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Citation: Yamaoki, R.; Uno, S.;

Kimura, S. Microbial Control in the

Primary Packaging of Pills Using

Ionizing Radiation and Its Effect on

Characteristic Constituents for

Quality Control in Irradiated Pills.

Processes 2022, 10, 300. https://

doi.org/10.3390/pr10020300

Academic Editor: Alina Pyka-Pajak

Received: 27 December 2021

Accepted: 30 January 2022

Published: 2 February 2022

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processes

Article

Microbial Control in the Primary Packaging of Pills UsingIonizing Radiation and Its Effect on Characteristic Constituentsfor Quality Control in Irradiated PillsRumi Yamaoki * , Satoshi Uno and Shojiro Kimura

Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Nasahara, Takatsuki City,Osaka 569-1094, Japan; philos.meta@gmail.com (S.U.); shojiro.kmr@ares.eonet.ne.jp (S.K.)* Correspondence: yamaoki@gly.oups.ac.jp; Tel.: +81-72-690-1020

Abstract: Pharmaceutical products that mix natural raw materials are subject to unavoidable contam-ination with microorganisms from the environment and animals. This study focused on the effect ofradiation on the quality of primary packaged pills, which are crude drug products. The pills, whichwere 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 ofbacteria were reduced to 103 CFU/g or less by 6 kGy of irradiation. The counts of the spore-formingbacteria Bacillus megaterium, B. cereus, and Brevibacillus brevis in the pills were reduced to not over100 CFU/g after 10 kGy irradiation. Although some of the cinnamaldehyde in the pills was oxidizedto cinnamic acid, the decomposition of swertiamarin, berberine, glycyrrhizin, and cinnamaldehydein the pills after 10 kGy irradiation were within the analytical accuracy by high-performance liquidchromatography. Gamma-ray or EB treatment at the final production of crude drug preparations waswithin the permissible standard value for the non-aqueous preparations for oral administration, withno statistically significant change in the indicator ingredients of crude drugs.

Keywords: pills; gamma rays; electron beams; microorganisms; primary packaging; natural products;quality control; survival counts; indicator ingredients; radiolysis

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 Chinesemedicine (TCM) and Kampo medicine for oral administration and have been used as aconvenient dosage form for carrying. Pills are traditionally produced by mixing crude drugpowders and additives, which are formed into spheres, coated, and dried. Thus, pills aredosage forms that can make the best use of the pharmacological effect of the combinedaction of each water- and fat-soluble component in crude drugs. Moreover, the use ofpills 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 derivedfrom the environment or animals because of the use of natural products. As a harmonizedstandard, for the European Pharmacopoeia, the Japanese Pharmacopoeia (JP), and theUnited States Pharmacopeia [1], the permissible standard values for the microbiologi-cal quality of non-aqueous preparations for oral administration of herbal medicine are103 CFU/g or CFU/mL for the total number of aerobic microorganisms and 102 CFU/g orCFU/mL for the total number of fungi. In contrast, the microbial quality of pills containingcrude drugs may be 100 times more than the standard levels required for oral non-aqueouspreparations because it is difficult to reduce the level of adhering microorganisms whilesuppressing the influence on the active ingredients [1]. However, some reports of outbreaksof Bacillus cereus in hospitals pointed out that B. cereus contamination of medical linen

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causes bacteremia and sepsis [2,3]. It is important to keep the bioburden of the final productused 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 beenused in combination therapy for cancer treatment. Decoctions or injections with microbio-logical 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 theclinical efficacy of components in decoctions or injections of TCM or Kampo medicine. Forexample, the reduction of gastrointestinal toxicity of the anticancer agent by Huangqin De-coction and PHY906 or the relief of postoperative ileus in patients undergoing surgery forgastrointestinal cancer by Daikenchuto has been reported [4–6]. When pills made of mixedcrude drug powder can control the bioburden at a level same as that of non-aqueous prepa-rations for oral administration, applying pills to immunocompromised patients should befurther developed.

Irradiation methods using gamma rays (γ-rays) or electron beams (EB) are used tosterilize medical devices and sterile preparations as well as crude drugs or herbal medicines,which result in less decomposition of crude drug components, volatilization of essentialoil components, and changes in color tone because the radiation method is a non-heatingtreatment. In addition, it is possible to sterilize the primary packaged drug, or the finalpackaged drug once radiation permeability is guaranteed. Microbicidal treatment at theprimary package or final product is effective for suppressing the propagation of microor-ganisms in the product in store and maintaining the long-term microbiological quality ofthe product post-treatment. However, when exposing the drugs to ionizing radiation, it isnecessary to investigate the effect of radiation [7]. To obtain a more appropriate bactericidaleffect, 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 andcrude drug preparations as a countermeasure against microorganism contamination [8,9].Many studies on the chemical and microbial qualities of irradiated herbs or medicinalplants have been reported [10–12]. However, there are limited reports on the effect ofirradiation treatment on herbal medicinal products. This study focused on the microbialcontrol in a primary packaged preparation for pills using ionizing radiation. The irradiationdose needs to be determined by the survival rate of microorganisms adhering to drugsand the residual rate of contained components—the balance between benefits and risksobtained there. Thus, the primary packaged pills were irradiated with γ-rays or EB atroom temperature in air, and the reduction of microorganisms, including spore-formingbacteria, in the pills and the radiolysis of characteristic constituents for quality control ofcrude drugs mixed in the pills were examined.

2. Materials and Methods2.1. 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%): Swertiaherb extract, powdered Phellodendron bark, powdered Cinnamon bark, powdered Gly-cyrrhiza (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 pillwas a 4 mm diameter sphere with a density of 1.1 g/cm3. Sample pills were dried at105 ◦C for 6 h and weighed accurately was obtained to determine the percentage loss ondrying [13] (p. 135). The loss on drying of pills was 3.21± 0.11%. The 30 pills for individualconsumption were provided in a small sachet as the primary package, and the sachet wasmade from laminated polyethylene terephthalate (PET), polyethylene (PE), and aluminumfoil. 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.

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2.2. Irradiation

The primary packing pills were laid flat, with no more than 5-mm thickness, sealedin polyethylene bags, and irradiated with γ-rays or EB. Irradiation, ranging from 1.0 to100 kGy, was applied at 22± 2 ◦C in the atmosphere. Gamma irradiation was performed ina source vessel of 4.2 PBq 60Co (0.05–18 kGy/h, Koga Isotope, Shiga, Japan). Electron beamirradiation was performed using a 5-MeV Dynamitron EB accelerator (4 kGy/s, RadiationDynamics, Japan Electron Beam Irradiation Service, Osaka, Japan). Post-irradiation sampleswere stored at room temperature (22± 2 ◦C, 50± 10% relative humidity) for up to 2 monthsbefore testing. The absorbed dose was measured using a cellulose triacetate dosimeter (FujiPhoto 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, andthe mean values were calculated. The absorbed dose was calculated from the difference inthe 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 mLof phosphate buffer (pH 7.2), and this method was repeated twice for a 5 min sonicationand 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 incubatedfor 5 d at 30–35 ◦C for determination of the TAMC and for 7 d at 20–25 ◦C for determinationof 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 with1 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 colonywere observed using a phase-contrast microscope (BX41, Olympus, Tokyo, Japan), andthe colonies were determined to be formed of either Gram-positive or Gram-negativebacteria [15]. For the detection of the spore-forming bacteria, Petri dishes were filled with1 mL of diluted sample solution and 0.3% yeast extract agar media (ca. 45 ◦C) and wereincubated for 5 d at 30–35 ◦C [16]. Each colony isolated by streaking was incubated for1 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 forquality control using the indicator ingredients swertiamarin, berberine, cinnamaldehyde,cinnamic acid, and glycyrrhizic acid in the pill were conducted through high-performanceliquid chromatography (HPLC) following the extraction conditions stated in Table 1. Thesample pills were crushed into a fine powder and mixed thoroughly. An accurately weighedportion of the powder was transferred to a centrifuge tube containing the dilution phase(mobile phase, 30 mL for detection of swertiamarin, berberine, cinnamaldehyde, andcinnamic acid or dilute ethanol, 40 mL for detection of glycyrrhizic acid) and sonicatedfor 5 min. This solution was centrifuged (1300× g, 20 ◦C) for 5 min, and the supernatantwas collected. The separated extraction residues were re-extracted from each dilution. Theextracted solutions were mixed, and each dilution was added to make the total volume100 mL (for berberine, cinnamaldehyde, cinnamic acid, and glycyrrhizic acid) or 200 mL (forswertiamarin). Then, the solution was filtered through a 0.47 µm filter. The separation ofthe indicator ingredients from the pills was performed thrice. Chromatographic separationswere performed using a C-VP HPLC system comprising LC-10ATvp, SPD-10Avp, andShim-pack CLC-ODS(M) ϕ 4.6 mm × 250 mm (Shimadzu, Kyoto, Japan) or COSMOSIL

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5C18-PAQ ϕ 4.6 mm × 150 mm (Nacalai Tesque, Kyoto, Japan). The injection volumewas 20µL. The signal from the detector was measured as the peak area using a dataprocessing system (C-R8A, Shimadzu, Kyoto, Japan). Calibration curves were preparedwith each dilution of the chemical constituents: swertiamarin (≥99.0%, Fujifilm WakoChemical, 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%, FujifilmWako Chemical, Osaka, Japan). The amount of each indicator ingredient was determinedusing the absolute calibration curve method.

Table 1. Conditions for the high-performance liquid chromatography (HPLC) separation and detec-tion 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 phase1/15M

KH2PO4·CH3CN·SDS1:1:1 g/L

KH2PO4·CH3CN65:35, pH = 2.5

H2O·CH3CN9:1

1M H3PO4·CH3CN5:3

Columntemperature 30 °C 30 °C 30 °C 30 °C

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

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 com-pared between the non-irradiated and irradiated samples using the Welch’s t-test. Statisticalsignificance was set at p < 0.05.

3. Results3.1. Microorganisms in the Pills

The TAMC of the sample pills was 104 CFU/g (average 1.6 × 104 CFU/g). The TYMCof the pills was less than 10 CFU/g. Specified microorganisms (bile-tolerant Gram-negativebacteria, Escherichia coli, Salmonella) were not detected in the pill. The counts of microbesin the sample pills conformed to the acceptance criteria for crude drugs, which are takendirectly without the extraction process and are directly consumed crude drug preparationscontaining powdered crude drug. The standardized acceptance criterion for TAMC is105 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 wasGram-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 areshown in Table 2. The main spore-forming bacilli were Bacillus megaterium, B. cereus, andBrevibacillus brevis and were 7.0 × 102–3.8 × 103 CFU/g.

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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 0.9Brevibacillus brevis 16.7 B. thiaminolyticus 0.9

B. cereus 6.5 B. thuringiensis 0.9B. licheniformis 1.9 Unanalyzed 37

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

3.2. Bactericidal Effect of Ionizing Radiation

Because of γ-ray or EB irradiation, most bacterial strains in the sample pills wereradiosensitive. The determined D10 values for the microbiota found in the samples, theabsorbed dose required to reduce the bacterial population by 90%, were calculated fromthe linear fit on the semi-logarithmic graph in Figure 1a. The D10 values of the pills were3.7 kGy for γ-ray irradiation and 4.8 kGy for EB irradiation, and the ratio of the D10 valueof EB to that of γ-ray irradiation was 1.3. In the survival curves of the genus of the sporespecies by γ-ray irradiation, a ‘shoulder’ appeared in the low-dose range before the linearslope started on B. cereus, as shown in Figure 1b. Radiation sensitive microorganismsmostly show an exponential survivor curve [17] (p. 20). The ‘shoulder’ in the low-doserange before the linear slope started may be explained by multiple targets and/or certainrepair processes being operative at low doses. The D10 values of the spore-forming bacteriain the pills were 5.5 kGy for B. megaterium, 3.0 kGy for Brevibacillus brevis, and 7.3 kGy forB. cereus.

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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.

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.

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.

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 1. Survival curves of microbes in the sample pills irradiated by ionized radiation. (a) Survivalcurves of the total aerobic microbial count (TAMC) related with irradiations. Gamma rays arerepresented by blue circles with a line of y = 1.3 × 104e−0.63x. The electron beam is represented bygreen circles with a line of y = 1.7 × 104e−0.50x. (b) Survival curves of the genus of spore species byγ-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 brevisis represented by gray triangles with a line of y = 1.8 × 103e−0.77x.

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

SD value (kGy) = D10 × logN0

NSAL(1)

where N0 is the number of microorganisms before irradiation, and NSAL is the target limitvalue 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 primarypacking pills were 4.1 kGy for γ-ray irradiation and 5.7 kGy for EB irradiation. The SDvalues in the primary packing pills for decreasing to 102 CFU/g on the genus of sporespecies were 8.7 kGy irradiation for B. megaterium, 3.7 kGy for Brevibacillus brevis, and6.2 kGy for B. cereus.

3.3. Effect of Irradiation on Indicator Ingredients

Figure 2 shows the HPLC chromatograms of the indicator ingredients in the samplepills. The contents of indicator ingredients in the non-irradiation pills (control value) were9.13± 1.71 mg/g pill for swertiamarin, 10.9± 0.19 mg/g pill for berberine, 0.17 ± 0.01 mg/gpill for cinnamic acid, 4.20 ± 0.04 mg/g pill for cinnamaldehyde, and 6.61 ± 0.10 mg/g pillfor glycyrrhizic acid, as shown in Table 3. The content of the indicator ingredients for eachamount of crude drug components (Phellodendron bark, Swertia herb, and Glycyrrhiza) inthe 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 pillsappeared to satisfy the standardized contents of crude drugs in JP (1.2% for berberine inPhellodendron bark, 2.0% for swertiamarin in Swertia herb, and 2.0% glycyrrhizic acid inGlycyrrhiza) [18] (p. 1938, p. 1996, p. 1862).

The HPLC chromatograms of the indicator ingredients in 100 kGy irradiated samplepills 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 irradiatedpills was almost unchanged (Table 3). In contrast, the detection of swertiamarin, cin-namaldehyde, and cinnamic acid in the irradiated pills differed slightly from that in thecontrol. The value of swertiamarin decreased in response to the increasing absorbed doseof 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 detectedin the sample pills fluctuated greatly, including that in the control (RSD, 0.187). The largestandard deviation appeared to owe to the non-uniform content among pills, given thatswertiamarin components were mixed as an extract of Swertia japonica in the pills. Thevalue of cinnamaldehyde in the 20 kGy irradiated pills was decreased by 4% (p < 0.05) ascompared to that of the control value. The value of cinnamic acid in the 60 kGy irradiatedpills showed a 12% increase (p < 0.05) as compared to that of the control value.

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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.

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

Figure 2. High-performance liquid chromatography (HPLC) chromatograms of the indicator in-gredients 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 3. Analysis of the indicator ingredients in the sample pills at varying irradiation doses.

Irradiation Swertiamarin Berberine Cinnamic Acid Cinnamaldehyde GlycyrrhizinDose (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).

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4. Discussion

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

When the pharmaceutical products are exposed to ionizing radiation, the radiationdose per unit time (dose rate) may cause differences in the effect of radiation. A variety ofexcitons 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 ofrecombination between radicals is high because the number of radicals generated within aunit time is extremely large. For that, in EB irradiation, it is deduced that the probabilityof hit to DNA of cells from radicals decreases. Therefore, the D10 values of TAMC in thesample pills irradiated with EB appeared to be approximately 1.3 times that of γ-rays. Asimilar tendency has been reported for the D10 values of black pepper after EB or γ-rayirradiation [19].

Owing to the high dose rate of EB, the processing time for the bactericidal effect of EBirradiation is shorter than γ-ray irradiation, but the radiolucency is lower than γ-ray. Thematerial used in the primary packaging of the sample pills was laminated PET film (density1.38 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, and0.95 cm for aluminum by calculation of R (g/cm2) = 0.542 × E − 0.133 (E > 0.8 MeV), as perthe Feather’s rule. Even when assuming each thickness of the PET and PE was 0.1 mm, theattenuation rate of 5-MeV EB to each thickness of the primary packaging materials wasestimated as 1.1% for PET, 0.7% for PE, and 0.2% for aluminum foil when the absorptioncurve 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 asample 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 whenthe primary packaging pills were laid on a flat surface and exposed to radiation. Withirradiation treatment of the boxed final products, which are packed dozens, γ-irradiationwill be more effective.

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

On the other hand, there is concern about the degradation of plant components andcell tissues, which are components of the matrix, with increasing exposure to ionizingradiation. In this study, we found little influence of ionizing radiation on some indicator

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ingredients derived from plant extracts or volatile components. First, the detection ofswertiamarin mixed as the extract of Swertia herb in the sample pills showed a decreasingtendency in response to absorbed doses. The decrease in swertiamarin in irradiated pillsmight be because of ions or radicals generated by ionized radiation hit with high probabilityon molecules of swertiamarin because the extracts include less matrix such as plant cellsand cellulose. In others, cinnamaldehyde detected in the sample pills decreased slightlywhen irradiated with 20 kGy or more; in contrast, cinnamic acid showed an increasingtendency with irradiation. The increase in the detection of cinnamic acid in irradiated pillsis because of the radio-oxidation of a part of cinnamaldehyde. Cinnamaldehyde is presentin 80–90% of the essential oil component of cinnamon and is known to easily volatilizeand fluctuate greatly depending on the treatment conditions [24], not only by ionizedradiation. Cinnamaldehyde is reported to be easily oxidized at low temperatures becauseof 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 fromirradiated raw materials by the degradation of the cell tissue by ionizing radiation [10–12].However, there was no clear increase in the extractability of the indicator ingredients in theirradiated 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 tothe combination of various ingredients. On the premise, it is impossible to standardize allthe components that make up a multi-component system in such drugs, some componentsare designated as quantitative indicator ingredients, and the contents are specified andstandardized. The quantitative indicator ingredients in the sample pills do not necessarilyrepresent the active ingredients for the drug. However, it is predicted that the influenceof another component by irradiation is similar, considering the biosynthesis of naturalproducts. Therefore, the change in the components of the stomachic drug pills with thispackaging condition (<5% loss on drying) by approximately 10 kGy irradiation for microbialcontrol 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 theradiolysis percent of bilirubin and cholic acids (cholic acid, deoxycholic acid, and lithocholicacid) in the 5% and 10% hydrous content pellets at γ irradiation of 774 C/kg was less than5%, 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 pillsshould be further investigated.

However, pills are molded using a mixture of active and inert ingredients. Thedisintegration rate of pills or the absorption rate of drugs varies depending on the structureof the molecules of additives and the viscosity of the components in crude drugs. Theprimary component of dry plant tissue is carbohydrates. In addition, polysaccharidesare also used as a popular binder for pills. Thus, there is a high probability that theprimary radiation reaction in the sample pills is the action on sugar molecules bearing aglucose unit. The cleavage or crosslinking of the molecular chain of the polymer is possiblycaused by irradiation. Alcohols, including sugars, cause a hydrogen abstraction reaction byirradiation, which occurs at the carbon bearing hydroxyl group, and forms a hydroxylalkylradical [27]. It has been reported that the main chain of polysaccharides, including celluloseor starches, is cleaved by ionizing radiation [28–30]. In the future, the physicochemicalinfluence of additives by irradiation should be investigated, including disintegration testsfor pills.

5. Conclusions

The effect of ionizing radiation on primary packaged pills of crude drug productswas investigated. The survival counts of microbes on the primary packaged pills werereduced to 103 CFU/g or less by 6 kGy irradiation. The counts of the spore-formingbacteria Bacillus megaterium, B. cereus, and Brevibacillus brevis in the pills were reduced to

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not over 102 CFU/g after 10 kGy irradiation. The degradation of swertiamarin, berberine,glycyrrhizin, and cinnamaldehyde, which are indicator ingredients contained in irradiatedsample pills, was within the analytic accuracy, although some of the cinnamaldehyde wasoxidized to cinnamic acid. Through ionizing radiation treatment at the final production ofcrude drug preparations, the bioburden satisfied the permissible standard value for thenon-aqueous preparations for oral administration, without loss of the components of crudedrugs. The findings of this study could be a reference for preparing herbal pharmaceuticalproducts for immunocompromised patients. In addition, it will be necessary to investigatethe effects of material properties on pharmaceutical preparations, including disintegrationin 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.; investi-gation, 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.; fundingacquisition, 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.

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