Tokishakuyakusan Research Articles

Efficacy of tokishakuyakusan and mecobalamin on post-infectious olfactory dysfunction: A prospective multicenter study

ObjectiveTo determine if tokishakuyakusan (TSS) is effective for treating post-infectious olfactory dysfunction (PIOD) compared with vitamin B12 (mecobalamin). MethodsWe conducted a randomized, nonblinded clinical trial. Patients with PIOD enrolled in 17 hospitals and clinics from 2016 to 2020 were randomly divided into two groups, and we administered TSS or mecobalamin for 24 weeks. Their olfactory function was examined using interviews and T&T olfactometry. The improvement of olfactory dysfunction was assessed following the criteria of the Japanese Rhinologic Society. ResultsOverall, 82 patients with PIOD were enrolled in this study. In the TSS and mecobalamin groups, 39 patients completed the medication regimen. In the TSS and mecobalamin groups, olfactory dysfunction was significantly improved based on self-reports and olfactory test results. The improvement rate of olfactory dysfunction was 56% in the TSS group and 59% in the mecobalamin group. Early intervention within 3 months produced a better prognosis than the treatment initiated after 4 months. Furthermore, age and sex differences were not observed. Both medications produced no severe adverse events. ConclusionThe present study showed that TSS and mecobalamin might be useful for treating PIOD.

A Chinese Medicine, Tokishakuyakusan, Increases Bovine Oviductal Tonus via G Protein-Coupled Estrogen Receptor 1.

Early embryo and sperm transport through the oviductal isthmus depends on the contraction and relaxation of the smooth muscle layers. Dysfunction of the oviduct transport is considered to be one of the causes of infertility. For human infertility, Chinese medicine is used in East Asia. Although there are many clinical reports regarding Tokishakuyakusan (TSS), there is little scientific evidence that it affects infertility. In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation via the G protein-coupled estrogen receptor 1 (GPER1). We collected bovine oviductal isthmic tissues at four stages of the estrous cycle, classified based on a macroscopic observation of the ovary. The Magnus method was used to monitor longitudinal contractility (frequency, contraction force, and tonus). The effects of TSS solution, GPER1 agonist (G-1), and antagonist (G-15) on oviductal contractility were examined. The protein expression level of GPER1 in the oviductal isthmic smooth muscle of each estrous stage was assessed by Western blotting. Although TSS did not affect frequency and contraction force, the tonus was significantly increased by TSS or G-1 at all stages (p < 0.05), and the effect was especially highest at days 1-4 after ovulation. The addition of G-15 significantly suppressed the TSS-induced increase of oviductal tonus at all stages (p < 0.05). There was no significant difference in GPER1 protein expression among the estrous stages. TSS affects oviductal contractility by increasing tonus via GPER1, and it may accelerate gamete and early embryo transport by contracting the oviducts longitudinally.

Tokishakuyakusan ameliorates lowered body temperature after immersion in cold water through the early recovery of blood flow in rats

Ethnopharmacological relevance‘Cold feeling’ is a subjective feeling of unusual coldness that aggravates fatigue, stiffness, and other symptoms, thereby reducing quality of life. Tokishakuyakusan (TSS) is a Kampo medicine reported to improve cold feeling and is used to treat symptoms aggravated by cold feeling. However, the mechanism of action of TSS is unclear. Cold feeling may involve reduced blood flow and subsequent inhibition of heat transport. Therefore, elucidating the effects of TSS on blood flow is one of the most important research topics for clarifying the mechanism of action of TSS. Aim of the studyWe aimed to evaluate the effect of TSS on recovery from lowered body temperature by the immersion of rats in cold water and to clarify the involvement of blood flow in the action of TSS. Materials and methodsAfter female Wistar rats underwent 9 days of low room temperature stress loading (i.e. room temperature of 18 °C), they were subjected to immersion in cold water (15 °C) for 15 min. Body surface temperature, rectal temperature, and plantar temperature were measured before and after immersion in cold water. Blood flow was measured before and after immersion in cold water without low room temperature stress loading. TSS (0.5 g/kg or 1 g/kg) or the vehicle (i.e. distilled water) was orally administered once daily for 10 days for the measurement of body temperature or once 30 min before immersion in cold water for the measurement of blood flow. In addition, we examined the effect of TSS on calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) cells, the effect of TSS ingredients on transient receptor potential (TRP) channels, and the effect of TSS ingredients on the membrane potential of vascular smooth muscle cells and evaluated the mechanism of the effects of TSS on blood flow. ResultsBody temperature and blood flow decreased after immersion in cold water and then recovered over time. A comparison of body temperature at each timepoint or area under the curve showed that TSS (1 g/kg) accelerated the recovery of body surface temperature, rectal temperature, and blood flow. TSS significantly increased CGRP release from DRG cells, which disappeared after pretreatment with HC-030031 (a transient receptor potential ankyrin 1 [TRPA1] antagonist). The effects of seven TSS ingredients on TRP channels were examined. The agonistic effect on TRPA1 was observed for atractylodin, atractylodin carboxylic acid and levistolide A. Among the TSS ingredients, atractylodin carboxylic acid had significant hyperpolarising effects. ConclusionsThe mechanism by which TSS accelerates the recovery of lowered body temperature in rats after immersion in cold water may involve the acceleration of the recovery of lowered blood flow. Increased CGRP release from DRG cells by TSS, TRPA1 activation by TSS ingredients, and membrane potential changes in vascular smooth muscle cells caused by TSS ingredients are part of the mechanism of action of TSS. These findings may partly contribute to the interpretation of the beneficial effects of TSS on cold feeling.

Potency of Tokishakuyakusan in treating preeclampsia: Drug repositioning method by in vitro screening of the Kampo library.

IntroductionPreeclampsia therapy has not been established, except for the termination of pregnancy. The aim of this study was to identify a potential therapeutic agent from traditional Japanese medicine (Kampo) using the drug repositioning method.Materials and methodsWe screened a library of 74 Kampo to identify potential drugs for the treatment of preeclampsia. We investigated the angiogenic effects of these drugs using human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were performed to measure the levels of placental growth factor (PlGF) in conditioned media treated with 100 μg/mL of each drug. We assessed whether the screened drugs affected cell viability. We performed tube formation assays to evaluate the angiogenic effects of PlGF-inducing drugs. PlGF was measured after administering 10, 50, 100, and 200 μg/mL of the candidate drug in the dose correlation experiment, and at 1, 2, 3, 6, 12, and 24 h in the time course experiment. We also performed tube formation assays with the candidate drug and 100 ng/mL of soluble fms-like tyrosine kinase 1 (sFlt1). PlGF production by the candidate drug was measured in trophoblastic cells (BeWo and HTR-8/SVneo). The Mann-Whitney U test or one-way analyses of variance followed by the Newman-Keuls post-hoc test were performed. P–values < 0.05 were considered significant.ResultsOf the 7 drugs that induced PlGF, Tokishakuyakusan (TS), Shoseiryuto, and Shofusan did not reduce cell viability. TS significantly facilitated tube formation (P = 0.017). TS administration increased PlGF expression in a dose- and time-dependent manner. TS significantly improved tube formation, which was inhibited by sFlt1 (P = 0.033). TS also increased PlGF production in BeWo (P = 0.001) but not HTR-8/SVneo cells (P = 0.33).ConclusionsBy using the drug repositioning method in the in vitro screening of the Kampo library, we identified that TS may have a therapeutic potential for preeclampsia. Its newly found mechanisms involve the increase in PlGF production, and improvement of the antiangiogenic state.

Efficacy of Tokishakuyakusan add-on therapy with low-dose oral contraceptive pills on endometriosis patients with dysmenorrhea.

Tokishakuyakusan (TSS) is representative of popular Kampo medicines for Japanese women to treat dysmenorrhea, irregular menstruation and menopause. Current hormonal treatments and low-dose oral contraceptives (OCs) used as a first-choice drug for primary symptoms, however, have a high incidence of adverse events without improving patients' satisfaction. We evaluated the efficacy of TSS as additional treatment on the quality of life (QOL) of patients taking OCs with reduced therapeutic satisfaction. Twelve patients treated with OCs for endometriosis were enrolled. TSS (7.5 g/day) in combination with OCs were given for three menstrual cycles. The main outcome was the degree of QOL assessed using the Endometriosis Health Profile-30 (EHP-30). Secondary outcomes were the Menstrual Distress Questionnaire (MDQ), patient's satisfaction using a Likert scale and cold feeling-visual analogue scale (VAS). In EHP-30, significant changes by TSS with OCs in each menstrual cycle were not found, although the scores for pain and emotional well-being tended to decrease. In MDQ, water retention in the premenstrual phase was improved. With regard to cold feeling-VAS, 61.6 mm at pretreatment decreased to 31.3 mm at the endpoint. Average Likert scale values after TSS treatment declined from 4.3 to 2.6. Cold feeling and patient's satisfaction have improved. Although the influence of TSS add-on therapy on QOL was moderate, possible improvement of cold feeling and menstruation-related symptoms was suggested.

Recovery Over Time and Prognostic Factors in Treated Patients with Post-Infectious Olfactory Dysfunction: A Retrospective Study.

The aims of the present study were to clarify the time-course of olfactory recovery and the prognostic factors in PIOD patients treated with Toki-shakuyaku-san (TSS). A retrospective cohort study of patients with PIOD was conducted by reviewing patients' medical records. This study included patients who received TSS or a combination of TSS and zinc sulfate. Olfactory function was examined by T&T olfactometer at each 3-monthly follow-up visit. Patients with normal and mild olfactory dysfunction were excluded. Gender, age, treatment, duration of disease until the first visit and olfactory function scores of the T&T olfactometer at the first visit were analyzed as candidate clinical predictors of recovery. A total of 82 PIOD patients with ages ranging from 16 to 79 years were included. The mean duration of follow-up was 14.5 months (range 3-45 months). The number of patients with olfactory recovery increased for 24 months and the cumulative recovery rate was 77.3%. In about 60% of patients, olfactory recovery occurred within 6 months. Multivariate analysis showed that younger age (<65 years) and residual olfactory function were significantly associated with good olfactory recovery. We revealed recovery rates over time in patients with PIOD. The recovery of olfactory function often occurred during the early period (≤6 months). However, the number of patients with olfactory recovery increased for a long-term of 24 months after the first visit. Residual olfactory function and younger age were prognostic factors exactly. TSS may be a useful therapeutic agent for patients with PIOD. We believe that these results provide important information that is useful for counseling patients with PIOD.

The effects of tokishakuyakusan, a traditional Japanese medicine (kampo), ferulic acid and paeoniflorin, on human endometriotic stromal cells and peritoneal macrophages

ObjectivesTo evaluate the anti-inflammatory and anti-angiogenetic effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo), and its ingredients, ferulic acid (FA) and paeoniflorin (PA) on endometriotic stromal cells (ESC) and peritoneal macrophages. Study designEndometriotic tissues were obtained from 16 patients and peritoneal macrophages were obtained from 11 patients that had undergone laparoscopic surgery for ovarian endometriosis. ESC isolated from endometriotic tissues and peritoneal macrophages were cultured, and pre-treated with 300 μg/mL of TSS, 500 μM FA or 50 μM PA. ESC and peritoneal macrophages were then stimulated with IL-1β. Concentrations of IL-8 and VEGF protein in supernatants were then detected and measured using specific ELISAs. TSS (4 g/kg body weight) was orally administered to female Sprague-Dawley rats. The concentration of FA in plasma and uteri was measured using liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS/MS). ResultsTSS and FA but not PA decreased the secretion of inflammatory cytokine (IL-8) and angiogenic factor (VEGF) in ESC. TSS and FA also suppressed the secretion of inflammatory cytokine (IL-8) from peritoneal macrophages. FA was detected in plasma and in uterine tissues after the oral administration of TSS to rats. ConclusionsOur study demonstrates that TSS has anti-inflammatory and anti-angiogenic effects on endometriosis related cells by controlling inflammatory cytokine and growth factor secretion from cells, and these effects, at least partially, may be due to the direct effects of the TSS ingredient FA.

Tokishakuyakusan, a Kampo medicine, attenuates endometriosis-like lesions and hyperalgesia in murine with endometriosis-like symptoms.

How are the effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo) on murine endometriosis model? BALB/c mice were used for making the murine endometriosis model. Homogeneous uterus was surgically implanted with lipopolysaccharide (LPS) in peritoneal cavity. We administered 2weeks of TSS (1.0g/kg) orally. Upon treatment completion, we performed the hot plate test for all mice and collected blood samples before sacrifice. Then, the endometriosis-like lesions and uteri in the abdominal cavity were harvested. Concentrations of several cytokines in sera and cyst fluids were measured using Bio-Plex Suspension Array System. IL-33 localization was determined by immunohistochemistry. Gene expression of inflammatory cytokines in the endometriosis-like lesions or the eutopic endometrium was evaluated by real-time RT-PCR. After 14days of TSS treatment, the numbers of endometriosis-like cysts and cyst weight were significantly decreased. In TSS-treated mice, the latency against heat stimuli was extended. Inflammatory cytokine concentrations in sera were not changed by TSS treatment. TSS intake decreased IL-33 mRNA expression in endometriosis-like lesions and led to the tendency of attenuation of the elevated IL-33 synthesis in the cyst fluids of lesions. These results suggest the TSS ameliorated the hyperalgesia and lesion formation on the LPS-accelerated endometriosis-like model. TSS represents a possible ideal target of novel therapeutics for endometriosis patients with dysmenorrhea.

Tokishakuyakusan, a traditional Japanese medicine (Kampo) mitigates iNKT cell-mediated pregnancy loss in mice.

Tokishakuyakusan (TSS) is a traditional herbal medicine that has been used empirically to prevent recurrent pregnancy loss. Its mode of action remains unclear. With their potent capacity to produce cytokines, invariant natural killer (iNKT) cells are involved in the control of fetomaternal immunity in early gestation. This study aimed to clarify the effect of TSS on iNKT cell activities in a well-studied murine miscarriage model. Pregnant mice were fed 1% TSS-containing or control diet from the day of vaginal plug formation. Alpha-galactosylceramide (AGC) was administered intraperitoneally to the pregnant mice at day 9.5 postcoitus (pc) to stimulate iNKT cells. Peripheral cytokine levels were evaluated using cytokine arrays. The percentage of iNKT cells among splenocytes was examined by flow cytometric analysis. The incidence of pregnancy loss was assessed at day 12.5 pc. The ratio of fetal resorptions to total conceptuses was significantly higher in the group exposed to TSS (34%) than in controls (78%). A rapid and robust surge in inflammatory cytokines, including IFN-γ and TNF-α, was detected in the peripheral blood of control animals 2hours after AGC administration. This peripheral cytokine induction was significantly attenuated in the TSS-fed group compared with the control. The percentage of iNKT cells among total splenocytes was lower in the TSS-fed group than in controls. The findings in this study suggest that the inhibitory effects of TSS on pregnancy loss may involve immune modulation of iNKT cells during early pregnancy.

Tokishakuyakusan ameliorates spatial memory deficits induced by ovariectomy combined with β-amyloid in rats

Previously, we reported that ovariectomy (OVX) combined with β-amyloid peptide (Aβ) impaired spatial memory by decreasing extracellular acetylcholine (ACh) levels in the dorsal hippocampus. Here, we investigated the effect of tokishakuyakusan (TSS), a Kampo medicine, on the impairment of spatial memory induced by OVX combined with Aβ in rats. Repeated administration of TSS (300 mg/kg, p.o.) significantly decreased the number of errors in the eight-arm radial maze test. Though TSS had no effect on extracellular ACh levels at baseline, TSS significantly increased extracellular ACh levels in the dorsal hippocampus. These results suggest that TSS improves the impairment of spatial memory induced by OVX combined with Aβ by (at least in part) increasing extracellular ACh levels in the dorsal hippocampus.

Traditional Japanese formulas tokishakuyakusan and ogikenchuto suppress dermal sclerosis in bleomycin‐induced murine scleroderma

ABSTRACTAimSystemic scleroderma is a connective tissue disease of unknown etiology characterized by fibrosis of the skin and internal organs. There are few effective treatments for this disease. In this study, we examined the effectiveness of the traditional Japanese medicines keishibukuryogan (KBG), tokishakuyakusan (TSS), and ogikenchuto (OKT) in treating systemic scleroderma.MethodsDermal sclerosis was induced in mice by daily injection of bleomycin. The mice were randomly divided into five groups based on treatment: KBG, TSS, and OKT groups (250 mg/kg/day), and two groups given water only (the sham and control groups). The sham group received local phosphate‐buffered saline injections (without bleomycin), and the control group received local bleomycin injections (without treatment). Kampo treatment groups also received local bleomycin injections. A daily dose of 0.01 mL of either kampo solution or water/g·bodyweight was given orally (through a stomach tube) for 4 weeks.ResultsOn histology at week 4, significantly fewer mast cells had infiltrated into the dermis in the TSS and OKT groups than in the control group; dermal sclerosis was also less severe in these groups. Furthermore, at week 1, the KBG, TSS, and OKT groups had significantly reduced serum transforming growth factor β1 (TGF‐β1). OKT and TSS had significantly reduced the level of malondialdehyde in the skin tissue.Conclusion OKT and TSS suppressed the progression of dermal sclerosis by inhibiting mast cell infiltration, as well as by decreasing TGF‐β1 and oxidative stress.

Toki-shakuyaku-san, a Japanese kampo medicine, reduces colon inflammation in a mouse model of acute colitis

Toki-shakuyaku-san (TOKI) is a Japanese kampo medicine, which consists of a mixture of herbal medicines and considered to be a promising remedial agent due to its immunomodulatory and anti-inflammatory effects. We examined the beneficial effects of TOKI in inflammatory bowel disease associated with the inflammation of the intestinal barrier. A study was designed, using C57BL/6 female mice and were administered with 3% DSS in drinking water for 8days with or without 1g/kg/day TOKI orally for the last 3days and a normal group supplied with plain drinking water for 8days. TOKI treatment attenuated the clinical symptoms of acute murine colitis and also alleviated the inflammatory mechanism by reducing the inflammatory mediators, such as IL-1β, IL-2, TGF-β, RAGE and TLR2. It has also decreased the levels of CHOP, caspase12, cleaved caspase3 and cleaved caspase7 and thereby down-regulated the endoplasmic reticulum stress and apoptotic signaling induced by DSS. Moreover, the expression levels of cyclin D1 and c-kit have also confirmed the beneficial role of TOKI in colitis. All these data suggested that TOKI can be a promising agent for the treatment of colitis since it alleviates the disease progression and severity.

Keishibukuryogan, a Traditional Japanese Medicine, Inhibits Platelet Aggregation in Guinea Pig Whole Blood.

Effects of keishibukuryogan (KBG) on platelet aggregation were investigated. To ensure the specificity of KBG, tokishakuyakusan (TSS) and kamisyoyosan (KSS), which are known to have platelet aggregation-inhibiting effects, and rikkunshito (RKT) and shakuyakukanzoto (SKT), which are considered to be devoid of such effects, were used for comparison. The platelet aggregation of each test drug was measured by the screen filtration pressure method using whole blood of guinea pigs and expressed as a collagen-induced pressure rate (%) or a collagen concentration required for 50% increase in the pressure rate (PATI value). KBG suppressed the collagen-induced whole blood pressure rate increase and increased the PATI value, like TSS and KSS. Neither RKT nor SKT showed these effects. The Moutan cortex and Cinnamomi cortex, the constituent crude drugs of KBG, showed KBG-like pressure rate suppression and PATI-increasing effects. Furthermore, paeonol, a representative component of Moutan cortex, and aspirin which is known to have platelet aggregation-inhibiting activity (COX-1 inhibitor) also showed similar effects. These results suggest that the platelet aggregation-inhibiting activity of the constituent crude drugs Moutan cortex and Cinnamomi cortex is involved in the improving effects of KBG on impaired microcirculation and that paeonol plays a role in these effects.

A Simultaneous Determination of Principal Compounds in Tokishakuyakusan by High-Performance Liquid Chromatography with Diode Array Detector

We developed a simultaneous analysis method using high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD) for six principal compounds (atractylenolide III, alisol A, alisol B, paeoniflorin, ferulic acid and (Z)-ligustilide) in a traditional Japanese (Kampo) medicine, tokishakuyakusan (TSS). The HPLC separation was conducted on a reversed-phase TSK-gel ODS-80TS column (4.6 i.d. × 250 mm, 5 µm) at 40°C with a 0.1% phosphoric acid-acetonitrile gradient system. The DAD detection wavelength was set at 205, 232 and 330 nm. Calibration curves for the compounds showed linear regressions with correlation coefficients of >0.999. The intra- and inter-day precision (i.e., the relative standard deviation) were in the range of 0.50-1.55 and 0.70-1.80%, respectively. The average recovery yields of the compounds ranged from 98.3 to 103%. The present results will contribute to shorter analysis times with less organic solvent compared with the individual analysis of each compound for the evaluation of TSS. The application of the established method to TSS will also provide helpful information for the further pharmacological and clinical studies.

The traditional kampo medicine tokishakuyakusan increases ocular blood flow in healthy subjects.

The aim of this study was to examine the effects of oral administration of kampo medical formulas on ocular blood flow (OBF). A crossover protocol was used to randomly administer five grams of yokukansan, tokishakuyakusan (TSS), keishibukuryogan, or hachimijiogan to 13 healthy blinded subjects (mean age: 37.3 ± 12.3 years). The mean blur rate, a quantitative OBF index obtained with laser speckle flowgraphy, was measured at the optic nerve head before and 30 minutes after administration. Blood pressure (BP) and intraocular pressure (IOP) were also recorded. No significant changes were observed in mean BP or IOP after the administration of any of the kampo medical formulas. There was a significant increase in OBF 30 minutes after administration of TSS (100% to 103.6 ± 6.9%, P < 0.01). Next, TSS was administered to 19 healthy subjects (mean age: 32.0 ± 11.0 years) and OBF was measured before and 15, 30, 45, and 60 minutes after administration. Plain water was used as a control. OBF increased significantly after TSS administration compared to control (P < 0.01) and also increased from 30 to 60 minutes after administration compared to baseline (P < 0.05). These results suggest that TSS can increase OBF without affecting BP or IOP in healthy subjects.

Screening of Preventive Effects of Herbal Prescriptions in Rat Model of Arterial Thrombosis Induced by Ferric Chloride

Objectives: This study examined the effects of 8 types of herbal prescriptions prescribed to alleviate dementia symptoms in a rat model of arterial thrombosis induced by ferric chloride (FeCl3). Methods: Thirty minutes before 35% FeCl3 treatment, SD rats were intraperitoneally injected with the 8 types of herbal prescriptions, respectively. We tested the effects of the herbal prescriptions on time to occlusion (TTO) in an arterial thrombosis model using a laser Doppler flow meter. In addition, thrombus weight (TW) and collagen fiber damages were evaluated in the same condition. Results: Herbal prescriptions showed the following rank-order based on their TTO: Chong-myung-tang (CMT) > modified Jangwonhan 02 (LMK02) > Toki-shakuyaku-san (TSS) Oren-gedoku-to (OGT) Yokukansan (YKS). In particular, CMT (100 mg/kg, i.p.) and LMK02 (100 mg/kg, i.p.) delayed the TTO the most (14.83 ± 0.98 and 13.67 ± 1.03 min, respectively) compared with the vehicle group (7.95 ± 0.78 min, P<0.001). In addition, CMT, LMK02, and OGT treatment (0.63 ± 0.01, 0.66 ± 0.02 and 0.67 ± 0.01 mg/mm, respectively) significantly reduced thrombus weight compared with the vehicle treatment (0.78 ± 0.03, P<0.001) and also alleviated collagen fiber damage (CMT; 28.40 ± 2.22%, LMK02; 30.79 ± 4.07%, OGT; 26.20 ± 1.48%) in the vessels injured by FeCl3. Therefore, CMT and LMK02 showed the greatest preventive activity in rat model of arterial thrombosis induced by FeCl3. Conclusions: These results provide experimental evidence for traditional use of herbal prescriptions, suggesting that CMT and LMK02 extracts could be used to prevent vascular injury and thrombosis in the early stages of dementia.

Maternal Administration of the Herbal Medicine Toki-Shakuyaku-San Promotes Fetal Growth and Placental Gene Expression in Normal Mice

Toki-shakuyaku-san (TSS), an herbal formula based on traditional Chinese medicine, is commonly used in obstetrics. To examine the effects of TSS on the normal mouse fetus and placenta, TSS was administered to normal pregnant mice and their placentas and fetuses were studied. First, the effects of maternal TSS treatment on implantation were investigated. Administration of TSS from gestation day 0.5 (G0.5) to G6.5 showed that litter size was not altered at embryonic day 11.5 (E11.5), but the number of resorbed fetuses was slightly decreased. Then, to investigate effects on fetal and placental growths after implantation, TSS was administered from G5.5. At E14.5, the body weight of fetuses from TSS-treated dams was significantly increased. Gene expression of insulin-like growth factor 2 (Igf2), one of the most important modulators of fetal growth, was significantly increased in the placentas and fetuses of TSS-treated dams. In addition, the expression of particular placental developmental genes and nutrient transporter genes was significantly increased in TSS-treated placentas. At E18.5, after longer-term administration of TSS, fetal and placental weights were not altered, but the expression of the placental developmental and nutrient transporter genes remained elevated compared with controls. These results suggest that maternal TSS treatment in normal mice enhances the expression of Igf2, placental developmental genes and nutrient transporter genes, resulting in increased fetal weight. No obvious changes were observed in the expression of these genes after longer-term maternal TSS treatment.

Effect of Toki-Shakuyaku-San on Regional Cerebral Blood Flow in Patients with Mild Cognitive Impairment and Alzheimer's Disease

The aim of this study was to examine the effect of toki-shakuyaku-san (TSS) on mild cognitive impairment (MCI) and Alzheimer's disease (AD) using single-photon emission computed tomography (SPECT). All subjects were administered TSS (7.5 g/day) for eight weeks. SPECT and evaluations using the Mini Mental State Examination (MMSE), Neuropsychiatric Inventory, and Physical Self-Maintenance Scale were performed before and after treatment with TSS. Three patients with MCI and five patients with AD completed the study. No adverse events occurred during the study period. After treatment with TSS, regional cerebral blood flow (rCBF) in the posterior cingulate was significantly higher than that before treatment. No brain region showed a significant decrease in rCBF. TSS treatment also tended to improve the score for orientation to place on the MMSE. These results suggest that TSS could be useful for treatment of MCI and AD.

A Chinese Herbal Medicine, Tokishakuyakusan, Reduces the Worsening of Impairments and Independence after Stroke: A 1‐Year Randomized, Controlled Trial

In post-stroke patients, the recurrence of stroke and progression of impairments lead to a bedridden state and dementia. As for their treatments, only anti-hypertension and anti-coagulation therapies to prevent the recurrence of stroke are available. In Asia, post-stroke patients with impairments are often treated with herbal medicine. The present study evaluated the effectiveness of tokishakuyakusan (TS) in improving the impairment and independence in post-stroke patients. Thirty-one post-stroke patients (mean age = 81.4 years) were recruited and enrolled. Participants were randomly assigned to the TS group (n = 16) or non-treatment (control) group (n = 15) and treated for 12 months. Impairments were assessed using the Stroke Impairment Assessment Set (SIAS). Independence was evaluated using the functional independence measure (FIM). For each outcome measure, mean change was calculated every 3 months. The results were that impairments according to SIAS did not significantly change in the TS group. In contrast, SIAS significantly worsened in the control group. There was a significant difference between the two groups. In each term of SIAS, affected lower extremity scores, abdominal muscle strength, function of visuospatial perception, and so forth. in the TS group were better than those in the control group. Independence according to FIM did not change significantly in the TS group. In contrast, FIM significantly worsened in the control group. There was also a significant difference between the two groups. In conclusion, TS was considered to suppress the impairments of lower limbs and to exert a favorable effect on cerebral function for post-stroke patients.

The relation between clinical effects of Tokishakuyakusan and the identity of Paeonia lactiflora materials

To investigate the relation between the clinical effects and the quality of crude drugs, we focused on Tokishakuyakusan (TS), consisted of 6 crude drugs. We prepared two kinds of TS containing either medicinal cultivar of Paeonia lactiflora (MTS) or ornamental one (OTS). Other components were the same. First, we assessed the clinical effects of two TS formulations by cross-over study among the anemia patients. Second, we investigated the chemical differences between them by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Mössbauer analysis. The clinical effects of these formulations (3 g/day for 8 weeks) were tested in the cross-over study consisted of 12 women patients who were diagnosed as having anemia (Hb ≤ 11 g/dl) and consented to participate to this study. Both TS formulations were effective for anemia symptoms as shown by the improvement of several hematological parameters, whereas their comprehensive effects were distinguishable by Genetic Algorithm Partial Least Squares (GA-PLS) analysis. There were no significant differences in organic ingredients and Fe content measured by ultra performance liquid chromatography (UPLC) and ICP-MS, respectively. Interestingly, Mössbauer spectra of Fe ion were remarkably different between two formulations. Fe ion in MTS was only one form, but that in OTS was at least two forms. This study suggested that clinical effects of TS formulation reflect the quality of Paeoniae Radix.