Comparative Study Of Microbial Limit Test And Criteria Of TCM Decoction Pieces Among United States Pharmacopeia，European Pharmacopeia，Japanese Pharmacopeia And Chinese Pharmacopeia

ABSTRACT

OBJECTIVE：To compare the difference of microbiological limit test and criteria of TCM decoction pieces among 43 edition of United States Pharmacopeia (USP43),10.0 edition of European Pharmacopeia (EP10.0),17 edition of Japanese Pharmacopeia (JP17) and 2020 edition of Chinese Pharmacopeia (ChP2020),and to provide refernce for the revision and improvement of microbiological standards for TCM decoction pieces in China. METHODS：The differences in the microbial enumeration tests method (including sampling and sample preparation,selection of bacteria and culture medium,count of microorganisms and heat-resistant bacteria,etc.),tests for specified microorganisms(including sample pretreatment,enrichment, separation and identification,etc.)and microbial related limit criteria were compared among USP43,EP10.0,JP17 and ChP2020. RESULTS & CONCLUSIONS：In terms of microbiological examination of TCM decoction pieces,USP43,EP10.0,JP17 had their own independent provisions. Chp2020 added"general rule 1108". In terms of inspection items,in addition to the total aerobic bacteria count and total combined yeasts and molds count,ChP2020 and EP10.0 provided three methods for the inspection of control bacteria (bile-resistant Gram-negative bacteria, Escherichia coli, Salmonella). On the basis, JP17 supplemented Staphylococcus aureus test;However,USP43 added Clostridium test method and put forward the concept of objectionable microorganisms risk assessment;ChP2020 also added a new method for counting heat-resistant bacteria. In terms of microbial limit criteria,USP43 was the most detailed in the classification of TCM decoction pieces,which was more strict than EP10.0 and JP17; ChP2020 had not set up a unified limit for the inspection of control bacteria of TCM decoction pieces. ChP2020 revised the "microbial limit standard for TCM extracts and TCM decoction pieces",but it was not perfect compared with the Pharmacopoeia of the United States,Europe and Japan. It is suggested that according to the current situation of microbial contamination and control of TCM decoction pieces,the microbial limit test and criteria of TCM related products in Pharmacopoeia should be gradually improved,and the microbial limit level of corresponding products should be reasonably refined.

KEYWORDS TCM decoction piece; Pharmacopeia; Microbial limit test;Limit criteria;Comparative study

Chinese herbal medicines are products that can be directly used in TCM clinical practice after processing of Chinese herbal medicines. Most of them are derived from natural plants, animals or minerals and usually carry a large number of microorganisms [1-2]. The processing process of most Chinese herbal medicines is simple (such as cleaning, frying, etc.), and the sterilization is incomplete, which leads to the residual pathogenic microorganisms [3-4]. In addition, during transportation and storage, contact with operators and external non-clean environments also increases the possibility of Chinese herbal medicines being contaminated by pathogenic microorganisms, thereby affecting their safety [5]. The microbial contamination of Chinese herbal medicines has not received enough attention, and there is a lack of research on the changes in microbial populations during their processing, which has led to the lack of microbial safety evaluation of Chinese herbal medicines. Many potential pathogenic microorganisms (such as heat-resistant bacteria and bacterial toxins) have not been effectively controlled, increasing the risk of immunocompromised patients using Chinese herbal medicines [6]. Although the 2015 edition of the Chinese Pharmacopoeia (Volume 1) includes a variety of identification and inspection methods for the analysis of the physical and chemical properties of Chinese herbal medicines, the specific inspection methods and limit standards for related microorganisms have been missing for a long time. At the same time, in the general rules of the 2015 edition of the Chinese Pharmacopoeia (Volume 4), except for Chinese herbal medicines that are directly imported, no microbial inspection items for other forms of Chinese herbal medicines are specified [7].

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Chinese medicine is called herbal medicine in Europe, crude medicine in Japan, and nutritional supplements in the United States.

Although its concept is not exactly the same as that of Chinese herbal medicines in my country [8-9], the European Pharmacopoeia 10.0 (EP10.0) [10], the Japanese Pharmacopoeia 17 (JP17) [11] and the United States Pharmacopoeia 43 (USP43) [12] all clearly include microbial inspection methods and limit standards for related products (herbal medicines, crude medicines or nutritional supplements). The 2020 edition of the Chinese Pharmacopoeia (ChP2020) (Volume 4) [13] added the "Microbial Limit Test Method for Chinese Herbal Medicine Pieces" (General Rule 1108), but the relevant microbial limit standards are still incomplete, and there is no unified and clear regulation on the microbial limit standards for decoction-type Chinese herbal medicine pieces. This paper compares the differences between USP43, EP10.0, JP17 and ChP2020 in terms of microbial test methods and limit standards for Chinese herbal medicine pieces, understands the revision direction of the microbial standards for Chinese herbal medicine pieces, and discusses the necessity of establishing microbial limits for Chinese herbal medicine pieces according to their risk levels, in order to provide a reference for the revision and improvement of relevant standards for microbial control of Chinese herbal medicine pieces.

1 Microbial counts of Chinese herbal medicines

After comparing the microbial testing methods related to Chinese herbal medicines in EP10.0, JP17, USP43 and ChP2020, the author found that although the first three pharmacopoeias have been internationally coordinated and integrated in the microbial testing of non-sterile products, the three have their own independent chapters on the inspection of microorganisms in Chinese herbal medicines, such as EP10.0 "2.6.12" and "2.6.31", JP17 "5.02", USP43 "2021" and "2022", while ChP2020 has added "General Principle 1108" for the detection of microorganisms in Chinese herbal medicines. In terms of testing items, the four pharmacopoeias all specify the inspection methods for total aerobic microbial count (TAMC) and total combined yeasts and moulds count (TYMC). The main differences are reflected in sampling and sampling, bacterial species and culture medium, and microbial counting methods. See Table 1 for details.

1.1 Sampling and sampling

Chinese herbal medicines are of various types, with different shapes, textures and sizes. In addition, microbial contamination is uneven, so the representativeness of sampling and inspection is crucial[14]. The four pharmacopoeias have obvious differences in the provisions of "sampling and sampling". Among them, EP10.0 "2.6.12"[10] and USP43 "2021"[12] have the same requirements for sampling of Chinese herbal medicines as chemical drugs, with a sampling volume of 10 g or 10 mL. JP17 "5.02" [11] stipulates four sampling methods for Chinese herbal medicines: (1) For small-volume or powdered samples, take 50-250 g of sample and mix it; (2) For large-volume samples, take 250-500 g of sample and mix it and chop it; (3) For single samples of not less than 100 g, take at least 5 or 500 g, chop it and mix it; (4) For samples in the form of solutions or preparations, mix them and take samples directly. Take 10 g or 10 mL of the mixed sample as the sample for inspection. ChP2020 "General Rule 1108" [13] states that "unless otherwise specified, test samples shall be drawn in accordance with the method for sampling medicinal materials and decoction pieces ('General Rule 0211'). For large-packaged decoction pieces, 100 to 500 g of samples shall be drawn from each batch and mixed; for independent small-packaged decoction pieces, 100 to 500 g of samples shall be drawn from each package according to the packaging volume; the test volume shall be 25 g or 25 mL unless otherwise specified, and the test volume may be reduced for precious varieties or varieties with lower density."

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1.2 Selection of strains and culture media

In terms of strain selection, USP43 lists five microorganisms including Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC16404 for culture media suitability and method suitability testing [12]. EP10.0 and JP17 replaced Escherichia coli with Pseudomonas aeruginosa ATCC 9027, and gave the above-mentioned strains the equivalent strain numbers of the National Collections of Industrial, Food and Marine Bacterial (NCIMB), the Collection de L'Institut Pasteur of Institut Pasteur (CIP), and the NITE Biological Resource Center (NBRC). This provision is consistent with the coordination proposal of the International Council for Harmonization (ICH) [10-11].
The strains of ChP2020 are the same as those of EP10.0 and JP17, but only CMCC strains are allowed [13].
In terms of the selection of culture media, in addition to using TSA culture media and SDA culture media as conventional counting culture media, considering that Chinese herbal medicines often contain a large amount of insoluble substances and a high probability of bacterial and fungal contamination, the above factors may affect the TAMC and TYMC counting results. JP17 also stipulates that a certain concentration of TTC can be added to TSA culture media to distinguish bacterial colonies and other impurities; if fungal growth affects the TAMC counting results of the sample, amphotericin B can be added to TSA culture media to inhibit fungal growth; if fungi spread and grow in SDA culture media containing antibiotics, rose bengal can be added to inhibit the spread of the phenomenon [11]. ChP2020 stipulates that when TYMC counting is affected by bacterial growth, SDA culture media containing antibiotics (such as chloramphenicol, gentamicin) or other selective culture media (such as rose bengal sodium agar medium) can be used [13].
However, neither USP43 nor EP10.0 explicitly mentions relevant requirements.

1.3 Microbial counting method

In terms of microbial counting methods, EP10.0 and JP17 both specify four counting methods, namely membrane filtration method, plate counting method (pouring, coating) and MPN method[10-11]; USP43 does not specify the plate counting method (pouring)[13], while ChP2020 only specifies the plate counting method (pouring)[13]. Since the MPN method has low accuracy and is extremely unreliable for fungal counting, the MPN method is only suitable for situations where other counting methods are not applicable, and can only be used for TAMC counting. In terms of method applicability, a method applicability investigation should be carried out before testing. USP43 requires that the recovery rate of the applicable bacteria count is greater than 70%[12], and the other three pharmacopoeias all adopt the requirement of a recovery factor of 2[10-11, 13].
The requirements for the culture temperature of TAMC counting and TYMC counting are consistent in each pharmacopoeia, but the regulations on the culture time and the appropriate range of colony counting are slightly different.

1.4 Heat-resistant bacteria count

Chinese herbal medicines are generally heated before consumption. Although heating can reduce the bioburden of the sample and reduce the risk of pathogenicity, heating cannot kill the heat-resistant bacteria in the Chinese herbal medicines. It can also easily lead to product spoilage and accumulation of biotoxins. Therefore, ChP2020 has added a heat-resistant bacteria count item, which is defined as "the general term for microorganisms detected by the total aerobic count method after being placed in a water bath (98-100°C) for 30 minutes" [13]. This method is aimed at decoction-type Chinese herbal medicines and evaluates the residual microorganisms in such Chinese herbal medicines after heating treatment; the other three pharmacopoeias do not include relevant methods.