Preparation of Goreisan Suppository and Pharmacokinetics of trans-Cinnamic Acid after Administration to Rabbits

Abstract

Goreisan suppository is prepared as a hospital preparation, and successfully used for the treatment of diarrhea and vomiting in young children with common cold. While clinical efficacy of the suppository has been reported, few studies have been carried out to clarify the preparation procedure and pharmacokinetics of the suppository. In this study, trans-cinnamic acid (CA) was used as a representative substance of goreisan constituents, and assayed by HPLC-UV. We investigated the properties of goreisan suppositories prepared using various sizes of pulverized goreisan extract granules, in vitro dissolution profiles using the reciprocating dialysis tube method, and pharmacokinetics in rabbits compared with those for goreisan enema. Mass and content uniformity tests on the suppositories of three size fractions, 0–75, 75–150, and 150–300 µm, showed good acceptance for all kinds of suppository. Storage stability at 4°C was maintained until 4 months. In vitro dissolution of CA from the suppository was proportional to time until 45 min, and slower than that from the enema. Finally, 80% of CA had dissolved at 60 min. Pharmacokinetic study in rabbits revealed that the area under the plasma concentration–time curve from 0 to 120 min (AUC_{0–120 min}) of the suppository was twice that of the enema. Moreover, from a study in rabbits using CA injection and CA suppository, we revealed that CA was rapidly and well absorbed from the rectum, showing 84% absolute bioavailability. Thus, we illustrated the defined preparation procedure of the suppository and the superiority of the suppository over the enema. This study will support evidence that the suppository is fast-acting and efficacious in clinical use.

Goreisan, a five-ingredient powder with Poria, is an herbal formula consisting of Polyporus, Alismatis Rhizoma, Sclerotium Poriae Cocos, Atractylodes macrocephalae Rhizoma, and Cinnamomi Cortex, and its herbal extract granule is accepted as a Kampo medicine in Japan.¹⁾ Goreisan is used generally for patients with the following health conditions and symptoms: low urination, edema, diarrhea, vomiting, dizziness, and hangover. In particular, goreisan is highly effective for preventing diarrhea and vomiting in young children with common cold.²⁾ In terms of oral use of this herbal medicine, however, it is hardly accepted by infants and young children owing to its unpleasant taste and odor.²⁾ Therefore, goreisan extract granules are changed to suppositories by pharmacists in hospital pharmacies.^3,4) The goreisan suppository is reported to be effective for clinical use in children.^5,6)

There are, however, some issues to clarify for the goreisan suppository, concerning its properties and pharmacokinetic behavior. The goreisan suppositories are prepared from powdered goreisan extract granules and suppository bases. In the preparation procedure, there is a difference in size fractionation of powdered goreisan extract granules among hospitals. Size fractionation would influence the properties of the suppositories, such as content uniformity and dissolution behavior. On the other hand, goreisan suppository is known to be fast-acting.³⁾ However, the pharmacokinetics of goreisan suppository is unclear. Besides, enema of goreisan is also used for the treatment of acute gastroenteritis in children with vomiting.^7,8) The enema is prepared by suspending powdered goreisan extract granules in warm water or normal saline. However, it is unknown whether there are differences between the goreisan suppository and the enema in terms of their pharmacokinetics.

Although the clinical efficacy of goreisan rectal preparations has been reported, data on the physical properties and pharmacokinetics of the preparations are still lacking because goreisan contains many active pharmaceutical ingredients that include well-known and unknown substances. In general, Kampo, namely Japanese traditional medicine, contains many constituents. Actually, it is impossible to determine all constituents of the medicine. Similarly, although the goreisan extract granule contains many constituents, the granule is well regulated as an ethical medicine.⁹⁾ In this study, we selected trans-cinnamic acid (CA) as a representative substance of goreisan constituents, which is one of the principal active pharmaceutical ingredients,¹⁰⁾ because it can be accurately detected using HPLC. For instance, Kampo medicine maouto suppository is evaluated using l-ephedrine as an indicative substance.¹¹⁾

In this study, we investigated the effects of the size of powdered goreisan on the content uniformity and dissolution behavior of goreisan suppositories by using the detection of CA. Then, the pharmacokinetics of CA after administration of the goreisan suppository and goreisan enema in rabbits was investigated to obtain fundamental basic information on its clinical efficacy. At present, no report focusing on the rectal absorption of CA appears to be available. Therefore, we also elucidated the rectal absorption of CA in rabbits using pure CA suppository and injection.

MATERIALS AND METHODS

Materials

Goreisanryo extract fine granules (Kotaro Pharmaceutical Co., Ltd., Osaka, Japan) were used as the goreisan. Hard fat Witepsol H-15 (Vosco, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) was used as a suppository base. Normal saline was obtained from Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan. HPLC-grade acetonitrile and methanol (MeOH) were purchased from Honeywell International Inc. (U.S.A.). CA and p-hydroxypropyl benzoic acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and used as a standard and an internal standard (IS), respectively. All other chemicals were of reagent grade.

Preparation of Goreisan Suppository

The goreisan suppository was prepared using a fusion method. Briefly, goreisan extract granules were ground and sieved with a 50 mesh sieve (opening: 300 µm). Using 100 and 200 mesh sieves (opening: 150 and 75 µm, respectively), the obtained goreisan powders were classified in three sizes of 0–75, 75–150, and 150–300 µm, named L, M, and H, respectively. Then, the goreisan powder was mixed well with suppository base Witepsol H-15 melted at 60°C at a weight ratio of 1 : 1. The resultant mixture was poured into plastic containers (1.75 mL, Kanae Co., Ltd., Osaka, Japan), and was allowed to solidify at room temperature and stored at 4°C until use. The suppository weighted 2 g and contained 1 g of powdered goreisan.

Preparation of Goreisan Enema

Goreisan extract granule was ground and sieved with a 50 mesh sieve (opening: 300 µm). Then, one gram of the powdered goreisan was suspended in 10 mL of normal saline by hand.

Preparation of CA Formulations

CA suppository was prepared by the fusion method. Namely, 10 mg of CA was mixed well with 1 g of Witepsol H-15 melted at 60°C. Then, the mixture was poured into a plastic container (1.75 mL) and was allowed to solidify at room temperature and stored at 4°C. The CA suppository contained 10 mg of CA. CA injection was prepared by dissolving 1 mg of CA into normal saline to make a final volume of 10 mL, and then filtered with a membrane filter (0.22 µm, Milex-GV, Millipore, U.S.A.).

Determination of CA in the Suppositories and Goreisan Granules

A suppository or one gram of goreisan granules was dissolved in 40 mL of MeOH–water (1 : 1, v/v) mixture at 50°C.¹¹⁾ One milliliter of p-hydroxy propylbenzoic acid MeOH solution (IS solution, 0.7 mg/mL) was added to the solution. Then, the resultant solution was centrifuged (3500 rpm, 5 min). The supernatant was filtered with a membrane filter (0.45 µm, Minisart RC15, Sartorius Stedim Biotech GmbH, Germany) and was introduced onto HPLC.

In Vitro Dissolution Test

Dissolution of CA from the suppository and enema was investigated at 37±0.5°C using the reciprocating dialysis tube method with tapping¹²⁾ (HZ-11D, Miyamoto Riken Industries Co., Ltd., Osaka, Japan). The dissolution medium was 1 L of 50 mM phosphate buffer (pH 7.4), and the membrane was 20 cm of dialysis membrane (No. 27, Wako Pure Chemical Industries, Ltd.). Up and down stroke speed was 40 rpm and stroke width was 55 mm. Sampling was conducted according to the experimental plan, and CA concentration was assayed using HPLC. The test was repeated three times.

Animals

Japanese white rabbits (male, Japan SLC Inc., Shizuoka, Japan) weighing 2.2±0.3 kg were housed with free access to a normal diet (ORC-4, Clea Japan, Tokyo, Japan) and water, and were maintained on a 12-h light/12-h dark cycle in a room with controlled temperature (22–24°C) and humidity (30–50%). The animals were fasted for 24 h prior to experimentation, but continued to have free access to water during this time. The study was performed in accordance with the guidelines of the Experimental Animal Ethics Committee of the University of Shizuoka.

Pharmacokinetic Study in Rabbits

Rabbits were administered with a suppository or enema containing 1 g of goreisan by insertion into the anus, which was then closed using a clip to prevent leakage. For CA rectal absorption study, rabbits were administered with a CA suppository (10 mg) rectally, and intravenously injected with CA (100 µg) into a marginal ear vein according to crossover design with at least one week washout period. After administration, blood samples were collected from the auricle vein of the other ear at designated times. Plasma samples were isolated from the whole blood by centrifugation and stored at −20°C until determination of the concentration of CA.

Assay of CA in the Plasma of Rabbits

The plasma concentration of CA was determined using HPLC.¹³⁾ Briefly, 5 µL of IS solution (1 µg/mL), 50 µL of 3% acetic acid solution, and 1 mL of MeOH–acetonitrile mixture (1 : 1, v/v) was added to the plasma sample of rabbits (100 µL). After centrifugation at 9000 rpm for 10 min, the supernatant fluid was separated and dried a using rotary evaporator. Then, the residue was solved into a mobile phase, and was introduced onto HPLC.

Determination of CA Using HPLC

HPLC (LC-10ADVp, Shimadzu Co., Ltd., Kyoto, Japan) was performed using the an analytical column (Inertsil ODS-100 V, 3 µm, 2.1 mmφ×150 mm, Tosoh Co., Tokyo, Japan) with a mobile phase (MeOH–acetonitrile–0.6% acetic acid aqueous solution=25 : 20 : 55, v/v) delivered at a flow rate of 0.2 mL/min. The wavelength of detection was 272 nm (SPD-10AVp, Shimadzu Co., Ltd.). CA concentration was determined using peak area for dissolution test and peak area ratio of CA and IS for the other studies.

Data Analysis

The linear trapezoidal method was used to calculate the area under the plasma concentration–time curve from 0 to 120 min (AUC_{0–120 min}) after intravenous and rectal administration in rabbits. In the case of rectal administration, the peak plasma concentration (C_max) and the time to the peak plasma concentration (T_max) were obtained by inspection. In the case of injection, the plasma concentration at time 0 (C₀) was calculated using linear regression from the first three points of the log plasma concentration–time curve. Results are expressed as mean±S.D. The differences of pharmacokinetic parameters between the two groups were tested by Student’s t-test. The p values <0.05 were considered statistically significant.

RESULTS

Properties of Goreisan Suppositories

Since there is inter-hospital difference in the preparation procedure, we examined the effect of size fraction on the properties of suppositories. To investigate the influence of size fractionation of goreisan powder on the properties, four kinds of goreisan suppository were prepared using L, M, H, and non-fractionized goreisan powders. The weight and CA content of the goreisan suppositories are listed in Table 1. The weight of all samples was within 1.97–2.04 g, conforming to the mass uniformity test of Japanese Pharmacopeia 16th Edition (JP16). Moreover, the CA content of all samples was good, varying in the range of 186–209 µg. Content uniformity test of JP16 was also conducted for each sample, resulting in its adoption. On the other hand, raw material, that is, goreisanryo extract fine granules, contained 191±2.0 µg (mean±S.D., n=3) of CA per gram. Therefore, the suppositories contained 99.5–107% of goreisan powder. These results indicated that the preparation procedure was suitable to maintain the quality of the suppositories. Besides, there was no significant difference in weight and CA content among the four kinds of suppository prepared at different size fractions. This indicated that size would not influence the preparation procedure.

Table 1. Weight and CA Content of the Goreisan Suppositories

Sample name	Size fraction of powder	Weight (g)	CA content (µg)
L-sup	0–75 µm	1.99±0.02	192±1.9
M-sup	75–150 µm	2.03±0.01	205±3.6
H-sup	150–300 µm	1.98±0.01	191±3.9
Suppository*	0–300 µm	2.00±0.00	190±3.8

* Not fractionized. Each value represents the mean±S.D. (n=10). No significant difference (p<0.05) was detected among suppositories prepared from various goreisan powders (one-way ANOVA followed by Tukey’s multiple comparison).

Stability during Storage

In hospital preparation, storage life is important for planning the production cycle. Therefore, we investigated the stability during storage at 4°C using determination of the CA content in the suppositories. The results are presented in Fig. 1. The residual CA percent against the initial content was 99.0±3.9% after storage for 4 months. A relatively high level of CA remained in the suppositories, indicating that the storage life was at least 4 months.

Fig. 1. Residual Percent of CA in the Suppositories after Storage at 4°C Compared with the Initial Amount

Each value represents the mean±S.D. (n=3). No significant differences against the initial amount were observed (Student’s t-test).

In Vitro Dissolution from Suppository

In general, the particle size of drug powder influences the dissolution rate. Therefore, we examined L-sup, M-sup, and H-sup for the dissolution behavior of CA. Dissolution profiles are presented in Fig. 2. Dissolution profiles were linear with respect to time until 45 min, finally reaching about 90% dissolution. The dissolution profiles of CA from the three kinds of suppository were similar to each other, being almost superimposable. There was no influence of the size fraction on the dissolution behavior, indicating no need for fractionation in the preparation procedure.

Fig. 2. Release Profiles of CA from Goreisan Suppositories

Release tests were performed in 1 L of phosphate buffer (pH 7.4) at 37±0.5°C using the reciprocating dialysis tube method with tapping. Each value represents the mean±S.D. (n=3).

Pharmacokinetics of CA after Rectal Administration of Goreisan Suppository and Enema

After rectal administration of goreisan suppository or enema at a dose of 1 g/body to four rabbits, the plasma concentration of CA was determined over 120 min. The mean plasma concentration–time profiles are presented in Fig. 3. When the goreisan formulations were administered via the rectal route, plasma CA levels reached the maximal level at 10–15 min after dosing, and decreased to near-zero level at 90 min. The observation of the profile of the enema showed that plasma CA was eliminated quickly. The pharmacokinetic parameters of CA after rectal administration of goreisan suppository and enema are summarized in Table 2. No significant difference in C_max and T_max was observed between the two formulations. AUC_{0–120 min} obtained after suppository and enema administration were 1.30 and 0.586 µg·h/mL, respectively. Thus, pharmacokinetic analysis revealed that the suppository showed significantly higher bioavailability than the enema.

Fig. 3. Plasma Concentration–Time Curves of CA after Rectal Administration of Goreisan Preparations at a Dose of 1 g Goreisan/Body in Rabbits

Each value represents the mean±S.D. (n=4). Asterisks show significant differences between the two formulation at * p<0.05 and ** p<0.01 (Student’s t-test).

Table 2. Pharmacokinetic Parameters of Goreisan Rectal Formulations in Rabbits

Parameter	Suppository	Enema
Cmax (µg/mL)	2.52±0.66	2.09±1.3
Tmax (h)	0.292±0.14	0.167±0.0
AUC0–120 min (µg·h/mL)	1.30±0.36*	0.586±0.27

Each value represents the mean±S.D. (n=4). The asterisk shows a significant difference between the two formulations at * p<0.05 (Student’s t-test).

To confirm the difference of the AUC_{0–120 min}, the in vitro dissolution behavior of CA from the enema was examined. The suppository gradually melted within 10 min from the start of the test due to external heat condition (37°C), and dispersed in the dialysis tube. The goreisan powders, which were released from the suppository or the enema, were retained in the dialysis tube and some sank at the lower position in the tube. The dissolution profile is shown in Fig. 4. The dissolution rate of CA from the enema was faster than that of the suppository. Totals of 63 and 80% of CA were dissolved in the medium at 30 and 60 min, respectively. It appears that there is no state of release from the base in the case of the enema compared with the suppository. The faster dissolution would cause rapid absorption and elimination of CA in plasma.

Fig. 4. Release Profiles of CA from Goreisan Suppository and Enema

Release tests were performed in 1 L of phosphate buffer (pH 7.4) at 37°C using the reciprocating dialysis method. Each value represents the mean±S.D. (n=3).

Bioavailability of CA after Rectal Administration

To confirm the rectal absorption and bioavailability of CA after rectal administration, we carried out pharmacokinetic study using pure CA suppository and CA injection. The average plasma level–time curves of CA after intravenous and rectal administration are shown in Fig. 5. The plasma levels are presented as the plasma concentration divided by the dose because there were differences in the dose between the formulations due to difficulty in manufacturing the injection with a high dose. The plasma levels of CA from the CA suppository peaked at 5 min, and then gradually returned to the baseline level after 60 min. In contrast, plasma CA levels after injection decreased rapidly, reaching the baseline at 45 min. This indicated that CA was continuously absorbed until about 60 min. Pharmacokinetic parameters are summarized in Table 3. T_max of the suppository was about 6 min, indicating rapid absorption of CA from rectum. C_max of the suppository showed about 28% of C₀, and bioavailability calculated from AUC_{0–120 min} obtained after rectal administration and AUC_{0–120 min} obtained after intravenous administration was about 84%. These observations indicated that CA was well absorbed from the rectum.

Fig. 5. Plasma Level Profiles of CA Normalized Dose after Intravenous and Rectal Administration in Rabbits

Each value represents the mean±S.D. (n=4).

Table 3. Pharmacokinetic Parameters of CA Concentration Divided by Dose after Intravenous and Rectal Administration in Rabbits

Parameter	Injection	Suppository
C0 (ng/mL/dose)	144±75	—
Cmax (ng/mL/dose)	—	41.0±16
Tmax (h)	—	0.104±0.036
AUC0–120 min (ng·h/mL/dose)	20.4±10	17.1±7.5

Plasma concentration of CA was divided by dose to compare the two formulations directly. Each value represents the mean±S.D. (n=4).

Moreover, the absorption rate of CA from the rectum was evaluated by a deconvolution method¹⁴⁾ from the time-courses of CA level after dosing of injection and suppository, and is presented in Fig. 6. Half of the total uptake amount was rapidly absorbed in the first 12 min. Thereafter, the CA absorption continued to about 60 min. It appears that CA shows good absorbability from the rectum.

Fig. 6. Absorption–Time Curve of CA from CA Suppository in Rabbits

Cumulative absorption percent was obtained from intravenous and rectal administration data using deconvolution method. Each value represents the mean±S.D. (n=4).

DISCUSSION

This study clarified the effect of size fractionation of powdered goreisan extract granules on product uniformity of goreisan suppository and in vitro CA dissolution from the suppository. The three kinds of suppository prepared from the goreisan powders L, M, and H were adopted after JP16 mass and content uniformity tests (Table 1), and then showed similar dissolution profiles of CA (Fig. 2). These results indicate that the size fraction of powdered goreisan does not affect the quality of the suppositories. According to the manufacture’s information on goreisan extract granules,¹⁵⁾ the dissolution of CA from goreisan extract granules is quite rapid, reaching 100% dissolution at 5 min when a dissolution test was conducted in water at a speed of 100 rpm using the JP16 paddle method. Thus, size fractionation of powdered goreisan in the range of 0–300 µm was not needed before mixing with suppository base.

Then, we elucidated the pharmacokinetics of CA after rectal administration of the goreisan suppository as well as the goreisan enema, indicating that AUC_{0–120 min} of the suppository was twice that of the enema (Fig. 3, Table 2). In some cases, goreisan enema is used instead of suppository.¹⁶⁾ However, AUC_{0–120 min} of the enema was quite different from that of the suppository, suggesting that they differ in their clinical efficacy. Of course, further research is expected to clarify the difference between the two formulations. In addition, compared with the enema, the suppository is advantageous in that no cumbersome handling is required.¹⁷⁾ Therefore, pharmacists should be able to prepare the suppository easily if rectal formulation of goreisan is needed.

Moreover, we revealed the rectal absorption of pure CA in rabbits. To our knowledge, there is no report of an investigation of the rectal absorption of CA. To clarify the pharmacokinetics of CA suppository, we determined the time-course of plasma concentrations of CA after administration rectally and intravenously to rabbits (Fig. 5). Absolute bioavailability of the CA suppository was estimated to be 84% from the AUC_{0–120 min} of the two formulations, suggesting good absorbability (Table 3). Then, the cumulative absorption of CA from the suppository was calculated by a deconvolution method. This profile showed that about 80% of the total uptake amount was absorbed by 30 min (Fig. 6). The high bioavailability is probably due to the avoidance of hepatic first-pass elimination¹⁸⁾ by CA absorbed from the rectum, and the lower contribution of metabolic enzymes and transporters in the intestine to the absorption of CA from the rectum. Further investigations are needed to clarify the absorption mechanism of CA upon rectal administration of goreisan suppository because intestinal absorption of CA in rats is reported to be affected by the metabolic enzyme CYP 3A4¹⁹⁾ and the transporter gp-1.²⁰⁾ This result suggested that CA in goreisan suppository was rapidly absorbed rectally, implying the rapid onset of a medical effect.

In this study, we evaluated the physical and pharmacokinetic properties of rectal formulations using CA as a representative of the main constituents in goreisan. This is a limitation of this study. In general, Kampo medicines consist of several herbal drugs, and contain many active pharmaceutical ingredients. It is not necessarily the case that all of the ingredients show similar properties and pharmacokinetics. Therefore, it might be necessary to clarify the pharmacokinetics of all ingredients in Kampo medicines. However, we considered that the study using a representative of the main constituents was meaningful to give us an indication of the physical and pharmacokinetic properties. Therefore, these results provide undisputed evidence that the formulation and preparation procedure are appropriate and support the clinical efficacy of goreisan suppository.

For some hospital preparations, the defined preparation procedure, evidence of the storage life, and the pharmacokinetics still remain unclear. Therefore, pharmacists should elucidate such information as well as the clinical efficacy of hospital preparations, to achieve safe and effective pharmacotherapy using them.

In conclusion, we confirmed that there is no need for fractionation under 300 µm of powdered goreisan extract granules in the preparation procedure. The residual content of CA in the suppository was maintained at 100% during 4 months of storage at 4°C. The in vitro dissolution rate of CA from the suppository was proportional to time until 45 min, reaching to 80% dissolution. Although the suppository and the enema showed similar C_max and T_max, the AUC_{0–120 min} of the suppository was twice that of the enema. Rectal absorption of CA was rapid and continued up to 60 min, reaching 80% of absolute bioavailability. These results support the clinical evidence that goreisan suppository is fast-acting and highly effective for diarrhea and vomiting in children.

REFERENCES

• 1) TSUMURA Goreisan Extract Granules for Ethical Use. Package insert, 4th ver., 2007.
• 2) Hashimoto H. Efficacy of goreisan for dizziness in children associated with acute gastroenteritis. Sci. Kampo Med, 25, 178–180 (2001).
• 3) Nishi K, Tataka K, Asano S, Tanaka O, Matsui H, Oshima M, Matsuda Y, Okada N, Sakurai M, Kanamoto I, Kurose K. Effects of goreisan suppository on vomiting in children—comparison with domperidone suppository. J. Jpn. Soc. Hosp. Pharm., 34, 1173–1176 (1998).
• 4) Jingami S, Fukunaga E. Investigation of the administration and clinical usefulness of suppositories containing goreisan. J. Jpn. Soc. Hosp. Pharm., 41, 1543–1546 (2005).
• 5) Yoshida M. Efficacy of goreisan suppository for vomiting in young children. Jpn. J. Orient. Med., 28, 36–38 (2000).
• 6) Yoshida M. Efficacy of goreisan suppository. Jpn. Pediatr. Soc. Orient. Med., 19, 13–17 (2003).
• 7) Fukutomi O. Study of goreisan enema for the treatment of acute enterogastritis in children associated with vomiting. Evolv. Kampo, 1, 25–28 (2005).
• 8) Fukutomi O, Sakurai S, Nada K, Orii T. Study of effect of goreisan enema on acute gastroenteritis of children. J. Trad. Med., 23, 151–155 (2006).
• 9) 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.” Eisei Shikenjo Hokoku, 105, 7–12 (1987).
• 10) Kotaro goreisanryo extract fine granules. Interview form, 5th ed., 2007.
• 11) Nishimura N, Doi N, Uemura T, Taketani T, Hayashi G, Kasai T, Kanai R, Yamaguchi S, Iwamoto K, Naora K. Pharmaceutical analysis and clinical efficacy of Kampo medicine, maoto, extract suppository against pediatric febrile symptoms. Yakugaku Zasshi, 129, 759–766 (2009).
• 12) Itoh S, Teraoka N, Matsuda T, Okamoto K, Takagi T, Oo C, Kao HD. Reciprocating dialysis tube method: Periodic tapping improved in vitro release/dissolution testing of suppositories. Eur. J. Pharm. Biopharm., 64, 393–398 (2006).
• 13) Li P, Zhang Y, Xiao L, Jin X, Yang K. Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study. Anal. Bioanal. Chem., 389, 2259–2264 (2007).
• 14) Veng-Pedersen P. An algorithm and computer program for deconvolution in linear pharmacokinetics. J. Pharmacokinet. Biopharm., 8, 463–481 (1980).
• 15) TSUMURA Goreisan Extract Granules for Ethical Use. Interview form, 2nd ed., 2006.
• 16) Miyakawa S, Ito K, Haruki E. Summary of a questionnaire on goreisan suppository and enema. Jpn. Pediatr. Soc. Orient. Med., 22, 41–43 (2007).
• 17) Iwakoshi H, Takai M, Ishida M, Imai N, Masamura S, Fukutomi O, Matsuo N, Orii T. Investigation of goreisan enema on vomiting in children—practice for outpatients and patients at home. Jpn. J. Nursing, 65, 488–490 (2001).
• 18) de Boer AG, Moolenaar F, de Leede LG, Breimer DD. Rectal drug administration: clinical pharmacokinetic considerations. Clin. Pharmacokinet., 7, 285–311 (1982).
• 19) Ikehata M, Ohnishi N, Matsumoto T, Kiyohara Y, Maeda A, Kawakita T, Takara K, Yokoyama T. Effects of sairei-to on the pharmacokinetics of nifedipine in rats. Phytother. Res., 22, 12–17 (2008).
• 20) Li W, Sun X, Ji Y, Nan L. Studies on intestinal absorption mechanism and influential factors of cinnamic acid in rats. ICME Int. Confer., 1–5 (2009).