, the lecithin is soybean lecithin.

[0015] In another preferred embodiment, the traditional Chinese medicine is an antibacterial traditional Chinese medicine.

[0016] In another preferred embodiment, the traditional Chinese medicine is a heat-clearing and detoxifying traditional Chinese medicine.

[0017] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, Compound Berberine Tablets, Niuhuang Jiedu Tablets / Pills, Huanglian Shangqing Pills, Sanhuang Tablets, Yiqing Capsules, Yinqiao Jiedu Tablets, Qingjiang Tablets, Qingkailing Granules, Qingre Jiedu Oral Liquid, Lanqin Oral Liquid, Pudilan Anti-inflammatory Oral Liquid, Bairui Granules, Daochi Pills, and Niuhuang Qingxin Pills.

[0018] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, and Compound Berberine Tablets.

[0019] In another preferred embodiment, the microorganisms detected by the microbial limit test are aerobic bacteria.

[0020] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, Escherichia coli, Salmonella, and bile salt-resistant Gram-negative bacteria.

[0021] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0022] In a second aspect, the present invention provides a diluting composition for microbial limit detection, the diluting composition comprising (i) a diluent and (ii) a neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations as described in the first aspect of the present invention.

[0023] In another preferred embodiment, the diluent is selected from the group consisting of water, buffer solution, physiological saline, and peptone water.

[0024] In another preferred embodiment, the buffer solution is a TSB buffer solution.

[0025] In another preferred embodiment, the buffer solution is a pH 7.0 sodium chloride-peptone buffer solution.

[0026] In another preferred embodiment, the buffer solution contains the following components in weight percentages: 0.5-4% tryptone, 0.1-0.6% soybean papain hydrolysate, 0.1-0.6% beef extract, 0.1-0.6% yeast extract, 0.2-1% sodium chloride, 0.1-0.5% dipotassium hydrogen phosphate, 0.1-0.5% potassium dihydrogen phosphate, 0.002-0.01% CaCl2, and 0.1-0.5% glucose or 0.1-0.5% anhydrous glucose.

[0027] In another preferred embodiment, the buffer solution contains the following components in weight percentages: 1.7% tryptone, 0.3% soybean papain hydrolysate, 0.3% beef extract, 0.3% yeast extract, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.195% potassium dihydrogen phosphate, 0.005% CaCl2, and 0.25% glucose or 0.23% anhydrous glucose.

[0028] In another preferred embodiment, the diluent composition contains the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin, 0.1-0.5% sodium pyruvate, 0.5-4% tryptone, 0.1-0.6% soybean papain hydrolysate, 0.1-0.6% beef extract, 0.1-0.6% yeast extract, 0.2-1% sodium chloride, 0.1-0.5% dipotassium hydrogen phosphate, 0.1-0.5% potassium dihydrogen phosphate, 0.002-0.01% CaCl2, and 0.1-0.5% glucose or 0.1-0.5% anhydrous glucose.

[0029] In another preferred embodiment, the diluted composition contains the following components in weight percentages: 0.7% Tween-80, 0.15% lecithin, 0.307% glutathione, 7.9% cyclodextrin, 0.2% sodium pyruvate, 1.7% tryptone, 0.3% soybean papain hydrolysate, 0.3% beef extract, 0.3% yeast extract, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.195% potassium dihydrogen phosphate, 0.005% CaCl2, and 0.25% glucose or 0.23% anhydrous glucose.

[0030] In another preferred embodiment, the diluting composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin, 2 g / L sodium pyruvate, 17 g / L trypsin, 3 g / L soybean papain hydrolysate, 3 g / L beef extract, 3 g / L yeast extract, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 1.95 g / L potassium dihydrogen phosphate, 0.05 g / L CaCl2, and 2.5 g / L glucose or 2.3 g / L anhydrous glucose.

[0031] In another preferred embodiment, the diluting composition comprises TSB buffer and the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin and 0.1-0.5% sodium pyruvate.

[0032] In another preferred embodiment, the diluting composition comprises a pH 7.0 sodium chloride-peptone buffer and the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin, and 0.1-0.5% sodium pyruvate.

[0033] In another preferred embodiment, the lecithin is soybean lecithin.

[0034] In another preferred embodiment, the traditional Chinese medicine is an antibacterial traditional Chinese medicine.

[0035] In another preferred embodiment, the traditional Chinese medicine is a heat-clearing and detoxifying traditional Chinese medicine.

[0036] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, Compound Berberine Tablets, Niuhuang Jiedu Tablets / Pills, Huanglian Shangqing Pills, Sanhuang Tablets, Yiqing Capsules, Yinqiao Jiedu Tablets, Qingjiang Tablets, Qingkailing Granules, Qingre Jiedu Oral Liquid, Lanqin Oral Liquid, Pudilan Anti-inflammatory Oral Liquid, Bairui Granules, Daochi Pills, and Niuhuang Qingxin Pills.

[0037] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, and Compound Berberine Tablets.

[0038] In another preferred embodiment, the microorganisms detected by the microbial limit test are aerobic bacteria.

[0039] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, Escherichia coli, Salmonella, and bile salt-resistant Gram-negative bacteria.

[0040] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0041] In a third aspect, the present invention provides the use of the neutralizing composition as described in the first aspect of the present invention or the diluting composition as described in the second aspect of the present invention for the detection of microbial limits in traditional Chinese medicine preparations.

[0042] In another preferred embodiment, the traditional Chinese medicine is an antibacterial traditional Chinese medicine.

[0043] In another preferred embodiment, the traditional Chinese medicine is a heat-clearing and detoxifying traditional Chinese medicine.

[0044] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, Compound Berberine Tablets, Niuhuang Jiedu Tablets / Pills, Huanglian Shangqing Pills, Sanhuang Tablets, Yiqing Capsules, Yinqiao Jiedu Tablets, Qingjiang Tablets, Qingkailing Granules, Qingre Jiedu Oral Liquid, Lanqin Oral Liquid, Pudilan Anti-inflammatory Oral Liquid, Bairui Granules, Daochi Pills, and Niuhuang Qingxin Pills.

[0045] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, and Compound Berberine Tablets.

[0046] In another preferred embodiment, the microorganisms detected by the microbial limit test are aerobic bacteria.

[0047] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, Escherichia coli, Salmonella, and bile salt-resistant Gram-negative bacteria.

[0048] In another preferred embodiment, the microbial limit test detects microorganisms selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0049] In a fourth aspect, the present invention provides a method for detecting the microbial limits of traditional Chinese medicine preparations, comprising the following steps: (S1) The traditional Chinese medicine sample is mixed and diluted with the diluting composition as described in the second aspect of the present invention in a certain proportion to obtain the test solution; and (S2) Take the test solution, add culture medium, culture and count the control bacteria.

[0050] In another preferred embodiment, the method further includes a method suitability verification step: (Z1) The test solution is mixed with the positive control bacteria to obtain a mixture, which is then added to a culture medium, cultured, and the positive control bacteria are counted. (Z2) Mix the buffer solution with the positive control bacteria, add to the culture medium, incubate, and count the positive control bacteria; and (Z3) Calculate the strain recovery rate.

[0051] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:2 to 1:50.

[0052] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:10.

[0053] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:50.

[0054] In another preferred embodiment, the traditional Chinese medicine is a heat-clearing and detoxifying traditional Chinese medicine.

[0055] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, Compound Berberine Tablets, Niuhuang Jiedu Tablets / Pills, Huanglian Shangqing Pills, Sanhuang Tablets, Yiqing Capsules, Yinqiao Jiedu Tablets, Qingjiang Tablets, Qingkailing Granules, Qingre Jiedu Oral Liquid, Lanqin Oral Liquid, Pudilan Anti-inflammatory Oral Liquid, Bairui Granules, Daochi Pills, and Niuhuang Qingxin Pills.

[0056] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Compound Berberine Tablets, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, and Gardenia and Golden Flower Pills.

[0057] In another preferred embodiment, the control bacteria are aerobic bacteria.

[0058] In another preferred embodiment, the control bacteria are microorganisms selected from the group consisting of: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, Escherichia coli, Salmonella, and bile salt-tolerant Gram-negative bacteria.

[0059] In another preferred embodiment, the control bacteria are microorganisms selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0060] In another preferred embodiment, the positive control bacteria are aerobic bacteria.

[0061] In another preferred embodiment, the positive control bacteria are selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, or combinations thereof.

[0062] In another preferred embodiment, the culture medium is tryptic soy agar (TSA) or tryptic soy liquid medium.

[0063] In another preferred embodiment, step (S2) is as follows: take 1 ml / plate or 0.2 ml / plate × 5 plates of test solution into a sterile culture dish, add tryptic soy agar (TSA) medium, culture, and count the control bacteria.

[0064] In another preferred embodiment, step (Z1) is as follows: the test solution is mixed with the positive control bacteria for 5-15 minutes to obtain a test solution-bacterial mixture; the test solution-bacterial mixture is taken at 1 ml / plate or 0.2 ml / plate × 5 plates and placed in a sterile culture dish, tryptic soy agar (TSA) medium is added, and the culture is carried out and the positive control bacteria are counted.

[0065] In another preferred embodiment, the bacterial count in the test solution bacterial mixture is less than 100 cfu / ml.

[0066] In another preferred embodiment, step (Z2) is as follows: the buffer solution is mixed with the positive control bacteria for 5-15 minutes to obtain a buffer-bacterial mixture; 1 ml of the buffer-bacterial mixture is taken into a sterile culture dish, tryptic soy agar (TSA) medium is added, and the positive control bacteria are cultured and counted.

[0067] In another preferred embodiment, the bacterial count in the buffer solution mixture is less than 100 cfu / ml.

[0068] In another preferred embodiment, the culture temperature is 25-40°C, more preferably 30-35°C, such as 32.5°C.

[0069] In another preferred embodiment, the culture time is 48-96 hours, more preferably 55-90 hours, even more preferably 60-80 hours, and most preferably 70-75 hours, such as 72 hours.

[0070] In another preferred embodiment, the buffer solution is selected from the group consisting of: 0.9% sodium chloride saline, TSB, or pH 7.0 sodium chloride-peptone buffer.

[0071] In another preferred embodiment, the strain recovery rate is the ratio of the number of colonies A1 counted in step (Z1) to the number of colonies A0 counted in step (Z2), i.e., A1 / A0.

[0072] In another preferred embodiment, the strain recovery rate of the method is 0.5-2.0, more preferably 0.7-2.0, and even more preferably 0.8-1.5.

[0073] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0074] Through extensive and in-depth research and screening, the inventors unexpectedly discovered a neutralizing agent composition for microbial limit testing of traditional Chinese medicine (TCM) preparations, as well as a diluent composition (such as a bio-stabilized broth) containing this neutralizing agent composition. Specifically, the neutralizing agent composition includes Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate. Using this neutralizing agent composition, the antibacterial properties of TCM preparations containing antibacterial components, such as Dabaidu capsules, can be effectively neutralized, resulting in a recovery rate of nearly 1.0 for various low-level challenging microorganisms, especially Staphylococcus aureus and Bacillus subtilis, meeting the requirements of the Chinese Pharmacopoeia (0.5-2). Furthermore, by using the bio-stabilized broth of this invention in conjunction with the culture medium dilution method, many TCM preparations that previously required membrane filtration to eliminate antibacterial properties can now be directly tested for aerobic bacteria using the plate method, reducing dilution steps, simplifying the testing process, and improving the detection rate and accuracy of aerobic bacteria testing for microbial limit. Based on this, the inventors completed this invention.

[0075] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0076] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary from the enumerated value by no more than ±1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0077] As used herein, the terms “containing,” “including,” and “comprising” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made up of” or “made up of.”

[0078] Dabaidu Capsules The ingredients of Dabaidu Capsules are rhubarb, dandelion, tangerine peel, momordica seed, angelica dahurica, trichosanthes root, honeysuckle, phellodendron bark, frankincense (processed), angelica sinensis, red peony root, licorice root, snake slough (wine-processed), dried toad (processed), centipede, scorpion, and mirabilite, totaling 17 components. Dabaidu Capsules have antibacterial properties covering bacteria, fungi, viruses, and drug-resistant bacteria, making it a model of traditional Chinese medicine compound formulas for combating complex infections.

[0079] Based on previous in vitro drug sensitivity tests, cell structure studies, and clinical relevance analyses, Dabaidu Capsules achieve broad-spectrum antibacterial activity through multiple antibacterial mechanisms of 17 traditional Chinese medicines. Research indicates that its antibacterial mechanisms include the following four types: disruption of microbial structure (cell wall / membrane) (e.g., rhubarb (anthraquinones, emodin, aloe-emodin), scorpion (scorpion venom peptides), mirabilite (sodium sulfate)); interference with key metabolisms (protein / DNA synthesis) (e.g., centipede (peptide antibacterial substances), dried toad (bufotoxin lactones, indole alkaloids), trichosanthes root (trichosanthes pollen protein)); inhibition of drug resistance pathways (e.g., licorice (glycyrrhizin chalcone A / C), red peony root (baicalin, ellagic acid)); and synergistic anti-inflammatory and antibacterial effects (e.g., frankincense (resin acids, volatile oils), snake slough (antimicrobial peptides)).

[0080] Twain-80 Tween-80 (polysorbate-80) is a highly efficient nonionic surfactant that can play a dual role of dispersion and neutralization. Its molecular structure contains hydrophilic polyoxyethylene chains and hydrophobic oleic acid groups, which can significantly reduce the surface tension of liquids, refine and uniformly suspend poorly soluble substances, prevent agglomeration and precipitation, and ensure system homogeneity.

[0081] Furthermore, Tween-80 possesses specific chemical neutralization capabilities, effectively adsorbing and inactivating common antibacterial components such as phenols, halogenated phenols, and aldehydes through hydrophobic interactions or hydrogen bonding. Tween-80 can eliminate the interference of residual toxicity on microbial detection, creating a non-toxic environment for the resuscitation of sensitive strains.

[0082] Lecithin Lecithin is a natural zwitterionic emulsifier, rich in phosphatidylcholine, which is key to maintaining system stability. Its unique amphiphilic structure can form a robust protective film at the oil-water interface, preventing droplet aggregation and system stratification, and ensuring long-term uniform dispersion of all components.

[0083] Functionally, lecithin is mainly used to neutralize or eliminate antibacterial components with surface activity or lipophilicity, such as quaternary ammonium salts, biguanides, or phenolic derivatives. It loses its bactericidal activity by electrostatically binding its negatively charged phosphate groups to positively charged quaternary ammonium cations, or by disrupting micelles through lipid exchange.

[0084] Glutathione Glutathione (GSH) is the most critical non-enzymatic antioxidant in cells. It is composed of glutamic acid, cysteine ​​and glycine, and its active sulfhydryl group (-SH) has a strong reducing ability.

[0085] Glutathione can directly scavenge exogenous free radicals (such as superoxide anions and hydroxyl radicals) and peroxides, blocking the chain reaction triggered by oxidative stress. This is crucial for protecting enzymes, DNA structures, and cell membrane lipids that are extremely sensitive to oxidation, effectively preventing the inactivation or degradation of active ingredients due to oxidation. Furthermore, glutathione can participate in intracellular redox cycles, maintaining the reduced state of the intracellular environment and protecting bacteria from the killing effects of exogenous oxidative toxins, thereby maximizing the maintenance of bacterial metabolic activity and structural integrity, and improving detection sensitivity.

[0086] Sodium pyruvate Sodium pyruvate is a key metabolic intermediate with both antioxidant and energy-supplying functions. As an α-keto acid, it can non-enzymatically decompose harmful oxides such as hydrogen peroxide, reducing the threat of oxidative damage to cells.

[0087] Furthermore, pyruvate is the entry point for the tricarboxylic acid cycle (TCA cycle), allowing it to directly enter microbial mitochondria or cytoplasm to participate in energy metabolism. For damaged, oxidized, chemically stressed, or dormant microorganisms, sodium pyruvate provides a rapidly available carbon source and energy precursor, promoting ATP synthesis and accelerating cell repair and proliferation. This "detoxification + energy supply" mechanism significantly improves the recovery and survival rates of microorganisms under stress, serving as a crucial support for maintaining metabolic activity.

[0088] Cyclodextrin The protective effect of cyclodextrins against bacteria primarily stems from their unique "molecular inclusion" mechanism. Many antibacterial agents (such as phenols or some antibiotics) possess hydrophobic groups, and cyclodextrins utilize their hydrophobic cavities to specifically encapsulate these free antibacterial molecules, forming inclusion complexes. This process significantly reduces the concentration of free antibacterial agents in solution, preventing them from effectively contacting and penetrating the bacterial cell membrane, thereby "neutralizing" the antibacterial toxicity at a physicochemical level. Furthermore, this inclusion mechanism stabilizes easily inactivated enzymes or signaling molecules secreted by bacteria, preventing their destruction by inhibitory factors in the environment. By isolating toxins and stabilizing the microenvironment, cyclodextrins provide a crucial recovery window for damaged bacteria, ensuring they can restore metabolic activity during detection and avoiding false negative results caused by residual inhibitors.

[0089] Diluted Composition The present invention provides a diluting composition, namely the diluting composition as described in the second aspect of the present invention, the diluting composition comprising (i) a diluent and (ii) a neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations as described in the first aspect of the present invention.

[0090] The neutralizing agent composition comprises: (a) Tween-80, (b) lecithin, (c) glutathione, (d) cyclodextrin and (e) optional sodium pyruvate, wherein the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin and sodium pyruvate is about 3-10:0.5-4:1-6:40-140:0-5.

[0091] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 3-10:0.5-4:1-6:40-140:1-5.

[0092] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 5-9:1-3:2-4:60-100:1-3.

[0093] In another preferred embodiment, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin, and sodium pyruvate in the neutralizing agent composition is about 7:1.5:3:8:2.

[0094] In another preferred embodiment, the neutralizing agent composition comprises: 3-10 g / L Tween-80, 0.5-4 g / L lecithin, 50-200 mM glutathione, 40-140 g / L cyclodextrin, and 1-5 g / L sodium pyruvate.

[0095] In another preferred embodiment, the neutralizing agent composition comprises: 5-9 g / L Tween-80, 1-3 g / L lecithin, 80-150 mM glutathione, 60-100 g / L cyclodextrin and 1-3 g / L sodium pyruvate.

[0096] In another preferred embodiment, the neutralizing agent composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin and 2 g / L sodium pyruvate.

[0097] In another preferred embodiment, the diluent is selected from the group consisting of water, buffer solution, physiological saline, and peptone water.

[0098] In another preferred embodiment, the buffer solution is a TSB buffer solution.

[0099] In another preferred embodiment, the buffer solution is a pH 7.0 sodium chloride-peptone buffer solution.

[0100] In another preferred embodiment, the buffer solution contains the following components in weight percentages: 0.5-4% tryptone, 0.1-0.6% soybean papain hydrolysate, 0.1-0.6% beef extract, 0.1-0.6% yeast extract, 0.2-1% sodium chloride, 0.1-0.5% dipotassium hydrogen phosphate, 0.1-0.5% potassium dihydrogen phosphate, 0.002-0.01% CaCl2, and 0.1-0.5% glucose or 0.1-0.5% anhydrous glucose.

[0101] In another preferred embodiment, the buffer solution contains the following components in weight percentages: 1.7% tryptone, 0.3% soybean papain hydrolysate, 0.3% beef extract, 0.3% yeast extract, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.195% potassium dihydrogen phosphate, 0.005% CaCl2, and 0.25% glucose or 0.23% anhydrous glucose.

[0102] In another preferred embodiment, the diluent composition contains the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin, 0.1-0.5% sodium pyruvate, 0.5-4% tryptone, 0.1-0.6% soybean papain hydrolysate, 0.1-0.6% beef extract, 0.1-0.6% yeast extract, 0.2-1% sodium chloride, 0.1-0.5% dipotassium hydrogen phosphate, 0.1-0.5% potassium dihydrogen phosphate, 0.002-0.01% CaCl2, and 0.1-0.5% glucose or 0.1-0.5% anhydrous glucose.

[0103] In another preferred embodiment, the diluted composition contains the following components in weight percentages: 0.7% Tween-80, 0.15% lecithin, 0.3% glutathione, 7.9% cyclodextrin, 0.2% sodium pyruvate, 1.7% tryptone, 0.3% soybean papain hydrolysate, 0.3% beef extract, 0.3% yeast extract, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate, 0.195% potassium dihydrogen phosphate, 0.005% CaCl2, and 0.25% glucose or 0.23% anhydrous glucose.

[0104] In another preferred embodiment, the diluting composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin, 2 g / L sodium pyruvate, 17 g / L trypsin, 3 g / L soybean papain hydrolysate, 3 g / L beef extract, 3 g / L yeast extract, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 1.95 g / L potassium dihydrogen phosphate, 0.05 g / L CaCl2, and 2.5 g / L glucose or 2.3 g / L anhydrous glucose.

[0105] In another preferred embodiment, the diluting composition comprises TSB buffer and the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin and 0.1-0.5% sodium pyruvate.

[0106] In another preferred embodiment, the diluting composition comprises a pH 7.0 sodium chloride-peptone buffer and the following components in weight percentages: 0.3-1% Tween-80, 0.05-0.4% lecithin, 0.05-0.6% glutathione, 4-14% cyclodextrin, and 0.1-0.5% sodium pyruvate.

[0107] In another preferred embodiment, the lecithin is soybean lecithin.

[0108] Microbial limit test of antibacterial traditional Chinese medicine Microbial limit testing of traditional Chinese medicine is a core quality control step in assessing the biosafety of drugs according to the Chinese Pharmacopoeia. Addressing the challenge of false negatives caused by the complex matrix and natural antibacterial components (such as alkaloids, tannins, and other diverse ingredients) in traditional Chinese medicine, the key to the test lies in "neutralizing interference" and "repairing sublethal damage."

[0109] This invention provides a method for testing the microbial limits of antibacterial traditional Chinese medicine preparations, comprising the following steps: (S1) The traditional Chinese medicine sample is mixed and diluted with the diluting composition as described in the second aspect of the present invention in a certain proportion to obtain the test solution; and (S2) Take the test solution, add culture medium, culture and count the control bacteria.

[0110] In another preferred embodiment, the method further includes a method suitability verification step: (Z1) The test solution is mixed with the positive control bacteria to obtain a mixture, which is then added to a culture medium, cultured, and the positive control bacteria are counted. (Z2) Mix the buffer solution with the positive control bacteria, add to the culture medium, incubate, and count the positive control bacteria; and (Z3) Calculate the strain recovery rate.

[0111] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:2 to 1:50.

[0112] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:10.

[0113] In another preferred embodiment, the dilution ratio of the traditional Chinese medicine is 1:50.

[0114] In another preferred embodiment, the traditional Chinese medicine is a heat-clearing and detoxifying traditional Chinese medicine.

[0115] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, Zhizi Jinhua Pills, Compound Berberine Tablets, Niuhuang Jiedu Tablets / Pills, Huanglian Shangqing Pills, Sanhuang Tablets, Yiqing Capsules, Yinqiao Jiedu Tablets, Qingjiang Tablets, Qingkailing Granules, Qingre Jiedu Oral Liquid, Lanqin Oral Liquid, Pudilan Anti-inflammatory Oral Liquid, Bairui Granules, Daochi Pills, and Niuhuang Qingxin Pills.

[0116] In another preferred embodiment, the traditional Chinese medicine is selected from the group consisting of: Dabaidu Capsules, Compound Berberine Tablets, Pudilan Anti-inflammatory Tablets, Qingre Sanjie Capsules, Ruyi Huangjin Powder, Shuanghuanglian Oral Liquid, and Gardenia and Golden Flower Pills.

[0117] In another preferred embodiment, the control bacteria are aerobic bacteria.

[0118] In another preferred embodiment, the control bacteria are microorganisms selected from the group consisting of: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, Escherichia coli, Salmonella, and bile salt-tolerant Gram-negative bacteria.

[0119] In another preferred embodiment, the control bacteria are microorganisms selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger.

[0120] In another preferred embodiment, the positive control bacteria are aerobic bacteria.

[0121] In another preferred embodiment, the positive control bacteria are selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus niger, or combinations thereof.

[0122] In another preferred embodiment, the culture medium is tryptic soy agar (TSA) or tryptic soy liquid medium.

[0123] In another preferred embodiment, step (S2) is as follows: take 1 ml / plate or 0.2 ml / plate × 5 plates of test solution into a sterile culture dish, add tryptic soy agar (TSA) medium, culture, and count the control bacteria.

[0124] In another preferred embodiment, step (Z1) is as follows: the test solution is mixed with the positive control bacteria for 5-15 minutes to obtain a test solution-bacterial mixture; the test solution-bacterial mixture is taken at 1 ml / plate or 0.2 ml / plate × 5 plates and placed in a sterile culture dish, tryptic soy agar (TSA) medium is added, and the culture is carried out and the positive control bacteria are counted.

[0125] In another preferred embodiment, the bacterial count in the test solution bacterial mixture is less than 100 cfu / ml.

[0126] In another preferred embodiment, step (Z2) is as follows: the buffer solution is mixed with the positive control bacteria for 5-15 minutes to obtain a buffer-bacterial mixture; 1 ml of the buffer-bacterial mixture is taken into a sterile culture dish, tryptic soy agar (TSA) medium is added, and the positive control bacteria are cultured and counted.

[0127] In another preferred embodiment, the bacterial count in the buffer solution mixture is less than 100 cfu / ml.

[0128] In another preferred embodiment, the culture temperature is 25-40°C, more preferably 30-35°C, such as 32.5°C.

[0129] In another preferred embodiment, the culture time is 48-96 hours, more preferably 55-90 hours, even more preferably 60-80 hours, and most preferably 70-75 hours, such as 72 hours.

[0130] In another preferred embodiment, the buffer solution is selected from the group consisting of: 0.9% sodium chloride saline, TSB, or pH 7.0 sodium chloride-peptone buffer.

[0131] In another preferred embodiment, the strain recovery rate is the ratio of the number of colonies A1 counted in step (Z1) to the number of colonies A0 counted in step (Z2), i.e., A1 / A0.

[0132] In another preferred embodiment, the strain recovery rate of the method is 0.5-2.0, more preferably 0.7-2.0, and even more preferably 0.8-1.5.

[0133] The main advantages of this invention include: (1) This invention provides a neutralizing agent composition for microbial limit testing of antibacterial traditional Chinese medicine preparations, and a diluting composition (such as a bio-stabilizing broth) containing the neutralizing agent composition. This bio-stabilizing broth, combined with a culture medium dilution method, can effectively eliminate interference from the traditional Chinese medicine matrix at an initial release ratio of 1:10, resulting in recoveries of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger all approaching 1.0, meeting the requirements of the Chinese Pharmacopoeia.

[0134] (2) The bio-balanced broth of the present invention can be used for microbial limit testing of various antibacterial traditional Chinese medicines.

[0135] (3) The neutralizing agent composition of the present invention can optimize the pretreatment steps, and can be directly applied to the total aerobic bacteria count of various antibacterial traditional Chinese medicines without complicated multiple washing or special dilution, thus taking into account both detection efficiency and result accuracy.

[0136] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0137] Example 1: Verification of aerobic bacteria count using the standard agar plate method (as described in the pharmacopoeia) 1.1 Methods TSB: Dissolve 17.0 g tryptone, 3.0 g soybean papain hydrolysate, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, and 2.5 g glucose / 2.3 g anhydrous glucose in pure water, bring the volume to 1 L, and adjust the pH to 7.3 ± 0.2 (25 °C).

[0138] pH 7.0 Sodium Chloride-Peptone Buffer: Dissolve 3.56 g potassium dihydrogen phosphate, 5.77 g anhydrous disodium hydrogen phosphate, 4.30 g sodium chloride and 1.00 g peptone in pure water, bring the volume to 1 L, and adjust the pH to 7.0 (25 °C).

[0139] Preparation of test solution: Dissolve 10g of Dabaidu capsule sample in TSB or pH 7.0 sodium chloride-peptone buffer, add to 100ml to prepare 1:10 test solution, and take 1ml to 9ml of dilution to prepare 1:100 test solution.

[0140] Experimental group: Take 0.1 ml of appropriate concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger suspensions, respectively. Add them to two dilutions of the above two sample preparation solutions to make the final bacterial concentration <100 CFU / ml. After mixing, take 1 ml and add it to a sterile petri dish, and perform 5 replicates. Add approximately 15-20 ml of 45℃ tryptic soy agar (TSA) medium and incubate at 32.5℃ for 72 h.

[0141] Control group: Use TSB or pH 7.0 sodium chloride-peptone buffer instead of bacterial culture, and perform the same procedures as the experimental group. Two replicates are performed.

[0142] Bacterial control group: The test solution was replaced with 0.9% sodium chloride physiological saline, and other procedures were the same as those for the experimental group. Two replicates were performed.

[0143] 1.2 Experimental Results The results are shown in Table 1. At a 1:10 dilution, Dabaidu capsules exhibited strong inhibitory effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, with recoveries ranging from 0.01 to 0.16. However, its inhibitory activity against Candida albicans and Aspergillus niger was relatively low, with recoveries ranging from 0.7 to 0.99.

[0144] When the dilution ratio was increased to 1:100, the recovery rate of TSB as the diluent was acceptable (greater than 0.5), while the recovery rate of Bacillus subtilis was 0.16 (less than 0.5) and the recovery rate of Staphylococcus aureus was 0.6 (close to the lower limit of 0.5) when pH 7.0 sodium chloride-peptone buffer was used as the diluent.

[0145] In summary, the buffer solution used in the conventional agar plate method according to the pharmacopoeia cannot completely neutralize the inhibitory effects of traditional Chinese medicine preparations such as Dabaidu capsules on Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis. Especially when using pH 7.0 sodium chloride-peptone buffer, even at a 1:100 dilution, the recovery rates of Staphylococcus aureus and Bacillus subtilis remain extremely low. Therefore, it is necessary to further add neutralizing agents to neutralize the antibacterial components in traditional Chinese medicine preparations and improve the accuracy of microbial detection.

[0146] Table 1. Results of the Pharmacopoeia Plate Method Example 2: Compatibility Effects of Different Neutralizing Agents This embodiment verifies the neutralizing effect of different neutralizing agent combinations on the antibacterial activity of Dabaidu capsules. Through the recovery rate experiment of Bacillus subtilis and Staphylococcus aureus, a scientific and reliable neutralizing agent formula is determined.

[0147] 2.1 Methods Basic broth preparation: Mix and grind 17.0g tryptone, 3.0g soy papain hydrolysate, 3.0g beef extract powder, 3.0g yeast extract powder, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate, 1.95g potassium dihydrogen phosphate, 0.05g calcium chloride, and 2.5g glucose or 2.3g anhydrous glucose and dissolve in water, then bring the volume to 1L.

[0148] Preparation of test solution: Dissolve 10g of Dabaidu capsule sample into 100ml of buffer solution or broth as shown in Table 2 to prepare a 1:10 test solution.

[0149] Take 0.1 ml of Staphylococcus aureus and Bacillus subtilis suspensions of appropriate concentrations and add them to the above test solution to make the final bacterial concentration <100 CFU / ml. After mixing, take 0.2 ml / plate and add it to 5 sterile Petri dishes, making 2-3 replicates. Inject TSA at 45℃ and incubate at 32.5℃ for 72 hours before counting.

[0150] Bacterial control group: The test solution was replaced with 0.9% sodium chloride physiological saline, and other procedures were the same as those for the experimental group. Five replicates were performed.

[0151] Table 2. Buffer solution or broth 2.2 Results For Staphylococcus aureus, the results are shown in Table 3. The average values ​​of control groups ① and ② were 39.2 cfu / ml and 45 cfu / ml, respectively, which were used as the baseline for calculating the recovery rate. The recovery rate of the ordinary broth basal group was 1.00, indicating that the basal medium itself was suitable for the growth of Staphylococcus aureus. However, the recovery rates of the TSB and pH 7.0 sodium chloride-peptone control groups were extremely low (0.27 and 0.09), indicating that the Dabaidu capsules had a strong antibacterial effect against this bacterium, and a neutralizing agent must be used.

[0152] The recoveries of all single-factor neutralizers were unsatisfactory, mostly between 0.01 and 0.32. Only the recovery of 2e (sodium pyruvate) reached the acceptable value (0.76). Therefore, single-factor neutralizers cannot completely neutralize the antibacterial effect.

[0153] The recovery rate increased significantly with the increase of the neutralizing agent component. The recovery rate of the two-factor combination was between 0.19 and 0.69, while the recovery rate of the three-factor combination increased to 0.59-0.87, approaching or reaching the acceptable standard. The recovery rate of the four-factor combination (abcd) reached 1.45, indicating a strong neutralizing effect or a slight promoting effect on bacterial growth. The recovery rate of the five-factor combination (abcde) reached 1.82, indicating a very thorough neutralizing effect.

[0154] Furthermore, the recovery rate of the five-factor neutralizing agent was restored to above 0.76 after adding the TSB and pH 7.0 sodium chloride-peptone base solutions, proving that the neutralizing agent formulation itself is non-toxic to Staphylococcus aureus and can effectively neutralize the antibacterial components in the samples.

[0155] Table 3. Recovery rate of Staphylococcus aureus Note: 1. Recovery rates marked with * are obtained by dividing the average value by the average value of the Staphylococcus aureus solution control group ②, while unmarked recovery rates are obtained by dividing the average value by the average value of the Staphylococcus aureus solution control group ①. The specific calculation should be based on the experimental batch.

[0156] For Bacillus subtilis, the results are shown in Table 4. The average values ​​of control groups ① and ② were 124.8 CFU / ml and 88 CFU / ml, respectively, which were used as the baseline for calculating the recovery rate. The recovery rate of the broth-based group was 0.47, while the recoveries of the TSB and pH 7.0 sodium chloride-peptone control groups were only 0.25 and 0.24, respectively, indicating that the Dabaidu capsules also have significant inhibitory activity against Bacillus subtilis. A neutralizing agent must be used.

[0157] Similar to Staphylococcus aureus, the recovery rates of all single-factor neutralizers against Bacillus subtilis were generally poor, mostly between 0.14 and 0.68. The best single factor was 2d (cyclodextrin), with a recovery rate of 0.68. Therefore, single-factor neutralizers cannot completely neutralize the antibacterial effect.

[0158] Among the multifactor combinations, those containing Tween-80 (a), cyclodextrin (d), and sodium pyruvate (e) performed exceptionally well. For example, the recovery rate of the ae combination (Tween-80 + sodium pyruvate) reached 1.02, the recovery rate of the de combination reached 0.96, the recovery rate of the ade combination (Tween-80 + cyclodextrin + sodium pyruvate) was as high as 2.50, and the recovery rate of the bde combination (lecithin + cyclodextrin + sodium pyruvate) was 2.12. The five-factor combination (abcde) achieved a recovery rate of 1.34 (calculated based on control group ①), demonstrating its effective neutralization of the inhibitory effect of the sample on Bacillus subtilis.

[0159] Furthermore, the recovery rates of the five-factor neutralizing agent were 1.25% and 1.16% in TSB and pH 7.0 sodium chloride-peptone base solutions, respectively, which further verified that the neutralizing agent formulation was non-toxic to Bacillus subtilis and had a good neutralizing effect.

[0160] Table 4. Bacillus subtilis recovery rate Note: 1. Values ​​marked with * (marked as " / ") are missing and have been corrected for the mean. 2. Recovery rates marked with ★ are obtained by dividing the average value by the average value of the Bacillus subtilis solution control group ②. Recovery rates without labels are obtained by dividing the average value by the average value of the Bacillus subtilis solution control group ①. The specific calculation should be performed based on the experimental batch. In summary, the five-factor neutralizing agent formulation (0.7% Tween-80 + 0.15% lecithin + 10mM glutathione + 7.9% cyclodextrin + 0.2% sodium pyruvate) exhibits a significant and reliable neutralizing effect on the antibacterial components of Dabaidu capsules. This formulation restores the recovery rates of both test bacteria to acceptable ranges (>0.7), fully meeting the recovery rate requirements (0.5-2) for microbial counting method validation in the Chinese Pharmacopoeia. Furthermore, this neutralizing agent did not show inhibition of test bacterial growth in TSB and pH 7.0 sodium chloride-peptone media, meeting the safety requirements for neutralizing agent validation.

[0161] Example 3: Validation Experiment of Bio-Stable Broth as an Alternative to Pharmacopoeia Diluent (with Neutralizing Agent) 3.1 Methods 3.1.1 Accuracy Test Staphylococcus aureus and Bacillus subtilis were used as test bacteria, and five concentration gradients were set up for each, with bacterial concentration ranges of 5-100 cfu / ml (5, 25, 45, 65, 95 cfu / ml).

[0162] Preparation of bio-balanced broth: Mix and grind 17.0g tryptone, 3.0g soybean papain hydrolysate, 3.0g beef extract powder, 3.0g yeast extract powder, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate, 1.95g potassium dihydrogen phosphate, 0.05g calcium chloride, and 2.5g glucose or 2.3g anhydrous glucose. Dissolve in an appropriate amount of purified water to obtain 500ml of the mixture. Add 7.0g Tween-80, 1.5g lecithin, 79g cyclodextrin, and 2g sodium pyruvate, and bring the volume to 1000ml. Adjust the pH to 7.3±0.1 at 25℃ after sterilization. Before use, mix 1ml of 1M glutathione with the broth to prepare 100ml of bio-balanced broth with a final concentration of 10mM glutathione.

[0163] Alternative group (bioequilibrium broth): Add 10g of Dabaidu capsule sample to bioequilibrium broth to a final volume of 100ml to prepare a 1:10 sample dilution. Add 0.1ml of a suitable concentration of bacterial suspension (Staphylococcus aureus, Bacillus subtilis) to 9.9ml of sample preparation solution and mix well. Add 0.2ml / plate to 5 sterile Petri dishes. Inject into 45℃ TSA, incubate at 32.5℃ for 72h, and then count the bacteria. Perform 5 parallel experiments for each bacterial strain and 5 concentrations for each concentration.

[0164] Pharmacopoeia Group (with neutralizing agent): Take 10g of Dabaidu capsule sample and add TSB or pH 7.0 sodium chloride-peptone (abbreviated as "pH 7.0") (both with neutralizing agents of 0.7% Tween-80, 0.15% lecithin, 100mM glutathione, 7.9% cyclodextrin, and 0.2% sodium pyruvate) to 100ml to prepare a 1:10 sample dilution. Other experimental procedures are the same as in the alternative group, and each concentration and each bacterium is tested in 5 parallel trials.

[0165] Control group: Take 0.1 ml of bacterial suspension of appropriate concentration (Staphylococcus aureus, Bacillus subtilis) into 100 ml of 0.9% sodium chloride physiological saline, without adding any sample. Other experimental procedures are the same as the alternative group, and each concentration and each bacterium is tested in duplicate.

[0166] Control group: 10g of Dabaidu capsules were added to 100ml of bio-balanced broth to prepare a 1:10 sample dilution, without adding bacterial culture. Other experimental procedures were the same as the alternative group, and two parallel experiments were conducted.

[0167] 3.1.2 Repeatability Test Using the results of the accuracy test, the relative standard deviation (RSD) of five parallel tests at five different bacterial concentrations of Staphylococcus aureus and Bacillus subtilis were calculated for the two groups. The RSD of each test strain was statistically analyzed using the t-test (two-sample equal variance test).

[0168] 3.1.3 Intermediate precision test Take 10g of Dabaidu capsule sample and add it to 100ml of bio-balanced broth to prepare a 1:10 sample dilution. Take 9.9ml of sample preparation solution and add 0.1ml of bacterial suspension of appropriate concentration, shake well, and take 0.2ml / plate to add to 5 sterile culture dishes. Incubate at 32.5℃ for 72h and count the bacteria. There are 5 concentrations for each bacteria, and each concentration is tested in 5 parallel experiments. The strains and bacterial suspension preparation used in the intermediate precision test are the same as in "3.1 Accuracy Test".

[0169] Two people were asked to perform the above experiment and calculate the relative standard deviation of their counting results. A two-sample t-test with equal variances was used to perform statistical analysis on the relative standard deviation of each test bacteria.

[0170] 3.1.4 Specificity Test Alternative group (bioequilibrium broth): Add 10g of Dabaidu capsule sample to bioequilibrium broth to a final volume of 100ml to prepare a 1:10 sample dilution. Add 0.1ml of a suitable concentration of bacterial suspension to 9.9ml of the sample preparation solution, shake well, and allow the bioequilibrium broth to react with the sample for 5 minutes. Then, add 0.2ml / plate to 5 sterile culture dishes and incubate at 32.5℃ for 72h and 120h, respectively. Count the bacteria after each incubation. Perform three parallel experiments for each concentration. Considering the complexity of microorganisms in actual samples, different mixed bacterial combinations were also used in the experiments. Specific groups are shown in Table 7. Control group: Microorganisms were added to 0.9% sodium chloride physiological saline (as above). 1 ml of bacterial suspension of appropriate concentration was taken and placed in a sterile Petri dish, then infused with tryptic soy agar medium and incubated at 32.5℃ for 72 h. Each concentration and each type of bacteria was tested twice in parallel. The mixed bacterial combinations of different properties were the same as those in the substitution group.

[0171] Calculate the false positive rate and recovery rate for each test bacterium.

[0172] 3.1.5 Limit of Quantitation Test The data of the control group corresponding to the lowest bacterial suspension concentration group that simultaneously meets the requirements of accuracy test, repeatability test and intermediate precision test is the quantification of the method.

[0173] 3.1.6 Linearity Test and Range Concentration gradient data that simultaneously meet the requirements of accuracy testing, repeatability testing, and intermediate precision testing were selected. Linear regression analysis was performed with the mean of the surrogate group results as the dependent variable and the mean of the bacterial control group results as the independent variable, and the coefficient of determination R was calculated. 2 .

[0174] 3.1.7 Durability Test Alternative group (bioequilibrium broth): Add 10g of Dabaidu capsule sample to bioequilibrium broth to a final volume of 100ml to prepare a 1:10 sample dilution. Add 0.1ml of a suitable concentration of bacterial suspension to 9.9ml of the 1:10 sample preparation and mix well. Incubate the bacterial suspension with the sample preparation solution (5, 10, or 15 min). Take 0.2ml / plate of the sample preparation solution at different incubation times and add it to 5 sterile Petri dishes (3 replicates). Randomly divide the Petri dishes into 5 groups, with 5 plates in each group. Add TSA and incubate at 32.5℃ for 72h, then count the bacteria.

[0175] Bacterial suspension control group: The strains and bacterial suspension preparation used in the durability test are the same as those in the accuracy test.

[0176] 3.2. Results The average count results and their recovery rates of the substitution group, the pharmacopoeia control group (TSB and pH 7.0 NaCl-peptone buffer with added neutralizing agent), and the bacterial culture group are shown in Table 5. Statistical analysis of the recovery rates was performed using the F-test. The results showed no significant difference in the recovery rates between the substitution group and the bacterial culture group and the pharmacopoeia control group (P > 0.05), indicating that homogeneous variances can be used for a two-sample t-test of equal variances. The accuracy results are shown in Table 6. The t-test showed a significant difference in the recovery rates between the substitution group and the pharmacopoeia control group (P < 0.05), with the substitution group showing a better recovery rate than the pharmacopoeia group. This indicates that the substitution of the bio-stabilized broth can reduce the antibacterial components and effectively improve the recovery rate.

[0177] Table 5. Accuracy Results Table 6. Statistical analysis of accuracy results (α = 0.05) The results of the repeatability test showed no significant difference in RSD between the alternative group and the pharmacopoeia group (TSB with added neutralizing agent and pH 7.0-Nacl peptone buffer) (P > 0.05). The specific results are shown in Table 7.

[0178] Table 7. Statistical analysis of repeatability results (α = 0.05) The intermediate precision test results showed that there was no significant difference in the relative standard deviation of the results between the two inspectors (P>0.05), and the specific results are shown in Table 8.

[0179] Table 8. Intermediate Precision Results The specificity test results showed that the average false positive rate of each test strain and mixed strain culture was less than 5%, and the average recovery ratio was in the range of 0.5-2. The results are shown in Table 9. The results show that the alternative method has good specificity.

[0180] Table 9. Specificity Results Note: a. The false positive rate is the ratio of the difference between the results of the alternative group cultured for 72 hours and the results of the alternative group cultured for 120 hours to the results of the alternative group cultured for 120 hours.

[0181] b. The recovery rate is the ratio of the average of the results from the alternative group at two different culture times to the result from the bacterial culture group.

[0182] In this embodiment, the limits of quantification for the antibacterial activity of Dabaidu capsules against sensitive Staphylococcus aureus and Bacillus subtilis were compared between the alternative group and the pharmacopoeia group with added neutralizing agent, both based on 5 CFU / ml. The accuracy test showed a significant difference in the recovery rates between the alternative group and the pharmacopoeia control group (P < 0.05), indicating that the bio-stabilized broth of the alternative group can effectively reduce the antibacterial components and improve the recovery rate.

[0183] The linearity test results showed that the alternative method for Staphylococcus aureus detection yielded y = 1.4074x - 1.3467, R0 2 The value was 0.9996; the Bacillus subtilis test result was y = 1.3993x – 2.8053, R0 2 The coefficient of determination is 0.9982, and all coefficients of determination are greater than 0.9.

[0184] One-way ANOVA was used to examine the effect of different treatment times on the experimental results. The results are shown in Table 10. There were no significant differences in the experimental results under each condition (P>0.05).

[0185] Table 10. Statistical Analysis of Durability Results Example 4: Five-factor combination neutralizer can be used for microbial limit testing of various traditional Chinese medicines. 4.1 Methods Preparation of test solutions: Take 10g of each of the following samples: Compound Berberine Tablets, Pu Di Lan Anti-inflammatory Tablets, Qing Re San Jie Capsules, Ru Yi Huang Jin San, Shuang Huang Lian Oral Liquid, and Zhi Zi Jin Hua Pills. Dissolve each sample in 100ml of one of the following six buffer solutions: Steady Broth, TSB+abcde, pH 7.0 Sodium Chloride-Peptone Buffer (abbreviated as "pH 7.0")+abcde, Broth Base, TSB, and pH 7.0 Sodium Chloride-Peptone Buffer (abbreviated as "pH 7.0"), to prepare a 1:10 test solution.

[0186] Experimental group: Take 0.1 ml of appropriate concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Aspergillus niger suspensions respectively. Add each to 9.9 ml of the prepared test solution to make the final bacterial concentration <100 CFU / ml. After mixing, take 1 ml and add it to a sterile petri dish (1:10), and perform 5 replicates. For some bacteria with low recovery rates (recovery rate less than 0.7 at 1:10), take 0.2 ml / dish and add it to 5 sterile petri dishes, and perform 2 replicates (using Pu Di Lan Anti-inflammatory Tablets, Qing Re San Jie Capsules, Ru Yi Huang Jin San, Shuang Huang Lian Oral Liquid, and Zhi Zi Jin Hua Wan). If still unsatisfactory, dilute to 1:50, take 0.2 ml / dish and add it to 5 sterile petri dishes, and perform 2 replicates (using Compound Berberine Tablets). Inject TSA at 45℃, incubate at 32.5℃ for 72 h, and then count.

[0187] Bacterial suspension control group: Add 0.1 ml of a suitable concentration of bacterial suspension to 9.9 ml of 0.9% sodium chloride physiological saline, without adding any sample. Add 1 ml to a sterile culture dish, and perform the same procedure as the experimental group. Repeat for 5 samples.

[0188] 4.2 Results The results are shown in Tables 11 and 12. In the four traditional Chinese medicine samples—Pu Di Lan Anti-inflammatory Tablets, Qing Re San Jie Capsules, Shuang Huang Lian Oral Liquid, and Zhi Zi Jin Hua Pills—the five-factor neutralizing agent (0.7% Tween-80 + 0.15% lecithin + 100mM glutathione + 7.9% cyclodextrin + 0.2% sodium pyruvate)—whether used with the broth-based bio-balanced broth or added to TSB or pH 7.0 sodium chloride-peptone buffer—showed high recoveries (greater than 0.7). However, when using TSB or pH 7.0 sodium chloride-peptone buffer alone, the recoveries of Bacillus subtilis were low (less than 0.5) in most of the traditional Chinese medicine samples.

[0189] In compound berberine tablets, the recoveries of Staphylococcus aureus and Bacillus subtilis in all treatments at a dilution of 1:10 (0.2 ml / plate) were below 0.5%. However, the treatment with the addition of a five-factor neutralizing agent at a dilution of 1:50 (0.2 ml / plate) showed recoveries between 0.79 and 0.98, significantly higher than those using TSB or pH 7.0 sodium chloride-peptone buffer alone (recoveries between 0.56 and 0.63). In Ruyi Golden Powder, at a dilution of 1 ml / plate and a 1:10 ratio, the recovery rate of Staphylococcus aureus in all treatments was below 0.5%. However, at the same dilution and with 0.2 ml / plate, the recovery rate of the treatment group with the addition of the five-factor neutralizing agent reached 0.84-1.98, which was significantly higher than that of TSB or pH 7.0 sodium chloride-peptone buffer alone (with a recovery rate between 0.51-0.58). However, the recovery rate of Bacillus subtilis spores treated with the addition of the five-factor neutralizing agent reached 0.64-1.43, which was significantly higher than that of TSB or pH 7.0 sodium chloride-peptone buffer alone (with a recovery rate between 0.32-0.41). However, at a concentration of 0.2 ml / plate (i.e., 1:50 dilution of Ruyi Huangjin San and 1:250 dilution of Compound Berberine Tablets), the recovery rate of the five-factor neutralizing agent was between 0.79 and 0.98, significantly higher than that of using TSB or pH 7.0 sodium chloride-peptone buffer alone (recovery rate between 0.51 and 0.65). These results indicate that the five-factor neutralizing agent can effectively neutralize the antibacterial effect of traditional Chinese medicine preparations.

[0190] Table 11. Recovery rate of bacterial strains in traditional Chinese medicine samples under different treatments (1) Note: 1. Bold text and * indicate a recovery rate of less than 0.5%, which does not meet the requirements.

[0191] 2. This indicates that the sample dilution is 1:50.

[0192] 3. The recovery rate of Pseudomonas aeruginosa in compound berberine tablets is calculated per ml / plate.

[0193] Table 12. Recovery rate of bacterial strains in traditional Chinese medicine samples under different treatments (2) discuss: Traditional Chinese medicine (TCM) preparations present unique challenges for microbial limit testing due to their natural antibacterial components. Besides common oral preparations such as those for clearing heat and detoxifying (e.g., Niuhuang Jiedu tablets) and clearing heat and drying dampness (e.g., compound berberine tablets), these also include external powders (e.g., Bingpengsan), suppositories, ophthalmic preparations, and gynecological medications (e.g., Fuke Qianjin tablets). The antibacterial substances contained in these medicines are complex and diverse, including plant-derived volatile oils, alkaloids, flavonoids, animal-derived antimicrobial peptides, and metal ions from minerals.

[0194] According to the requirements of the Chinese Pharmacopoeia, all drugs must undergo microbial limit methodological validation. However, currently, different manufacturers often employ different validation strategies for the same product: some increase the dilution factor, some add different types of neutralizing agents, and some use membrane filtration combined with different rinsing solutions and volumes. This inconsistency in validation methods directly leads to a lack of comparability in test results, affecting the objectivity and consistency of quality evaluation. More importantly, as the sample dilution factor increases and the operational procedures become more complex, not only does the risk of microbial damage and cross-contamination increase, but the validation work for each product also becomes time-consuming and labor-intensive, resulting in redundant investment and significant waste of corporate resources.

[0195] To address this issue, this study, following the guidelines of the Chinese Pharmacopoeia 9201, innovatively employed a bio-stabilized broth containing the neutralizing agent composition of this invention as the sample preparation medium. Using Dabaidu capsules as a model drug, a comprehensive validation of the alternative method for counting aerobic microorganisms was conducted through plate counting combined with culture medium dilution. Staphylococcus aureus and Bacillus subtilis were selected as sensitive indicator bacteria, and the new method was scientifically evaluated from multiple dimensions, including accuracy, precision, specificity, limit of quantitation, linearity, range, and robustness. The results showed that the bio-stabilized broth, at a 1:10 dilution, achieved ideal recoveries (0.5-2.0), meeting the pharmacopoeia requirements. Statistical analysis revealed a significant difference between the new method and the pharmacopoeia method in the recovery rate of sensitive bacteria (P < 0.05), and the new method demonstrated superior performance in key indicators such as precision and specificity, indicating that this method significantly improved detection efficiency while ensuring accuracy. Mechanistic studies have shown that bio-balanced broths effectively eliminate antibacterial activity in samples through multiple synergistic effects, including chemical neutralization, nutritional enhancement, antioxidant protection, cell wall repair, and buffer system optimization, creating an environment more conducive to the survival and growth of microorganisms.

[0196] Using the neutralizing agent composition developed in this study, and the sample treatment with a bio-stabilized broth containing this neutralizing agent composition, the antibacterial components in various traditional Chinese medicines, such as Dabaidu capsules, can be effectively neutralized, significantly simplifying the sample pretreatment process. Compared with traditional methods, the new method not only improves the accuracy and repeatability of detection, but also achieves comparability of test results across laboratories and batches. The widespread application of this method will help solve the long-standing problem of inconsistent microbiological testing methods and difficulty in comparing results in the industry, providing technical support for establishing standardized procedures for microbiological testing of traditional Chinese medicines. By adopting a unified sample processing medium, the validation costs for enterprises to develop proprietary methods for different varieties can be significantly reduced, avoiding redundant investment, improving testing efficiency, and ultimately providing more reliable and consistent technical assurance for drug quality supervision and product release, promoting the improvement of quality standards for traditional Chinese medicine products and the healthy development of the industry.

[0197] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations, characterized in that, include: (a) Tween-80, (b) lecithin, (c) glutathione, (d) cyclodextrin and (e) optional sodium pyruvate, wherein the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin and sodium pyruvate is approximately 3-10:0.5-4:1-6:40-140:0-5.

2. The neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations as described in claim 1, characterized in that, In the neutralizing agent composition, the mass ratio of Tween-80, lecithin, glutathione, cyclodextrin and sodium pyruvate is about 7:1.5:3:8:

2.

3. The neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations as described in claim 1, characterized in that, The neutralizing agent composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin and 2 g / L sodium pyruvate.

4. The neutralizing agent composition for microbial limit detection of traditional Chinese medicine preparations as described in claim 1, characterized in that, The traditional Chinese medicine is an antibacterial traditional Chinese medicine, preferably a traditional Chinese medicine for clearing heat and detoxifying.

5. A dilution composition for microbial limit detection, characterized in that, The diluting composition comprises (i) a diluent and (ii) a neutralizing agent composition for microbial limit detection of traditional Chinese medicine as described in claim 1.

6. The dilution composition for microbial limit detection as described in claim 5, characterized in that, The diluent is selected from the group consisting of: water, buffer solution, physiological saline, and peptone water.

7. The dilution composition for microbial limit detection as described in claim 5, characterized in that, The diluted composition comprises: 7 g / L Tween-80, 1.5 g / L lecithin, 100 mM glutathione, 79 g / L cyclodextrin, 2 g / L sodium pyruvate, 17 g / L trypsin, 3 g / L soybean papain hydrolysate, 3 g / L beef extract, 3 g / L yeast extract, 5 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 1.95 g / L potassium dihydrogen phosphate, 0.05 g / L CaCl2, and 2.5 g / L glucose or 2.3 g / L anhydrous glucose.

8. Use of the neutralizing composition as claimed in claim 1 or the diluting composition as claimed in claim 5, characterized in that, Used for microbial limit testing of traditional Chinese medicine preparations.

9. A method for detecting the microbial limits of traditional Chinese medicine preparations, characterized in that, Including the following steps: (S1) The traditional Chinese medicine sample is mixed and diluted with the diluting composition as described in claim 5 in a certain proportion to obtain the test solution; and (S2) Take the test solution, add culture medium, culture and count the control bacteria.

10. The method for detecting the microbial limit of traditional Chinese medicine preparations as described in claim 9, characterized in that, The method also includes a method applicability verification step: (Z1) The test solution is mixed with the positive control bacteria to obtain a mixture, which is then added to a culture medium, cultured, and the positive control bacteria are counted. (Z2) Mix the buffer solution with the positive control bacteria, add to the culture medium, incubate, and count the positive control bacteria; and (Z3) Calculate the strain recovery rate.

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