2024 Volume 72 Issue 12 Pages 1055-1060
The main ingredients of Maobushisaishinto (MBST) are ephedrine (EP), methyl eugenol (ME), and aconitine (AC). The pharmacological effects are presumed to be due to the combined effects of these ingredients. In this study, we investigated the impact of the particles present in MBST suspensions on the absorption of the ingredients. Coarse, colloidal, and molecular dispersions were detected when MBST was dispersed in water at 25 and 70 °C. Regardless of temperature, the ratio of MBST in molecular dispersions was the highest, and the ratio of coarse dispersions was greater than that of colloidal dispersions. Particles ranging from 50 to 900 nm were observed in the colloidal dispersions prepared by treatment at 25 °C for 3 min. However, in 70 °C water, the mean particle size decreased, and the number of nanoparticles tended to increase. The levels of EP, ME, and AC in molecular dispersions were higher than those in coarse and colloidal dispersions, with no significant difference observed between the coarse and colloidal dispersions. On the other hand, in small intestinal penetration, the levels of EP, ME, and AC in colloidal dispersions were higher than those in the other two dispersions. Moreover, adding colloidal particles to the dissolved drug (molecular dispersions) increased the drug’s permeability through the small intestinal membrane. In conclusion, colloidal particles are produced when MBST is suspended. Furthermore, we showed that these colloidal particles enhance the absorption of the main ingredients of MBST.
A Kampo preparation is essentially a drug that combines two or more herbal medicines in specified quantities. These Kampo formulations are covered by insurance as easily handled extracts, with granules being the most common form. Granules are superior to other dosage forms in terms of portability, ease of administration, and long shelf life. According to a questionnaire survey, approximately 90% of physicians in Japan currently use Kampo preparations for some kind of treatment.1)
Extract granules are supposed to be taken with room temperature water or hot water, and in some cases, as a suspension.2) In general, precipitates are observed in the suspensions of extract granules, which are classified academically into three types of dispersions: coarse dispersions, colloidal dispersions, and molecular dispersions. Briefly, coarse dispersions refer to dispersed particles larger than 1 µm that cannot pass through filter paper or semipermeable membranes, while colloidal dispersions refer to dispersed particles of 1 nm to 1 µm that can pass through filter paper but not semipermeable membranes. Molecular dispersions are homogeneously dissolved particles of 1 nm or less in an ionic or molecular form that can pass through both filter paper and semipermeable membranes. Molecular dispersions are generally called solutions. However, it is still unclear in what proportions these dispersions are obtained when Kampo preparations are suspended, the distribution of the main ingredients, and their absorption in different dispersions. Therefore, it is important to clarify the effect of the distribution of the main ingredients on absorption.
Maobushisaishinto (MBST) has been used to treat the common cold, nasal allergy, bronchoconstriction, inflammation, pain, and bacterial infections in Japan.3–9) The main ingredients are ephedrine (EP), methyl eugenol (ME), and aconitine (AC), and the pharmacological effects of MBST are presumed to be due to the combined effects of these ingredients. In this study, we investigated whether colloids based on MBST are present in suspensions. Moreover, we demonstrated the effect of produced colloids on the absorption of MBST by measuring the intestinal penetration of the main ingredients, such as EP, ME, and AC.
MBST was purchased from SANWA SHOYAKU (Tochigi, Japan). EP and ME were provided by FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and AC was obtained from Sigma-Aldrich Co., LLC (Tokyo, Japan). Zero point eight micrometer Minisart® syringe filters and 0.025 µm MF-Millipore™ Membrane Filters were purchased from Sartorius Japan K.K. (Tokyo, Japan), and Merck Millipore (MA, U.S.A.), respectively. All other chemicals were of the highest purity commercially available.
AnimalSeven-week-old male Wistar rats were provided by Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan). They were housed under standard conditions (light from 7:00 to 19:00, dark from 19:00 to 7:00, room temperature 25 °C) and given a commercial diet (CE-2, Clea Japan Inc., Osaka, Japan) and water. These experiments were approved on 1 April, 2021 by Kindai University (code KAPS-31-014) and were performed by the guidelines of Kindai University and the Japanese Pharmacological Society.
Preparation of MBST DispersionsMBST per packet (about 1.5 g) was suspended in approximately 180 mL of room temperature water (25 °C) or hot water (70 °C) and stirred for 3 or 20 min. Next, the obtained dispersions were centrifuged (100 × g, 3 min), and the supernatant was filtered with an 800 nm filter to collect the coarse dispersion. The adsorption ratio of EP, ME, and AC solution onto the 800 nm filter were 2.5, 4.5, and 3.9%, respectively. The precipitate formed at that time was dried and weighed as the coarse dispersions. After that, the supernatant was filtered with a 25 nm filter to separate the colloidal and molecular dispersions. The concentrations of ingredients in the coarse, colloidal, and molecular dispersions were measured by the HPLC method described below. The MBST content in the collected samples (coarse, colloidal, and molecular dispersions) was calculated by measuring the weight after drying. Moreover, the particle size in colloidal dispersions was measured using the NANOSIGHT LM10 (Quantum Design Japan, Tokyo, Japan), and the measurement conditions were a viscosity of 0.98 mPa·s, a wavelength of 405 nm, and a measurement time of 60 s. Additionally, the number of nanoparticles was also measured using the NANOSIGHT LM10.
HPLCEP, ME, and AC in the dispersions were measured using an LC-20AT system (HPLC; Shimadzu Corp., Kyoto, Japan) with an Inertsil® ODS-3 column (GL Science Co., Inc., Tokyo, Japan) at 35 °C.10) One microgram per milliliter of propyl p-hydroxybenzoate was used as an internal standard; 50 µL of the sample and 100 µL of methanol containing 0.1 µg of propyl p-hydroxybenzoate were mixed, and 10 µL of the mixture was injected. The mobile phases for the measurement of EP, ME, and AC were 0.1 M KH2PO4/methanol (97/3, v/v), methanol/water/acetonitrile (25/30/45, v/v/v), and acetonitrile/water/phosphoric acid (25/75/0.1, v/v/v), respectively, and the flow rate was 0.2 mL/min. Wavelengths of 281, 260, and 230 nm were detected for EP, ME, and AC, respectively.
In Vitro Small Intestinal Membrane PermeabilityThe in vitro intestinal penetration of EP, ME, and AC in MBST dispersions was evaluated according to a previous study using methacrylate cells.11,12) The enterocyte membranes of 7-week-old Wistar rats were set in the methacrylate cells, and 3 mL of MBST dispersions and isotonic N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (+Glc) buffer (10 mM HEPES, 136.2 mM NaCl, 5.3 mM KCl, 1.0 mM KH2PO4, 1.7 mM CaCl2·2H2O, 5.5 mM glucose, pH 7.4) were added to the donor and reservoir sides, respectively. In this study, the components were diluted and prepared using purified water, and samples with identical concentrations of the target components were prepared for each dispersion (EP 300 µM, ME 30 µM, and AC 100 µM), and used for the measurements. The methacrylate cells were kept at 37 °C in a thermostatic bath, and 50 µL samples were taken from the reservoir side at 1, 2, 4, 6, and 8 h. EP, ME, and AC levels were measured using the HPLC method described above and expressed as a ratio to the initial concentration on the donor side (%).
In the evaluation of the effect of colloidal particles on intestinal penetration of MBST dispersions, dispersions (EP 300 µM, ME 30 µM, and AC 100 µM) containing 0.1 × 1012, 0.5 × 1012, 1 × 1012, and 2 × 1012/mL colloidal particles were used as samples for the in vitro intestinal penetration experiment. The colloidal particles were collected from colloidal dispersions. Moreover, the amount of colloid particles was determined based on the amount in standard suspensions when the MBST was administered as described in the “Preparation of MBST dispersions” section.
Statistical AnalysisStatistical comparisons were performed using the Student’s t-test and Tukey–Kramer test with JMP (SAS Institute Inc., Cary, NC, U.S.A.). p < 0.05 was considered significant.
Figure 1 shows the MBST contents in coarse, colloidal, and molecular dispersions resulting from MBST dispersion in water. Regardless of temperature treatment (25 and 70 °C) and duration (3 and 20 min), the ratio of MBST in molecular dispersions was the highest, with the ratio of coarse dispersion being higher than that of colloidal dispersion (Figs. 1A–D). However, the ratios of coarse, colloidal, and molecular dispersions were similar between the 3 min and 20 min treatments. Increased temperature reduced the ratio of coarse dispersions and enhanced the ratio of molecular dispersions (Fig. 1D).
(A, B) Weight of MBST in coarse, colloidal, and molecular dispersions prepared by treatment at 25 °C for 3 min (A) and 20 min (B). (C) Weight of MBST in coarse, colloidal, and molecular dispersions prepared by treatment at 70 °C for 3 min. (D) Content rate of MBST in coarse, colloidal, and molecular dispersions prepared by treatment at 25 or 70 °C for 3 min and 20 min. Data are shown as the means ± standard error (S.E.) of 6 experiments. * p < 0.05, vs. coarse dispersions for each category. # p < 0.05, vs. colloidal dispersions for each category.
Figure 2 shows the particle distribution and number of MBST in colloidal dispersions. Particles ranging from 50 to 900 nm were observed in colloidal dispersions prepared at 25 °C for 3 min and 20 min, with no significant difference in nanoparticle number. However, at 70 °C for 3 min, the mean particle size of MBST decreased from 359.5 ± 19.6 to 239.6 ± 14.0 nm, and there was a tendency for an increase in nanoparticle number compared to the corresponding group at 25 °C.
(A, B) Particle distribution of MBST in colloidal dispersions prepared by treatment at 25 °C for 3 min (A) and 20 min (B). (C) Particle distribution of MBST in colloidal dispersions prepared by treatment at 70 °C for 3 min. (D) Particle number of MBST in colloidal dispersions prepared by treatment at 25 or 70 °C for 3 and 20 min. Data are shown as the means ± S.E. of 6 experiments.
Figure 3 shows the distribution of the main drugs (EP, ME, and AC) in each MBST dispersion (coarse, colloidal, and molecular dispersions). The levels of EP, ME, and AC in molecular dispersions were higher than those in coarse and colloidal dispersions, with no significant difference observed between coarse and colloidal dispersions.
The coarse, colloidal, and molecular dispersions (dis.) of MBST were prepared by treatment at 25 °C for 3 min. Data are shown as the means ± S.E. of 6 experiments. * p < 0.05, vs. coarse dispersions for each category. # p < 0.05, vs. colloidal dispersions for each category.
Figure 4 shows whether the intestinal penetration of EP, ME, and AC in coarse, colloidal, and molecular dispersions changed. EP, ME, and AC in colloidal dispersions exhibited higher membrane permeability compared to those in the other two dispersions (coarse and molecular dispersions). Moreover, the lowest permeability was observed in the molecular dispersions for all EP, ME, and AC. These results suggest that the presence of colloids may enhance the absorption of drugs. Therefore, we investigated the effect of the coexistence of colloidal particles on the intestinal penetration of EP, ME, and AC as shown in Fig. 5. The addition of colloidal particles to the dissolved drug (molecular dispersions) increased the drug’s permeability through the small intestinal membrane. Drug permeability tended to increase with the addition of 0.1–2 × 1012 particles/mL colloidal particles. Furthermore, even with as few as 0.1 × 1012 particles/mL colloidal additions, the permeability of drugs (EP, ME, and AC) through the intestinal membrane was significantly enhanced.
The coarse, colloidal, and molecular dispersions (dis.) of MBST were prepared by treatment at 25 °C for 3 min. The EP, ME, and AC levels were expressed as a ratio to the initial concentration on the donor side (%). Data are shown as the means ± S.E. of 6 experiments. * p < 0.05, vs. coarse dispersions for each category. # p < 0.05, vs. colloidal dispersions for each category.
The colloidal particles were collected from colloidal dispersions (dis.), and 0.1 × 1012, 0.5 × 1012, 1 × 1012, and 2 × 1012 particles/mL colloidal particles were added into molecular dispersions, and used as samples for the experiment of the in vitro intestinal penetration. Data are shown as the means ± S.E. of 6 experiments. * p < 0.05, vs. molecular dispersions for each category. # p < 0.05, vs. dispersions with 2 × 1012 particles/mL colloidal particles.
Multiple ingredients (e.g., EP, ME, and AC) contained in MBST are considered to contribute to its pharmacological actions. The status of MBST at the time of absorption significantly affects the bioavailability of these ingredients; however, it remains unclear how the particles present in MBST suspensions affect their absorption. In this study, we investigated the effect of insoluble substances on the absorption of MBST and found that the intestinal permeability of these main ingredients (EP, ME, and AC) of MBST was markedly enhanced in the presence of insoluble substances such as colloidal particles that occur when suspended in water.
First, we classified the status of MBST dispersions when suspended in water and the distribution of the main ingredients in each dispersion. Coarse, colloidal, and molecular dispersions were observed regardless of temperature. Although treatment time did not affect the ratio of coarse, colloidal, and molecular dispersions, the ratio of molecular dispersions tended to increase when suspended in 70 °C water compared to 25 °C water (Fig. 1). Additionally, the number of colloidal particles increased slightly, while particle size decreased in the group treated with 70 °C water (Fig. 2). This result suggests that MBST suspended in hot water may precipitate less compared to that in 25 °C water, making the product easier to administer.
Next, the distribution of EP, ME, and AC in coarse, colloidal, and molecular dispersions was investigated (Fig. 3). Although levels of EP, ME, and AC in coarse or colloidal dispersions were lower than those in molecular dispersions, EP, ME, and AC were attached to or enclosed within coarse and colloidal particles (Fig. 3). Moreover, the levels of EP, AC, and ME followed the order EP > AC > ME in all layers of MBST dispersions. The molecular weights of EP, ME, and AC are 165.24, 178.23, and 645.74, respectively. AC and ME are lipophilic, while EP is hydrophilic. Taken together, it was hypothesized that ingredients with lower molecular weight and hydrophobic properties are easily encapsulated in the coarse and colloidal particles of MBST.
Furthermore, we investigated the differences in intestinal absorption of the ingredients in the three dispersion layers (Fig. 4). For the three ingredients, intestinal penetration was highest in colloidal dispersions, followed by coarse and then molecular dispersions (Fig. 4). Particularly, penetration in colloidal dispersions was significantly higher compared to coarse and molecular dispersions. Therefore, we examined the relationship between the amount of colloids and drug absorption to determine whether the presence of colloidal particles enhances drug absorption (Fig. 5). The intestinal absorption of EP, ME, and AC was enhanced in the presence of colloidal particles. However, we did not observe a dependence on particle number, as the difference in intestinal penetration rate between 1 × 1012 and 2 × 1012 particles/mL colloidal particles was smaller than the difference observed with and without the presence of 0.1 × 1012 particles/mL colloidal particles. These results suggest that when MBST dispersions are orally administered, drug intake may be reduced unless precipitates are also ingested. Therefore, it is important to determine the composition of these precipitates, such as coarse and colloidal particles. Further studies are needed to evaluate the mechanism by which drug intestinal penetration is enhanced in the presence of coarse and colloidal particles. It has been reported that the endocytosis pathway is involved in the penetration of drug-loaded nanoparticles/hydrogels into cells and nuclei, and many researchers have elucidated the relationship between endocytosis and nanoparticle-based drug delivery.13–15) We also reported that co-instillation of nano-solid magnesium hydroxide enhances corneal permeability of hydrophilic drugs (such as dissolved timolol).16,17) Thus, the endocytosis pathway may also play a role in the enhanced intestinal penetration of drugs in MBST. Further studies analyzing the composition of coarse and colloidal particles, and investigating the involvement of endocytosis in the intestinal penetration of MBST by using endocytosis inhibitors, are currently being planned. Moreover, the evaluating of intestinal permeability using solutions with the actual contents is also important in further study.
In conclusion, colloidal particles are produced when MBST is suspended. Additionally, we demonstrated that these colloidal particles enhance the intestinal penetration of EP, ME, and AC, which are the main ingredients of MBST. The MBST contain numerous components in addition to their main active ingredients. Therefore, it is not possible to fully elucidate their effects by evaluating only the main ingredients. However, in this study, the MBST was assessed based on the dynamics of its three main ingredients, all of which demonstrated similar behavior. Furthermore, it is important to clarify the behavior of the components responsible for the medicinal effects of MBST. These findings provide valuable insights into the optimal administration of Kampo preparations, such as MBST.
The authors declare no conflict of interest.
