Microbial Control in the Primary Packaging of Pills Using Ionizing Radiation and Its Effect on Characteristic Constituents for Quality Control in Irradiated Pills

Abstract

Pharmaceutical products that mix natural raw materials are subject to unavoidable contamination with microorganisms from the environment and animals. This study focused on the effect of radiation on the quality of primary packaged pills, which are crude drug products. The pills, which were sealed in a sack for primary packaging laminated with polyethylene terephthalate, polyethylene, and aluminum foil, were irradiated by gamma rays or electron beam (EB). The survival counts of bacteria were reduced to 10³ CFU/g or less by 6 kGy of irradiation. The counts of the spore-forming bacteria Bacillus megaterium, B. cereus, and Brevibacillus brevis in the pills were reduced to not over 100 CFU/g after 10 kGy irradiation. Although some of the cinnamaldehyde in the pills was oxidized to cinnamic acid, the decomposition of swertiamarin, berberine, glycyrrhizin, and cinnamaldehyde in the pills after 10 kGy irradiation were within the analytical accuracy by high-performance liquid chromatography. Gamma-ray or EB treatment at the final production of crude drug preparations was within the permissible standard value for the non-aqueous preparations for oral administration, with no statistically significant change in the indicator ingredients of crude drugs.

1. Introduction

There are various dosage forms in herbal medicine products, including decoctions, injections, tablets, pills, and powders. Pills are a popular formulation in traditional Chinese medicine (TCM) and Kampo medicine for oral administration and have been used as a convenient dosage form for carrying. Pills are traditionally produced by mixing crude drug powders and additives, which are formed into spheres, coated, and dried. Thus, pills are dosage forms that can make the best use of the pharmacological effect of the combined action of each water- and fat-soluble component in crude drugs. Moreover, the use of pills reduces the load on the environment during manufacturing by reducing plant waste, including extraction residue.

It is unavoidable for herbal medicine to be contaminated with microorganisms derived from the environment or animals because of the use of natural products. As a harmonized standard, for the European Pharmacopoeia, the Japanese Pharmacopoeia (JP), and the United States Pharmacopeia [1], the permissible standard values for the microbiological quality of non-aqueous preparations for oral administration of herbal medicine are 10³ CFU/g or CFU/mL for the total number of aerobic microorganisms and 10² CFU/g or CFU/mL for the total number of fungi. In contrast, the microbial quality of pills containing crude drugs may be 100 times more than the standard levels required for oral non-aqueous preparations because it is difficult to reduce the level of adhering microorganisms while suppressing the influence on the active ingredients [1]. However, some reports of outbreaks of Bacillus cereus in hospitals pointed out that B. cereus contamination of medical linen causes bacteremia and sepsis [2,3]. It is important to keep the bioburden of the final product used in medical treatment low to eliminate the possibility of damaging the patient’s health.

Traditional Chinese medicine (TCM) or Kampo medicine in China or Japan has been used in combination therapy for cancer treatment. Decoctions or injections with microbiological qualities can be used as non-sterile substances for pharmaceutical use (decoctions) or sterilized pharmaceutical products (injections) because they are manufactured by boiling (decoctions) or filtering (injections). Recently, advances have been made in studies on the clinical efficacy of components in decoctions or injections of TCM or Kampo medicine. For example, the reduction of gastrointestinal toxicity of the anticancer agent by Huangqin Decoction and PHY906 or the relief of postoperative ileus in patients undergoing surgery for gastrointestinal cancer by Daikenchuto has been reported [4,5,6]. When pills made of mixed crude drug powder can control the bioburden at a level same as that of non-aqueous preparations for oral administration, applying pills to immunocompromised patients should be further developed.

Irradiation methods using gamma rays (γ-rays) or electron beams (EB) are used to sterilize medical devices and sterile preparations as well as crude drugs or herbal medicines, which result in less decomposition of crude drug components, volatilization of essential oil components, and changes in color tone because the radiation method is a non-heating treatment. In addition, it is possible to sterilize the primary packaged drug, or the final packaged drug once radiation permeability is guaranteed. Microbicidal treatment at the primary package or final product is effective for suppressing the propagation of microorganisms in the product in store and maintaining the long-term microbiological quality of the product post-treatment. However, when exposing the drugs to ionizing radiation, it is necessary to investigate the effect of radiation [7]. To obtain a more appropriate bactericidal effect, it is important to confirm quality deterioration of the object by irradiation in advance.

Here, we aimed to study the application of ionizing radiation to crude drugs and crude drug preparations as a countermeasure against microorganism contamination [8,9]. Many studies on the chemical and microbial qualities of irradiated herbs or medicinal plants have been reported [10,11,12]. However, there are limited reports on the effect of irradiation treatment on herbal medicinal products. This study focused on the microbial control in a primary packaged preparation for pills using ionizing radiation. The irradiation dose needs to be determined by the survival rate of microorganisms adhering to drugs and the residual rate of contained components—the balance between benefits and risks obtained there. Thus, the primary packaged pills were irradiated with γ-rays or EB at room temperature in air, and the reduction of microorganisms, including spore-forming bacteria, in the pills and the radiolysis of characteristic constituents for quality control of crude drugs mixed in the pills were examined.

2. Materials and Methods

2.1. Materials

The pills, an over-the-counter medicine for the digestive system in Japan, was selected. The components of the pills were four crude drugs (amount of ingredients, 80%): Swertia herb extract, powdered Phellodendron bark, powdered Cinnamon bark, powdered Glycyrrhiza (ratio of crude drug component, 1:2:2:1), medicinal carbon (amount of ingredients, 4%), and rice flour as inactive ingredients (amount of ingredients, 16%). The sample pill was a 4 mm diameter sphere with a density of 1.1 g/cm³. Sample pills were dried at 105 °C for 6 h and weighed accurately was obtained to determine the percentage loss on drying [13] (p. 135). The loss on drying of pills was 3.21 ± 0.11%. The 30 pills for individual consumption were provided in a small sachet as the primary package, and the sachet was made from laminated polyethylene terephthalate (PET), polyethylene (PE), and aluminum foil. The sample pills were obtained from wholesalers and stored at room temperature (22 ± 2 °C, 50 ± 10% relative humidity) for up to 6 months before testing.

2.2. Irradiation

The primary packing pills were laid flat, with no more than 5-mm thickness, sealed in polyethylene bags, and irradiated with γ-rays or EB. Irradiation, ranging from 1.0 to 100 kGy, was applied at 22 ± 2 °C in the atmosphere. Gamma irradiation was performed in a source vessel of 4.2 PBq ⁶⁰Co (0.05–18 kGy/h, Koga Isotope, Shiga, Japan). Electron beam irradiation was performed using a 5-MeV Dynamitron EB accelerator (4 kGy/s, Radiation Dynamics, Japan Electron Beam Irradiation Service, Osaka, Japan). Post-irradiation samples were stored at room temperature (22 ± 2 °C, 50 ± 10% relative humidity) for up to 2 months before testing. The absorbed dose was measured using a cellulose triacetate dosimeter (Fuji Photo Film, Tokyo, Japan) and a radiochromic dosimeter (Far West Technology, Goleta, CA, USA). The alanine-EPR dosimetry was used as a reference standard dosimetry system [14]. Four or six dosimeters were placed on both surfaces of samples to measure each dose, and the mean values were calculated. The absorbed dose was calculated from the difference in the absorbance before and after irradiation.

2.3. Microbial Tests

The total aerobic microbial count (TAMC) and total combined yeasts/molds count (TYMC) in the sample pills before and after irradiation were determined using the pour-plate method with casein soya bean digest agar medium and glucose peptone agar medium (Nihon Pharmaceutical, Tokyo, Japan). A sample was weighed (1 g) and was added to 9 mL of phosphate buffer (pH 7.2), and this method was repeated twice for a 5 min sonication and 5 min intermission. The suspension was serially diluted 10²-fold with phosphate buffer. The diluted solutions (1 mL) were poured into Petri dishes (φ 90 mm × 15 mm), agar media (ca. 45 °C) were added, and the mixtures were left to harden. These plates were incubated for 5 d at 30–35 °C for determination of the TAMC and for 7 d at 20–25 °C for determination of the TYMC. The tests were performed using at least two Petri dishes for each dilution.

To isolate the bacterial species in the sample pills, the Petri dishes were filled with 1 mL of a diluted solution of the sample and casein soya bean digest agar media (ca. 45 °C) and incubated for 1 d at 30–35 °C. The morphological characteristics of each colony were observed using a phase-contrast microscope (BX41, Olympus, Tokyo, Japan), and the colonies were determined to be formed of either Gram-positive or Gram-negative bacteria [15]. For the detection of the spore-forming bacteria, Petri dishes were filled with 1 mL of diluted sample solution and 0.3% yeast extract agar media (ca. 45 °C) and were incubated for 5 d at 30–35 °C [16]. Each colony isolated by streaking was incubated for 1 to 3 d at 30–35 °C and tested with a BBL Crystal Identification Kit (Becton-Dickinson, Tokyo, Japan).

2.4. Assay for the Determination of the Characteristic Constituent for Quality Control

The separation and assays for the determination of the characteristic constituent for quality control using the indicator ingredients swertiamarin, berberine, cinnamaldehyde, cinnamic acid, and glycyrrhizic acid in the pill were conducted through high-performance liquid chromatography (HPLC) following the extraction conditions stated in Table 1. The sample pills were crushed into a fine powder and mixed thoroughly. An accurately weighed portion of the powder was transferred to a centrifuge tube containing the dilution phase (mobile phase, 30 mL for detection of swertiamarin, berberine, cinnamaldehyde, and cinnamic acid or dilute ethanol, 40 mL for detection of glycyrrhizic acid) and sonicated for 5 min. This solution was centrifuged (1300× g, 20 °C) for 5 min, and the supernatant was collected. The separated extraction residues were re-extracted from each dilution. The extracted solutions were mixed, and each dilution was added to make the total volume 100 mL (for berberine, cinnamaldehyde, cinnamic acid, and glycyrrhizic acid) or 200 mL (for swertiamarin). Then, the solution was filtered through a 0.47 μm filter. The separation of the indicator ingredients from the pills was performed thrice. Chromatographic separations were performed using a C-VP HPLC system comprising LC-10ATvp, SPD-10Avp, and Shim-pack CLC-ODS(M) φ 4.6 mm × 250 mm (Shimadzu, Kyoto, Japan) or COSMOSIL 5C18-PAQ φ 4.6 mm × 150 mm (Nacalai Tesque, Kyoto, Japan). The injection volume was 20 μL. The signal from the detector was measured as the peak area using a data processing system (C-R8A, Shimadzu, Kyoto, Japan). Calibration curves were prepared with each dilution of the chemical constituents: swertiamarin (≥99.0%, Fujifilm Wako Chemical, Osaka, Japan), berberine hydrochloride (97.0–102.0%, Nacalai Tesque, Kyoto, Japan), cinnamaldehyde (≥98.0%, Fujifilm Wako Chemical, Osaka, Japan), cinnamic acid (≥99.5%, Fujifilm Wako Chemical, Osaka, Japan), and glycyrrhizic acid (≥99.0%, Fujifilm Wako Chemical, Osaka, Japan). The amount of each indicator ingredient was determined using the absolute calibration curve method.

2.5. Statistical Analysis

Statistical analyses were performed using Excel 2016 (Microsoft, Seattle, WA, USA). The mean and standard deviations were calculated for the results. The values were compared between the non-irradiated and irradiated samples using the Welch’s t-test. Statistical significance was set at p < 0.05.

3. Results

3.1. Microorganisms in the Pills

The TAMC of the sample pills was 10⁴ CFU/g (average 1.6 × 10⁴ CFU/g). The TYMC of the pills was less than 10 CFU/g. Specified microorganisms (bile-tolerant Gram-negative bacteria, Escherichia coli, Salmonella) were not detected in the pill. The counts of microbes in the sample pills conformed to the acceptance criteria for crude drugs, which are taken directly without the extraction process and are directly consumed crude drug preparations containing powdered crude drug. The standardized acceptance criterion for TAMC is 10⁵ CFU/g for TAMC and 10⁴ CFU/g for TYMC [1].

Eighty-six percent of the detected aerobic bacteria that adhered to the sample pills was Gram-positive, whereas 14% was Gram-negative. Bacilli were detected in 60% of spore-forming aerobic bacteria, as indicated by spore staining and morphological observations. The results of the isolation of spore-forming bacteria (rod-shaped bacterium) in the pills are shown in Table 2. The main spore-forming bacilli were Bacillus megaterium, B. cereus, and Brevibacillus brevis and were 7.0 × 10²–3.8 × 10³ CFU/g.

3.2. Bactericidal Effect of Ionizing Radiation

Because of γ-ray or EB irradiation, most bacterial strains in the sample pills were radiosensitive. The determined D₁₀ values for the microbiota found in the samples, the absorbed dose required to reduce the bacterial population by 90%, were calculated from the linear fit on the semi-logarithmic graph in Figure 1a. The D₁₀ values of the pills were 3.7 kGy for γ-ray irradiation and 4.8 kGy for EB irradiation, and the ratio of the D₁₀ value of EB to that of γ-ray irradiation was 1.3. In the survival curves of the genus of the spore species by γ-ray irradiation, a ‘shoulder’ appeared in the low-dose range before the linear slope started on B. cereus, as shown in Figure 1b. Radiation sensitive microorganisms mostly show an exponential survivor curve [17] (p. 20). The ‘shoulder’ in the low-dose range before the linear slope started may be explained by multiple targets and/or certain repair processes being operative at low doses. The D₁₀ values of the spore-forming bacteria in the pills were 5.5 kGy for B. megaterium, 3.0 kGy for Brevibacillus brevis, and 7.3 kGy for B. cereus.

The survival dose (SD) value (the required dose for decreasing the number of contaminating microorganisms to the target limit) was calculated using Equation (1):

(1)$SD value \left(kGy\right)=D_{10}\times \mathrm{l}\mathrm{o}\mathrm{g}\frac{N_0}{N_{SAL}}$

where N₀ is the number of microorganisms before irradiation, and N_SAL is the target limit value for microorganism contamination.

To meet the acceptance criteria (10³ CFU/g) for the microbiological quality of non-aqueous preparations for oral administration, the SD values of TAMC in the primary packing pills were 4.1 kGy for γ-ray irradiation and 5.7 kGy for EB irradiation. The SD values in the primary packing pills for decreasing to 10² CFU/g on the genus of spore species were 8.7 kGy irradiation for B. megaterium, 3.7 kGy for Brevibacillus brevis, and 6.2 kGy for B. cereus.

3.3. Effect of Irradiation on Indicator Ingredients

Figure 2 shows the HPLC chromatograms of the indicator ingredients in the sample pills. The contents of indicator ingredients in the non-irradiation pills (control value) were 9.13 ± 1.71 mg/g pill for swertiamarin, 10.9 ± 0.19 mg/g pill for berberine, 0.17 ± 0.01 mg/g pill for cinnamic acid, 4.20 ± 0.04 mg/g pill for cinnamaldehyde, and 6.61 ± 0.10 mg/g pill for glycyrrhizic acid, as shown in Table 3. The content of the indicator ingredients for each amount of crude drug components (Phellodendron bark, Swertia herb, and Glycyrrhiza) in the sample pills were estimated to be 4.1% for berberine, 7.1% for swertiamarin, and 5.1% for glycyrrhizic acid. The amounts of crude drug components mixed in the sample pills appeared to satisfy the standardized contents of crude drugs in JP (1.2% for berberine in Phellodendron bark, 2.0% for swertiamarin in Swertia herb, and 2.0% glycyrrhizic acid in Glycyrrhiza) [18] (p. 1938, p. 1996, p. 1862).

The HPLC chromatograms of the indicator ingredients in 100 kGy irradiated sample pills revealed a similar peak pattern to that of the control, and no new peak was detected, as shown in Figure 2. The detection of berberine and glycyrrhizic acid in the irradiated pills was almost unchanged (Table 3). In contrast, the detection of swertiamarin, cinnamaldehyde, and cinnamic acid in the irradiated pills differed slightly from that in the control. The value of swertiamarin decreased in response to the increasing absorbed dose of the pill, and the value of swertiamarin in 100 kGy irradiated pills had a 4% decrease (p > 0.05) as compared to that of the control value. The amount of swertiamarin detected in the sample pills fluctuated greatly, including that in the control (RSD, 0.187). The large standard deviation appeared to owe to the non-uniform content among pills, given that swertiamarin components were mixed as an extract of Swertia japonica in the pills. The value of cinnamaldehyde in the 20 kGy irradiated pills was decreased by 4% (p < 0.05) as compared to that of the control value. The value of cinnamic acid in the 60 kGy irradiated pills showed a 12% increase (p < 0.05) as compared to that of the control value.

4. Discussion

The number of microorganisms in pharmaceutical products varies depending on the level of contamination of the starting material and the condition from the time of manufacture to the time of administration. Microbial contamination in the manufacturing process of pills is because of the manufacturing environment, including dust generated by crushing natural product-derived raw materials, fluctuations in the bactericidal effect due to drying time and temperature, and an increase in the number of microorganisms by a high-water activity induced when water is added during the kneading stage. Therefore, the decontamination of microorganisms on the primary packaged product stage or the final product stage of the pharmaceutical product prevents the propagation of microbes from storage, and the microbiological quality of the product is useful for long-term maintenance.

When the pharmaceutical products are exposed to ionizing radiation, the radiation dose per unit time (dose rate) may cause differences in the effect of radiation. A variety of excitons are produced in the molecules by either the direct or the indirect action of radiation. In EB irradiation, it is a higher dose rate than γ-ray irradiation, and the probability of recombination between radicals is high because the number of radicals generated within a unit time is extremely large. For that, in EB irradiation, it is deduced that the probability of hit to DNA of cells from radicals decreases. Therefore, the D₁₀ values of TAMC in the sample pills irradiated with EB appeared to be approximately 1.3 times that of γ-rays. A similar tendency has been reported for the D₁₀ values of black pepper after EB or γ-ray irradiation [19].

Owing to the high dose rate of EB, the processing time for the bactericidal effect of EB irradiation is shorter than γ-ray irradiation, but the radiolucency is lower than γ-ray. The material used in the primary packaging of the sample pills was laminated PET film (density 1.38 g/cm³), PE (density, 0.92 g/cm³), and aluminum foil (thickness, 9 μm; density, 2.7 g/cm³). The maximum ranges on 5-MeV EB were estimated to be 1.9 cm for PET, 2.8 cm for PE, and 0.95 cm for aluminum by calculation of R (g/cm²) = 0.542 × E − 0.133 (E > 0.8 MeV), as per the Feather’s rule. Even when assuming each thickness of the PET and PE was 0.1 mm, the attenuation rate of 5-MeV EB to each thickness of the primary packaging materials was estimated as 1.1% for PET, 0.7% for PE, and 0.2% for aluminum foil when the absorption curve of incident mono-energetic electrons passing through a material was exponential. Thus, most interactions of EB occur in the pills because of the thickness of 4 mm on a sample pill with a density of 1.1 g/cm³ (the attenuation rate of 5-MeV EB for pill: ca. 16%). There was slightly difference in the bactericidal effect between γ-rays and 5-MeV EB when the primary packaging pills were laid on a flat surface and exposed to radiation. With irradiation treatment of the boxed final products, which are packed dozens, γ-irradiation will be more effective.

However, even when using γ-rays with high radiolucency, the composition of the medium or matrix surrounding the microorganism is suspected to contribute to the radiation resistance of microorganisms. The D₁₀ values for certain microorganisms can differ considerably in different media. It has been reported that radio sensitivity depends on the media type [20], and higher radiation doses are necessary to kill the microorganisms in soils than in pure cultures [21]. In this study, the D₁₀ values of the microbiota attached to γ-irradiated pills were higher than the D₁₀ values on the irradiated raw material, even if the number of adhered microorganisms were similar. For example, the D₁₀ value of TAMC in the irradiated pills (3.7 kGy) was higher than the D₁₀ value in Glycyrrhiza with the same level of TAMC (2.2 kGy) [22]. In addition, the D₁₀ values of the spore-forming bacteria in the pills were higher than those of spore-forming bacteria collected on filter paper [23]. The tendency of microorganisms to resist radiation in the sample pills suggests the radicals generated after irradiation is scavenged by some components in crude drugs or additives of the pharmaceutical product, that is due to a matrix effect.

On the other hand, there is concern about the degradation of plant components and cell tissues, which are components of the matrix, with increasing exposure to ionizing radiation. In this study, we found little influence of ionizing radiation on some indicator ingredients derived from plant extracts or volatile components. First, the detection of swertiamarin mixed as the extract of Swertia herb in the sample pills showed a decreasing tendency in response to absorbed doses. The decrease in swertiamarin in irradiated pills might be because of ions or radicals generated by ionized radiation hit with high probability on molecules of swertiamarin because the extracts include less matrix such as plant cells and cellulose. In others, cinnamaldehyde detected in the sample pills decreased slightly when irradiated with 20 kGy or more; in contrast, cinnamic acid showed an increasing tendency with irradiation. The increase in the detection of cinnamic acid in irradiated pills is because of the radio-oxidation of a part of cinnamaldehyde. Cinnamaldehyde is present in 80–90% of the essential oil component of cinnamon and is known to easily volatilize and fluctuate greatly depending on the treatment conditions [24], not only by ionized radiation. Cinnamaldehyde is reported to be easily oxidized at low temperatures because of its low activation energy, which leads to its deterioration during storage, transportation, and usage [25].

Some studies have reported an increase in the extractability of components from irradiated raw materials by the degradation of the cell tissue by ionizing radiation [10,11,12]. However, there was no clear increase in the extractability of the indicator ingredients in the irradiated sample pills. The reason may be that the pills contained the powder of herbs, and the plant cells in the powder were mechanically destroyed in the original process.

Most herbal medicines and crude drug products have medicinal properties owing to the combination of various ingredients. On the premise, it is impossible to standardize all the components that make up a multi-component system in such drugs, some components are designated as quantitative indicator ingredients, and the contents are specified and standardized. The quantitative indicator ingredients in the sample pills do not necessarily represent the active ingredients for the drug. However, it is predicted that the influence of another component by irradiation is similar, considering the biosynthesis of natural products. Therefore, the change in the components of the stomachic drug pills with this packaging condition (<5% loss on drying) by approximately 10 kGy irradiation for microbial control is within the analytic accuracy or the manufacturing error of sample pills. However, radiation reaction is influenced by the presence of oxygen or water. It was reported that the radiolysis percent of bilirubin and cholic acids (cholic acid, deoxycholic acid, and lithocholic acid) in the 5% and 10% hydrous content pellets at γ irradiation of 774 C/kg was less than 5%, and the radiolysis percent of that in the 20% hydrous contents pellets was 15–22% [26]. The radiation for over 10% hydrous contents pills, or for the deoxygenation packaging pills should be further investigated.

However, pills are molded using a mixture of active and inert ingredients. The disintegration rate of pills or the absorption rate of drugs varies depending on the structure of the molecules of additives and the viscosity of the components in crude drugs. The primary component of dry plant tissue is carbohydrates. In addition, polysaccharides are also used as a popular binder for pills. Thus, there is a high probability that the primary radiation reaction in the sample pills is the action on sugar molecules bearing a glucose unit. The cleavage or crosslinking of the molecular chain of the polymer is possibly caused by irradiation. Alcohols, including sugars, cause a hydrogen abstraction reaction by irradiation, which occurs at the carbon bearing hydroxyl group, and forms a hydroxylalkyl radical [27]. It has been reported that the main chain of polysaccharides, including cellulose or starches, is cleaved by ionizing radiation [28,29,30]. In the future, the physicochemical influence of additives by irradiation should be investigated, including disintegration tests for pills.

5. Conclusions

The effect of ionizing radiation on primary packaged pills of crude drug products was investigated. The survival counts of microbes on the primary packaged pills were reduced to 10³ CFU/g or less by 6 kGy irradiation. The counts of the spore-forming bacteria Bacillus megaterium, B. cereus, and Brevibacillus brevis in the pills were reduced to not over 10² CFU/g after 10 kGy irradiation. The degradation of swertiamarin, berberine, glycyrrhizin, and cinnamaldehyde, which are indicator ingredients contained in irradiated sample pills, was within the analytic accuracy, although some of the cinnamaldehyde was oxidized to cinnamic acid. Through ionizing radiation treatment at the final production of crude drug preparations, the bioburden satisfied the permissible standard value for the non-aqueous preparations for oral administration, without loss of the components of crude drugs. The findings of this study could be a reference for preparing herbal pharmaceutical products for immunocompromised patients. In addition, it will be necessary to investigate the effects of material properties on pharmaceutical preparations, including disintegration in the irradiated pills, to understand the influence of the absorption rate of drugs.

Author Contributions

Conceptualization, R.Y. and S.K.; methodology, R.Y., S.U. and S.K.; investigation, R.Y. and S.U.; validation, R.Y.; formal analysis, R.Y.; resources, R.Y. and S.K.; data curation, R.Y., S.U. and S.K.; writing—original draft preparation, S.U.; writing—review and editing, R.Y.; visualization, R.Y. and S.U.; supervision, R.Y. and S.K.; project administration, R.Y. and S.K.; funding acquisition, R.Y. and S.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Survival curves of microbes in the sample pills irradiated by ionized radiation. (a) Survival curves of the total aerobic microbial count (TAMC) related with irradiations. Gamma rays are represented by blue circles with a line of y = 1.3 × 104e−0.63x. The electron beam is represented by green circles with a line of y = 1.7 × 104e−0.50x. (b) Survival curves of the genus of spore species by γ-irradiation. Bacillus megaterium is represented by purple triangles with a line of y = 5.3 × 103e−0.48x. Bacillus cereus is represented by orange triangles with a line of y = 9.7 × 102e−0.36x. Brevibacillus brevis is represented by gray triangles with a line of y = 1.8 × 103e−0.77x.

Figure 2. High-performance liquid chromatography (HPLC) chromatograms of the indicator ingredients in the sample pills at non-irradiation (control) and after 100 kGy of EB irradiation. (a) Swertiamarin; (b) berberine; (c) cinnamaldehyde (C. Ald) and cinnamic acid (C. Acid); and (d) glycyrrhizin.

Table 1

Conditions for the high-performance liquid chromatography (HPLC) separation and detection of the characteristic constituents for quality control—berberine, cinnamaldehyde, cinnamic acid, swertiamarin, and glycyrrhizic acid, in the sample pills.

Analyte	Berberine	CinnamaldehydeCinnamic Acid	Swertiamarin	Glycyrrhizic Acid
Amount (pill)	0.2 g	0.2 g	0.5 g	0.5 g
Dilution	Eluent,30 mL→100 mL	Eluent,30 mL→100 mL	Eluent,30 mL→200 mL	Dilute ethanol,40 mL→100 mL
Column	Shim-pack CLC-ODS(M),φ 4.6 mm × 250 mm		COSMOSIL 5C18-PAQ,φ 4.6 mm × 150 mm
Mobile phase	1/15M KH2PO4·CH3CN·SDS1:1:1 g/L	KH2PO4·CH3CN65:35, pH = 2.5	H2O·CH3CN9:1	1M H3PO4·CH3CN5:3
Columntemperature	30 ℃	30 ℃	30 ℃	30 ℃
Flow rate	0.8 mL/min	1.0 mL/min	0.7 mL/min	0.8 mL/min
Detection	270 nm	280 nm	238 nm	254 nm

Table 2

Relative amounts of each genus of spore species (rod-shaped bacterium) in the sample pills.

Microorganisms	Amount (%)	Microorganisms	Amount (%)
Bacillus megaterium	35.2	B. sphaericus	0.9
Brevibacillus brevis	16.7	B. thiaminolyticus	0.9
B. cereus	6.5	B. thuringiensis	0.9
B. licheniformis	1.9	Unanalyzed	37

Unanalyzed involves spore-forming bacteria (cocci) and non-spore-forming bacteria.

Table 3

Analysis of the indicator ingredients in the sample pills at varying irradiation doses.

Irradiation	Swertiamarin	Berberine	Cinnamic Acid	Cinnamaldehyde	Glycyrrhizin
Dose (kGy)	(mg/g Pills)
Control	9.13 ± 1.71 a	10.9 ± 0.19 a	0.17 ± 0.01 a	4.20 ± 0.04 a	6.61 ± 0.10 a
1	9.20 ± 1.41 a	10.9 ± 0.34 a	0.17 ± 0.01 a	4.26 ± 0.08 a	6.60 ± 0.20 a
6	9.13 ± 1.31 a	10.8 ± 0.08 a	0.19 ± 0.01 a	4.16 ± 0.16 a	6.59 ± 0.21 a
10	9.05 ± 1.16 a	10.6 ± 0.86 a	0.18 ± 0.01 a	4.26 ± 0.12 a	6.58 ± 0.16 a
20	9.04 ± 2.31 a	11.1 ± 0.63 a	0.19 ± 0.01 a	4.04 ± 0.05 b	6.65 ± 0.05 a
60	8.87 ± 1.82 a	11.0 ± 0.11 a	0.19 ± 0.01 b	4.10 ± 0.08 a	6.70 ± 0.01 a
100	8.71 ± 1.64 a	11.1 ± 0.07 a	0.18 ± 0.005 a	4.05 ± 0.10 a	6.65 ± 0.04 a

Note: same letters (a, b) are not significantly different from each other (p = 0.05).

References

1. U.S. Pharmacopeia. Limits for Non-Sterile Products. Available online: https://www.usp.org/harmonization-standards/pdg/general-methods/limits-non-sterile-products (accessed on 1 November 2021).

2. Cheng, V.C.C.; Chen, J.H.K.; Leung, S.S.M.; So, S.Y.C.; Wong, S.C.; Wong, S.C.Y.; Tse, H.; Yuen, K.Y. Seasonal outbreak of bacillus bacteremia associated with contaminated linen in Hong Kong. Clin. Infect. Dis.; 2017; 64, pp. S91-S97. [DOI: https://dx.doi.org/10.1093/cid/cix044] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28475782]

3. Kato, K.; Matsumura, Y.; Yamamoto, M.; Nagao, M.; Ito, Y.; Takakura, S.; Ichiyama, S. Seasonal trend and clinical presentation of Bacillus cereus bloodstream infection: Association with summer and indwelling catheter. Eur. J. Clin. Microbiol. Infect. Dis.; 2014; 33, pp. 1371-1379. [DOI: https://dx.doi.org/10.1007/s10096-014-2083-1]

4. Wang, J.; Fan, H.; Wang, Y.; Wang, X.; Zhang, P.; Chen, J.; Tian, Y.; Zhang, W.; Xu, F.; Zhang, Z. Metabolomic study of Chinese medicine Huang Qin decoction as an effective treatment for irinotecan-induced gastrointestinal toxicity. RSC Adv.; 2015; 5, pp. 26420-26429. [DOI: https://dx.doi.org/10.1039/C5RA02581H]

5. Lam, W.; Bussom, S.; Guan, F.; Jiang, Z.; Zhang, W.; Gullen, E.A.; Liu, S.H.; Cheng, Y.C. Chemotherapy: The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity. Sci. Transl. Med.; 2010; 2, 45ra59. [DOI: https://dx.doi.org/10.1126/scitranslmed.3001270] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20720216]

6. Ishizuka, M.; Shibuya, N.; Nagata, H.; Takagi, K.; Iwasaki, Y.; Hachiya, H.; Aoki, T.; Kubota, K. Perioperative administration of traditional Japanese herbal medicine daikenchuto relieves postoperative ileus in patients undergoing surgery for gastrointestinal cancer: A systematic review and meta-analysis. Anticancer Res.; 2017; 37, pp. 5967-5974. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29061775]

7. World Health Organization. Annex 6 WHO Good Manufacturing Practices for Sterile Pharmaceutical Products; World Health Organization Technical Report Series, No. 961; World Health Organization: Geneva, Switzerland, 2011; pp. 273-276.

8. Yamaoki, R.; Kimura, S. Effectiveness of electron beam irradiation for microbial decontamination of turmeric powder (Curcuma longa Linne) and analysis of curcuminoid degradation. J. Food Process. Preserv.; 2018; 42, e13334. [DOI: https://dx.doi.org/10.1111/jfpp.13334]

9. Yamaoki, R.; Tsujino, T.; Kimura, S.; Mino, Y.; Ohta, M. Detection of organic free radicals in irradiated Foeniculi fructus by electron spin resonance spectroscopy. J. Nat. Med.; 2009; 63, pp. 28-31. [DOI: https://dx.doi.org/10.1007/s11418-008-0284-6]

10. Khattak, K.F.; Simpson, T.J.; Ihasnullah,. Effect of gamma irradiation on the extraction yield, total phenolic content and free radical-scavenging activity of Nigella staiva seed. Food Chem.; 2008; 110, pp. 967-972. [DOI: https://dx.doi.org/10.1016/j.foodchem.2008.03.003]

11. Gupta, P.; Garg, N.; Joshi, P. Effect of Gamma Irradiation on the Extraction Yield and Microbial Contamination of Medicinal Plants. Internet J. Food Saf.; 2011; 13, pp. 351-354.

12. Abolhasani, A.; Barzegar, M.; Sahari, M.A. Effect of gamma irradiation on the extraction yield, antioxidant, and antityrosinase activities of pistachio green hull extract. Radiat. Phys. Chem.; 2018; 144, pp. 373-378. [DOI: https://dx.doi.org/10.1016/j.radphyschem.2017.09.025]

13. Ministry of Health, Labour and Welfare. Tests for Crude Drugs, Loss on drying. The Japanese Pharmacopoeia Seventeenth Edition; Ministry of Health, Labour and Welfare: Tokyo, Japan, 2016; 135.Available online: https://www.mhlw.go.jp/file/06-Seisakujouhou-11120000-Iyakushokuhinkyoku/JP17_REV_1.pdf (accessed on 1 November 2021).

14. ISO/ASTM 51607 2013 Practice for Use of the Alanine-EPR Dosimetry System; International Organization for Standardization: Geneva, Switzerland, 2013.

15. Ryu, E. A Simple Method of Differentiation Between Gram-positive and Gram-negative Organisms without Staining. Kitasato Arch. Exp. Med.; 1940; 17, pp. 58-63.

16. Koshikawa, T. Recent Aspects of Radiation Sterilization. 5. Microbiological Methods and Validation for Radiation Sterilization of Medical Devices. Radioisotopes; 1998; 47, pp. 172-190. [DOI: https://dx.doi.org/10.3769/radioisotopes.47.172]

17. Mulder, R.W.A.W. The survivor curve. Salmonella Radiation of Poultry Carcasses; Spelderholt Poultry: Beekbergen, The Netherlands, 1982; 20.

18. Ministry of Health, Labour and Welfare. Crude Drugs and Related Drugs. (a) Phellodendron Bark, (b) Swertia Herb, (c) Glycyrrhiza. The Japanese Pharmacopoeia Seventeenth Edition; Ministry of Health, Labour and Welfare: Tokyo, Japan, 2016; (a) p.1938, (b) p.1996, (c) p.1862 Available online: https://www.mhlw.go.jp/file/06-Seisakujouhou-11120000-Iyakushokuhinkyoku/JP17_REV_1.pdf (accessed on 1 November 2021).

19. Ito, H.; Islam, M.S. Effect of dose rate on inactivation of microorganisms in spices by electron-beams and gamma-rays irradiation. Radiat. Phys. Chem.; 1994; 43, pp. 545-550. [DOI: https://dx.doi.org/10.1016/0969-806X(94)90165-1]

20. Van Gerwen, S.J.C.; Rombouts, F.M.; Riet, K.V.T.; Zwietering, M.H. A data analysis of the irradiation parameter for bacteria and spores under various conditions. J. Food Prot.; 1999; 62, pp. 1024-1032. [DOI: https://dx.doi.org/10.4315/0362-028X-62.9.1024]

21. Vela, G.R.; Wyss, O. Radiation Resistance of Soil Azotobacter. J. Bacteriol.; 1965; 89, pp. 1280-1285. [DOI: https://dx.doi.org/10.1128/jb.89.5.1280-1285.1965]

22. Kimura, S.; Taimatsu, M.; Kodani, N.; Ohnishi, T.; Okamoto, S. Radiation sterilization of the crude drug “glycyrrhiza”. Biocontrol. Sci.; 1997; 2, pp. 87-92. [DOI: https://dx.doi.org/10.4265/bio.2.87]

23. Koshikawa, T.; Sone, K.; Kobayashi, T. A simple identification method for spore-forming bacteria showing high resistance against γ-rays. Radioisotopes; 1993; 42, pp. 614-623. [DOI: https://dx.doi.org/10.3769/radioisotopes.42.11_614]

24. Suzuki, H.; Noguchi, M.; Harada, M. Search for a marker substance of Cinnamomi cortex leading to quality evaluation of a Chinese-medicinal formulation “Gorei-san”. Bull. Natl. Inst. Hyg. Sci.; 1987; 105, pp. 7-12.

25. Yu, C.; Li, Y.L.; Liang, M.; Dai, S.Y.; Ma, L.; Li, W.G.; Lai, F.; Liu, X.M. Characteristics and hazards of the cinnamaldehyde oxidation process. RSC Adv.; 2020; 10, pp. 19124-19133. [DOI: https://dx.doi.org/10.1039/C9RA10820C]

26. Kimura, S.; Sasaki, M.; Kondo, Y.; Jo, H.; Kanbashi, T.; Koide, H. Study on Radiosterilization of Crude Drug Pill Involving Bezoar Bovis Radiolysis of bilirubin and cholic acids in hydrous starch pellet. Radioisotopes; 1981; 30, pp. 669-673. [DOI: https://dx.doi.org/10.3769/radioisotopes.30.12_669]

27. Kawakishi, S. Oxidation and Depolymerization of Polysaccharides by Radical Reaction. J. Jpn. Soc. Starch Sci.; 1984; 31, pp. 161-170. [DOI: https://dx.doi.org/10.5458/jag1972.31.161]

28. Orozco, R.S.; Hernández, P.B.; Ramírez, N.F.; Morales, G.R.; Luna, J.S.; Montoya, A.J.C. Gamma irradiation induced degradation of orange peels. Energies; 2012; 5, pp. 3051-3063. [DOI: https://dx.doi.org/10.3390/en5083051]

29. Zhu, F. Impact of γ-irradiation on structure, physicochemical properties, and applications of starch. Food Hydrocoll.; 2016; 52, pp. 201-212. [DOI: https://dx.doi.org/10.1016/j.foodhyd.2015.05.035]

30. Lam, N.D.; Quynh, T.M.; Diep, T.B.; Binh, P.T.; Lam, T.D. Effect of gamma irradiation and pyrolysis on indigestible fraction, physicochemical properties, and molecular structure of rice starch. J. Food Process. Preserv.; 2021; 45, e15880. [DOI: https://dx.doi.org/10.1111/jfpp.15880]