Traditional Kampo Medicine Research Articles

Effects of Tokishakuyakusan on Regeneration of Murine Olfactory Neurons In Vivo and In Vitro.

Post-upper respiratory tract infection related olfactory dysfunction typically occurs due to neural damage after an upper respiratory tract infection associated with a common cold or influenza. At present, Tokishakuyakusan, a Japanese traditional Kampo medicine, has been found to be effective for post-viral olfactory dysfunction. However, the pharmacodynamics of Tokishakuyakusan in the treatment of post-viral olfactory dysfunction remains unresolved. We investigated the effects of Tokishakuyakusan on the regeneration of olfactory neurons and expression of nerve growth factor (NGF) in neural systems, using in vivo murine studies and in vitro cell culture studies. Eight-week-old BALB/C female mice were fed a pellet diet with or without Tokishakuyakusan. Degeneration of cells in olfactory epithelium was induced by intraperitoneal methimazole injection. Regeneration of olfactory neurons was observed by histological and immunohistochemical procedures. NGF expression in the olfactory bulb was measured by enzyme-linked immunosorbent assay. NGF gene and protein expression were measured using rat primary cultured astrocytes by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We found that olfactory marker protein, Ki-67, and NGF were more highly expressed in the olfactory epithelium during the regeneration period in mice receiving Tokishakuyakusan. In cultured astrocytes, Tokishakuyakusan as well as its individual components, Atractylodes lancea rhizome and Japanese angelica root, increased NGF expression. Screening assays revealed that NGF production was increased by atractylodin and levistolide A, which are ingredients in Atractylodes lancea rhizome and Japanese angelica root, respectively. These results suggest that Tokishakuyakusan promotes regeneration of olfactory neurons by increasing NGF expression in the olfactory bulb.

Hainosan (painongsan) suppresses the biofilm formation of Porphyromonas gingivalis and Prevotella intermedia in vitro

ABSTRACTAim Porphyromonas gingivalis and Prevotella intermedia are pathogenic bacteria that cause several periodontal diseases such as periodontitis. They frequently form biofilm to protect from threats such as antimicrobial agents. Hainosan (painongsan in Chinese) is a Japanese traditional kampo and traditional Chinese medicine formula used to treat acute and chronic purulent disease. The aim of the present study was to elucidate the mechanism of antibacterial effects such as anti‐biofilm activity, of hainosan extract (HNS) against Porphyromonas gingivalis and Prevotella intermedia.MethodsThe antibacterial and anti‐biofilm activity of HNS was examined on bacterial turbidity and colony‐forming unit analysis, transmission electron microscopy, microplate analysis, and hydrophobicity and auto‐aggregation analysis.ResultsHNS significantly suppressed the growth of Porphyromonas gingivalis and Prevotella intermedia. It also significantly inhibited the formation of biofilm from Porphyromonas gingivalis and Prevotella intermedia. The size and shape of HNS‐treated Porphyromonas gingivalis and Prevotella intermedia were significantly smaller and irregular. Furthermore, HNS reduced the bacterial auto aggregation and hydrophobicity of the bacterial cell surface. These antibacterial effects of HNS were greater than those of hangeshashinto, another Japanese traditional kampo medicine formula for oral cavity inflammation.ConclusionHNS may be useful for the treatment of Porphyromonas gingivalis and Prevotella intermedia‐induced periodontitis because of the inhibition of bacterial biofilm formation.

Kampo medicine for Frailty as Kidney deficiency

This review aims to understand the clinical efficacy of Japanese Traditional (Kampo) medicine for managing frailty-related disorders mainly based on Kidney deficiency. Kampo is Japanese traditional herbal medicine, and is the most frequently used alternative and complementary medicine in Japan. The aim of Kampo therapy is to improve patients’ condition whatever their diseases are. Kampo therapy is unique because its focus is patient’s condition not their disease. Moreover, it is widely used as Kampo formulas are covered by National Insurance System in Japan. Therefore, Kampo Medicine plays more and more important roles in closing the gap between modern Western Medicine and demand of patients. With the advent of an aging society, the concept of a fragile state of mind and body that leads to many health problems, “Frailty”, was proposed. It is not easy to prevent or cure frailty because it contains complexed factors. Here, Kampo medicine often obtains and analyzes a large amount of information through four examinations, and can grasp and deal with an aging state such as frailty. This article outlines the effects of representative formulas on frailty related to Kidney deficiency, like lumbago and arthralgia which often cause deterioration of ADL and QOL of patients. Our review showed some evidence for the usefulness of representative Kampo formulas for Kidney deficiency, hachimigan and goshajinkigan. Our review suggests that these formulas have the potential to be used for the management of aging-related and frailty symptoms, although further clinical studies in elderly patients should be conducted.

Macrophage-selective inhibitory effect of Kikyo-to, a Japanese traditional kampo medicine, on the viability of mouse macrophage RAW264.7 cells.

Macrophage-selective inhibitory effect of Kikyo-to, a Japanese traditional kampo medicine, on the viability of mouse macrophage RAW264.7 cells.

Drug interaction between shoseiryuto extract or catechins and fexofenadine through organic-anion-transporting polypeptide 1A2 In vitro

Objective: Fexofenadine is an anti-allergy drug frequently used to treat rhinitis, which is absorbed through organic-anion-transporting polypeptide (OATP) 1A2 in the intestine. Clinical studies have revealed that grapefruit juice inhibits OATP1A2 to reduce the absorption of fexofenadine. In Japanese traditional Kampo medicine, shoseiryuto, a formula composed of eight crude drugs, is frequently used to treat allergic rhinitis, especially cases of pollinosis. The objective of this study is to present the drug information regarding the interaction between shoseiryuto and fexofenadine through OATP1A2. Materials and Methods: We established human embryonic kidney 293 cells stably expressing OATP1A2 and evaluated the inhibitory effects of the extracts of shoseiryuto and its herbal components on the uptake of fexofenadine into the cells. Results: Shoseiryuto extract inhibited fexofenadine uptake in a concentration-dependent manner with an IC50 value of 238 μg/ml. The inhibitory titer of shoseiryuto was much lower than that of grapefruit juice, and it is predicted that shoseiryuto could not pharmacokinetically interact interaction with fexofenadine through OATP1A2. Among the eight herbal components of shoseiryuto, the extracts of the terrestrial stem of Ephedra sinica and the root and stolon of Glycyrrhiza uralensis inhibited fexofenadine uptake with IC50 values of 102 and 89 μg/ml, respectively. We isolated catechin as the active ingredient from the extract of the terrestrial stem of E. sinica. Catechins inhibited the uptake of fexofenadine in a concentration-dependent manner, and the IC50 values of epicatechin (EC) gallate, epigallocatechin (EGC) gallate, EGC, catechin, and EC were 11, 26, 41, 52, and 96 μM, respectively. Conclusion: These results would contribute to the safe and effective use of shoseiryuto in clinics. Abbreviations used: CYP: Cytochrome P450; JPXVII: The 17th Edition of the Japanese Pharmacopoeia; EC: Epicatechin; ECG: Epicatechin gallate; EGC: Epigallocatechin; EGCG: Epigallocatechin gallate; HBSS: Hanks' balanced salt solution; HEK: Human embryonic kidney; OATP: Organic-anion-transporting polypeptide; PBS: Phosphate-buffered saline; PHB: p-hydroxybenzoic acid butyl ester.

Effects of crushed Schisandra Fruit on the content of lignans in Kampo decoction.

Arboreous fruit of Schisandra chinensis Baillon, Schisandra Fruit (SF), is a crude drug used in Japanese traditional Kampo medicine. The marker compounds of SF for quality control are lignans, such as schizandrin (Sz) and gomisinA (GmA). Kampo formulation containing SF is usually prepared as decoctions in the dosage form of whole crude drug (W), as its size is small enough to measure using a spoon. However, in some traditional books, it has been described that SF must be used in the dosage form of crushed or cut pieces (Cr). In this study, we evaluated the transferring ratio of lignans from SF to the decoction, and the stability and taste of the decoctions of shoseiryuto (SST) and ninjin'yoeito (NYT) using each dosage form, i.e., Cr and W, of SF. The transferring ratio of Sz and GmA was significantly higher in the decoction prepared with the Cr form than that prepared using the W form in both SST and NYT. The concentration of Sz and GmA in the decoctions was stable when maintained at 4°C for 35 days. The taste of SST decoction prepared using the Cr form was more acidic, harsher, and bitterer than SST decoction prepared using the W form, and the taste of NYT decoction prepared using the Cr form was harsher than NYT decoction prepared using the W form. In conclusion, when SF is used in Kampo prescription, crushing the fruits and seeds can increase its effectiveness.

Rosmarinic Acid Derivatives' Inhibition of Glycogen Synthase Kinase-3β Is the Pharmacological Basis of Kangen-Karyu in Alzheimer's Disease.

Inhibition of glycogen synthase kinase 3β (GSK-3β) is considered to be the central therapeutic approach against Alzheimer’s disease (AD). In the present study, boiled water extracts of the Kangen-karyu (KK) herbal mixture and its constituents were screened for GSK-3β inhibitory activity. KK is used in traditional Kampo and Chinese medicines for improving cognitive function. The GSK-3β inhibition potential was evaluated by using the Kinase-Glo luminescent kinase assay platform. Furthermore, enzyme kinetics and in silico modeling were performed by using AutoDockTools to demonstrate the mechanism of enzyme inhibition. KK extract significantly inhibited GSK-3β in a concentration-dependent manner (IC50: 17.05 ± 1.14 μg/mL) when compared with the reference drug luteolin (IC50: 2.18 ± 0.13 μM). Among the six components of KK, extracts of Cyperi Rhizoma and Salviae Miltiorrhizae Radix significantly inhibited GSK-3β with IC50 values of 20.68 ± 2.50 and 7.77 ± 1.38 μg/mL, respectively. Among the constituents of the roots of S. miltiorrhiza water extract, rosmarinic acid, magnesium lithospermate B, salvianolic acid A, salvianolic acid B, and salvianolic acid C inhibited GSK-3β with IC50 values ranging from 6.97 to 135.5 μM. Salvianolic acid B was found to be an ATP-competitive inhibitor of GSK-3β and showed the lowest IC50 value (6.97 ± 0.96 µM). In silico modeling suggested a mechanism of action by which the hydrophobic, π–cation, and hydrophilic interactions of salvianolic acid B at ATP and substrate sites are critical for the observed GSK-3β inhibition. Therefore, one of the mechanisms of action of KK against AD may be the inhibition of GSK-3β and one of the active components of KK is the root of S. miltiorrhiza and its constituents: rosmarinic acid, magnesium lithospermate B, and salvianolic acids A, B, and C. Our results demonstrate the pharmacological basis for the use of KK against AD.

Effect of Hochuekkito (Buzhongyiqitang) on Nasal Cavity Colonization of Methicillin-Resistant Staphylococcus aureus in Murine Model.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infections are largely preceded by colonization with MRSA. Hochuekkito is the formula composing 10 herbal medicines in traditional Kampo medicine to treat infirmity and to stimulate immune functions. We evaluated the efficacy of hochuekkito extract (HET) against MRSA colonization using a nasal infection murine model. Methods: We evaluated the effects of HET as follows: (1) the growth inhibition by measuring turbidity of bacterial culture in vitro, (2) the nasal colonization of MRSA by measuring bacterial counts, and (3) the splenocyte proliferation in mice orally treated with HET by the 3H-thymidine uptake assay. Results: HET significant inhibited the growth of MRSA. The colony forming unit (CFU) in the nasal fluid of HET-treated mice was significantly lower than that of HET-untreated mice. When each single crude drug—Astragali radix, Bupleuri radix, Zingiberis rhizoma, and Cimicifugae rhizome—was removed from hochuekkito formula, the effect of the formula significantly weakened. The uptake of 3H-thymidine into murine splenocytes treated with HET was significantly higher than that from untreated mice. The effects of the modified formula described above were also significantly weaker than those of the original formula. Conclusions: Hochuekkito is effective for the treatment of MRSA nasal colonization in the murine model. We suggest HET as the therapeutic candidate for effective therapy on nasal cavity colonization of MRSA in humans.

KampoDB, database of predicted targets and functional annotations of natural medicines

Natural medicines (i.e., herbal medicines, traditional formulas) are useful for treatment of multifactorial and chronic diseases. Here, we present KampoDB (http://wakanmoview.inm.u-toyama.ac.jp/kampo/), a novel platform for the analysis of natural medicines, which provides various useful scientific resources on Japanese traditional formulas Kampo medicines, constituent herbal drugs, constituent compounds, and target proteins of these constituent compounds. Potential target proteins of these constituent compounds were predicted by docking simulations and machine learning methods based on large-scale omics data (e.g., genome, proteome, metabolome, interactome). The current version of KampoDB contains 42 Kampo medicines, 54 crude drugs, 1230 constituent compounds, 460 known target proteins, and 1369 potential target proteins, and has functional annotations for biological pathways and molecular functions. KampoDB is useful for mode-of-action analysis of natural medicines and prediction of new indications for a wide range of diseases.

Clinical practice guidelines for the use of traditional Korean medicine in the treatment of patients with traffic-related injuries: An evidence-based approach

IntroductionIn 2016, traditional Korean medicine (TKM) treatment for traffic-related injuries comprised 27.7% of Korea’s total car insurance medical coverage, and that proportion has been increasing annually. However, clinical practice guidelines (CPGs) that employ evidenced-based medicine (EBM) methodology for traffic-related injuries have not been established in TKM. Therefore, the aim of this study was to develop a CPG for the treatment of traffic-related injuries in TKM by conducting a systematic literature review following EBM. MethodsFirst, a network of experts was established to develop the CPG. Then, a systematic search of TKM treatments for traffic-related injuries was conducted using the Patients/Populations–Intervention–Comparison–Outcome method. To measure the methodological quality of the systematic reviews and meta-analyses, the assessment of multiple systematic reviews was used. The Cochrane collaboration’s risk of bias tool was used to assess the randomised controlled trials. ResultsThe CPG for the treatment of traffic-related injuries in TKM consisted of three parts: diagnosis, treatment, and prognosis. In total, 13 recommendations were developed, with two regarding diagnosis, 10 regarding treatment, and one regarding prognosis. ConclusionsThis CPG was developed by conducting a systematic literature review of randomised controlled trials. Therefore, this CPG can be adapted to different clinical status for traffic-related injuries. Specifically, it can be useful reference for traditional Chinese medicine and Kampo medicine practitioners using the same or similar interventions. In addition, the CPG can serve as an EBM model for countries unfamiliar with the complementary and alternative medicine treatment of traffic-related injuries.

Peroxisome proliferator-activated receptor-γ agonistic effect of Chrysanthemum indicum capitulum and its active ingredients

Objective: The capitulum of Chrysanthemum indicum (Compositae) is the source of a crude drug used in Japanese traditional Kampo and traditional Chinese medicine. Previous research showed that C. indicum flowers promote adipocyte differentiation through the activation of peroxisome proliferator-activated receptor (PPAR)-γ that may be an important mechanism for controlling systemic insulin resistance or other biological functions. Materials and Methods: The capitulum of C. indicum was extracted with methanol, and its PPAR-γ agonistic activity was measured using luciferase assay. The active ingredients were isolated by the activity-guided fractionations. Results: We isolated (Z)-tonghaosu and (E)-tonghaosu, and (E)-tonghaosu had PPAR-γ agonistic constituents by the activity-guided fractionation from the capitulum of C. indicum. The isomer (Z)-tonghaosu did not show PPAR-γ-agonistic activity. Conclusion: (E)-tonghaosu is one of the active ingredients as PPAR-γ agonist in the capitulum of C. indicum. Abbreviations used: ANOVA: One-way analysis of variance; DMEM: Dulbecco's modified minimum essential medium; FBS: Fetal bovine serum; HPLC: High-performance liquid chromatography; MCI: Methanol extract of the capitulum of Chrysanthemum indicum; NMR: Nuclear magnetic resonance; PPAR: Peroxisome proliferator-activated receptor.

A possible linkage between cholinergic and vascular endothelial growth factor (VEGF) systems in antidementia effects of traditional Chinese and Kampo medicines

A possible linkage between cholinergic and vascular endothelial growth factor (VEGF) systems in antidementia effects of traditional Chinese and Kampo medicines

Establishment of a Novel In Situ Rat Model for Direct Measuring ofIntestinal Drug Absorption: Confirmation of Inhibitory Effects of Daijokito onthe Absorption of Ranitidine

Daijokito (DJKT), a classical traditional Kampo and Chinese medicine, has been used to treat acute pancreatitis in China. In our previous study, DJKT was found to reduce the area under the plasma concentration-time curve (AUC) of ranitidine in humans. Therefore, we established a novel rat model to examine the direct absorption of ranitidine after daijokito administration. An in situ intestinal injection with portal vein sampling (IIPS) model was created to determine the rate of intestinal drug absorption. Rats were divided into two groups: the ranitidine group (R, n = 6) or the ranitidine and daijokito group (RD, n = 6). Blood was collected after intestinal injection of drugs. After the experiment, the concentrations of ranitidine were measured by LC/MS/MS analysis. The concentrations of ranitidine increased linearly with time in both groups. Compared with the R group, the concentrations of ranitidine in RD group significantly decreased throughout the experiment. Co-administration of ranitidine with DJKT resulted in significant decreases in intestinal absorption in rats. The reduction of the systemic ranitidine concentration by co-administration of DJKT may be due, at least in part, to the inhibition of intestinal absorption of ranitidine.

Inhibitory Effect of Japanese Traditional Kampo Formula Frequently Prescribed in Gynecological Clinics on CYP3A4.

Recently, the use of herbal medicines has become popular, and information on drug interactions between herbal medicines and chemical drugs is needed in clinics. In Japan, the number of patients taking Japanese traditional Kampo medicines has been increasing, and the proper drug information about herb-drug interaction is highly demanded. The most established herb-drug interaction is the case of grapefruit juice (GFJ) via the inhibition on CYP3A4 expressed in the small intestine. In the present study, we compared the inhibitory titer on CYP3A4 between the target Kampo products and GFJ used as positive control. We evaluated the inhibitory effects of GFJ and three extracts of Kampo formulas frequently used in gynecological clinics on CYP3A4 in vitro and calculated the related titer of one-time dosage of Kampo formulas to GFJ in order to predict its effect on clinics. Although the extracts of these three Kampo formulas and the most of crude drug components in the formulas exhibited the inhibitory effects on CYP3A4 in some levels, the possibilities of tokishakuyakusan and keishibukuryogan to cause drug interaction can be quite low; however, it is possible that the excessive dosage of kamishoyosan may cause drug interaction with the substrate of CYP3A4 in clinics.

Comparison between the Effects of Oral and Intramuscular Administration of Shin'iseihaito (Xinyiqingfeitang) in a Streptococcus pyogenes-Induced Murine Sinusitis Model.

Streptococcus pyogenes (S. pyogenes) is a species of Gram-positive coccoid bacteria having many virulence factors. Its capsule and exotoxins can cause upper respiratory tract infections such as sinusitis. The general treatment for S. pyogenes-induced sinusitis is administration of antibiotics such as penicillin and macrolides; however, a serious problem associated with these antibiotics is their attenuated effect. Shin'iseihaito (Xinyiqingfeitang), a formula of Japanese traditional Kampo medicine and traditional Chinese medicine, has been used for the treatment of sinusitis. In general, formulas of Japanese traditional Kampo medicine are orally administered. This is in contrast to certain formulas of traditional Chinese medicine, which are being recently administered intramuscularly or intravenously. Regarding these traditional Chinese medicine formulas, the injection methodology is reported to be more effective than oral intake. In this study, we compared the efficacy between orally and intramuscularly administered Shin'iseihaito against S. pyogenes-induced sinusitis. We evaluated the antibacterial effect of Shin'iseihaito extract (SSHT) against S. pyogenes by K-B disk diffusion assay. Furthermore, we investigated the nasal colonization of S. pyogenes, determined cytokine (TNF-α, IL-1β, and IL-6) levels, and conducted a splenocyte proliferative assay in a murine sinusitis model. SSHT displayed direct anti-S. pyogenes activity. Intramuscular administration of SSHT decreased the nasal colonization of S. pyogenes compared with oral administration. Thymidine uptake analysis revealed that the proliferation of splenocytes from S. pyogenes-infected mice under intramuscular SSHT treatment was upregulated compared to that of splenocytes from S. pyogenes-infected mice under oral SSHT treatment. We also found that TNF-α, IL-1β, and IL-6 levels in the nasal discharge from intramuscularly treated S. pyogenes-infected mice were lower than those from orally treated mice. Our findings suggest that intramuscular administration of Shin'iseihaito may be useful for the treatment of murine S. pyogenes-induced sinusitis.

Kamisyoyosan, a Japanese traditional Kampo medicine, ameliorates sex-dependent ADS-like behavior caused by decrease of brain allopregnanolone

Kamisyoyosan, a Japanese traditional Kampo medicine, ameliorates sex-dependent ADS-like behavior caused by decrease of brain allopregnanolone

Time-, Sex-, and Dose-Dependent Alterations of the Gut Microbiota by Consumption of Dietary Daikenchuto (TU-100).

Medications or dietary components can affect both the host and the host's gut microbiota. Changes in the microbiota may influence medication efficacy and interactions. Daikenchuto (TU-100), a herbal medication, comprised of ginger, ginseng, and Japanese pepper, is widely used in Japanese traditional Kampo medicine for intestinal motility and postoperative paralytic ileus. We previously showed in mice that consumption of TU-100 for 4 weeks changed the gut microbiota and increased bioavailability of bacterial ginsenoside metabolites. Since TU-100 is prescribed in humans for months to years, we examined the time- and sex-dependent effects of TU-100 on mouse gut microbiota. Oral administration of 1.5% TU-100 for 24 weeks caused more pronounced changes in gut microbiota in female than in male mice. Changes in both sexes largely reverted to baseline upon TU-100 withdrawal. Effects were time and dose dependent. The microbial profiles reverted to baseline within 4 weeks after withdrawal of 0.75% TU-100 but were sustained after withdrawal of 3% TU-100. In summary, dietary TU-100 changed mouse microbiota in a time-, sex-, and dose-dependent manner. These findings may be taken into consideration when determining optimizing dose for conditions of human health and disease with the consideration of differences in composition and response of the human intestinal microbiota.

Interactions between crude drug extracts used in Japanese traditional Kampo medicines and organic anion-transporting polypeptide 2B1

Ethnopharmacological relevanceThe use of herbal medicines has become popular worldwide, and the information on drug interactions between herbal medicines and chemical drugs is needed. Aim of the studyWe screened the inhibitory effects of crude drugs used in Kampo medicines used in Japan on organic anion-transporting polypeptide (OATP) 2B1 to predict potential interactions between Kampo medicines and chemical drugs used together. Materials and methodsWe chose 98 kinds of crude drugs frequently used as ingredients of Kampo formulations in Japan and prepared their boiling water extracts. We then screened their inhibitory effects on OATP2B1 by measuring the uptake of estrone 3-sulphate (E3S) by HEK293 cells stably expressing OATP2B1. ResultsAt the concentration of 100µg/ml, the extracts prepared from 12 kinds of crude drugs, Scuteralliae Radix, Arecae Semen, Aurantii Fructus Immaturus, Perillae Herba, Panacis Japonici Rhizoma, Moutan Cortex, Polygalae Radix, Rhei Rhizoma, Cannabis Fructus, Chrysanthemi Flos, Eriobotryae Folium, and Querci Cortex, suppressed the function of OATP2B1 by less than 20%. The extract of bofutsushosan, a representative Kampo formulation, inhibited OATP2B1 function with sufficient levels to suppress absorption of OATP2B1 substrates in clinics. We further evaluated the inhibitory effects of several ingredients containing Rhei Rhizoma, Perillae Herba, and Moutan Cortex on OATP2B1. ConclusionsBecause of crude drugs used in Kampo medicines might suppress absorption of OATP2B1 substrates, these results may contribute to the safe and effective use of Kampo medicine in clinics.A list of abbreviations: EC, (−)-epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, Epigallocatechin gallate; FBS, fetal bovine serum; grapefruit juice; HEK293, Human embryonic kidney; IC50, The half inhibitory concentration; OATP, organic anion-transporting polypeptide; β-PGG, penta-O-galloyl-β-D-glucose; t.i.d, 3 times a day

The Kampo medicine Yokukansan (YKS) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells.

Accumulating evidence indicates that neurotrophic factor-like substances involved in the induction of neurotrophic factor synthesis may aid in the treatment of neurological disorders, such as Alzheimer's disease. Yokukansan (YKS), a traditional Kampo medicine, has been used for the treatment of anxiety and mood disorders. In the present study, we aimed to identify the signaling pathways associated with YKS-mediated enhancement of nerve growth factor (NGF)-induced neurite extension in rat pheochromocytoma (PC12) cells. Akt and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation levels were assessed by western blot analysis, in the presence of YKS and following the treatment with TrkA inhibitor, K252a. YKS treatment (NGF+YKS 0.5 group) enhanced NGF-induced neurite outgrowth and phosphorylation/activation of Akt and ERK1/2 in PC12 cells. Moreover, YKS-induced effects were inhibited by the treatment with the TrkA receptor antagonist K252a (NGF+YKS 0.5+K252a group); no significant difference in neurite outgrowth was observed between K252a-treated (NGF+YKS 0.5+K252a group) and NGF-K252a-treated cells (NGF+K252a group). However, neurite outgrowth in K252a-treated cells (NGF+K252a and NGF+YKS 0.5+K252a group) reached only one-third of the level in NGF-treated cells (NGF group). NGF-mediated Akt phosphorylation increased by YKS was also inhibited by K252a treatment (NGF+YKS 0.5+K252a group), but no significant difference in ERK1/2 phosphorylation was observed between NGF-YKS-K252a- and NGF-treated cells (NGF group). Our results indicate that YKS treatment enhanced NGF-induced neurite outgrowth via induction of Akt and ERK1/2 phosphorylation, following the binding of NGF to the TrkA receptor. These findings may be useful in the development of novel therapeutic strategies for the treatment of Alzheimer's disease.

Impact of phosphorus and potassium fertilizers on growth and anthraquinone content in Rheum tanguticum Maxim. ex Balf

Abstract The dried root of Rheum tanguticum plays an important role in formulations and prescriptions in traditional Chinese medicine and Kampo medicine. Due to over-exploitation, R. tanguticum resources have decreased sharply in recent years. The main objective of our investigation (a 3-year field experiment) was to explore the effect of different levels of phosphorus (superphosphate) and potassium (potassium sulfate) fertilizer on the biomass (root fresh weight, root increment, and root dry weight), yield, dry matter content, and anthraquinone content of this plant at different harvesting stages (green stage, growth stage, and wilting stage) under alpine conditions. The root fresh weight and root dry weight increased significantly at the wilting stage following treatment with 90 kg P2O5/ha (100% and 59%, respectively) in 2016 and 75 kg K2O/ha (43% and 41%, respectively) in 2015 compared to the control. The yield of root dry weight obtained from three-year-old R. tanguticum plants was 9200 kg/ha when 90 kg P2O5/ha of phosphorus fertilizer was applied, and 10,400 kg/ha when 75 kg K2O/ha of potassium fertilizer was applied. This yield reached a maximum at the wilting stage. The anthraquinone content of two-year-old R. tanguticum plants had already reached the standard level of the Chinese Pharmacopoeia; however, three-year-old plants had double the anthraquinone content of two-year-old plants. Phosphorus and potassium fertilizers had no obvious influence on the anthraquinone content of R. tanguticum at the same harvesting stage.