Prolonging effects of Valeriana fauriei root extract on pentobarbital-induced sleep in caffeine-induced insomnia model mice and the pharmacokinetics of its active ingredients under conditions of glycerol fatty acid ester as emulsifiers.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana plant roots have traditionally been used to treat central nervous system-related disorders in European countries. Among this genus, the Japanese Pharmacopoeia registers the dried roots of V. fauriei Briq. (VF). However, insufficient pharmacological data are available for this species. AIM OF THE STUDY: We investigated the sedative effects of VF extract in a murine caffeine-induced insomnia model as well as the active ingredients and their pharmacokinetics to determine its basic pharmacological action mechanisms under conditions glycerol fatty acid ester is used as emulsifiers. MATERIALS AND METHODS: A murine insomnia model was created by caffeine. Samples derived from the ethanol extract of VF were administered per oral (p.o.), and caffeine was injected intraperitoneally (i.p.). Pentobarbital was injected i.p. and the sleep latency and duration were measured. To confirm the mechanism of action of VF, flumazenil, a specific γ-aminobutyric acid receptor type A (GABAA receptor) antagonist, was administered (i.p.) immediately prior to the sample administration. We examined the pharmacokinetic profiles of the active ingredients in the plasma, brain, urine, and feces of mice after the administration (p.o and intravenous (i.v.)) of VF samples. RESULTS: VF extract (5 g as VF/kg, p.o.) significantly shorten sleep latency and prolonged pentobarbital-induced sleep in caffeine-induced insomnia mice, partially mediated via the GABAergic nervous system, although a higher dose (10 g as VF/kg, p.o.) was required to exhibit the significant effects in normal mice. Kessyl glycol diacetate (KGD), the main constitutive compound in VF, did not shorten sleep latency but exhibited the same sleep prolonged effect at a dose related to VF extract. The concentration of kessyl glycol 8-acetate (KG8) in the plasma was higher than that of KGD in mice treated (p.o.) with VF extract. The profiles of brain concentrations of KGD and KG8 were similar to those in the plasma, and approximately 20% of those in the plasma were distributed throughout the brain. The excretions of KGD and KG8 in urine and feces was slightly detected, and an unknown large peak related to KG8 was detected in the urine of mice administered with VF extract by HPLC-MS/MS analysis. CONCLUSIONS: VF exhibits more sedative effects under stressed conditions, such as insomnia, and the major active ingredients are KGD and its metabolite KG8, which are distributed from the blood circulation into the brain by simple diffusion. KG8 is further metabolized into other metabolites that are easily excreted in the urine.

Historical Study for the Differences of Processing of Pinellia ternata Tuber Between China and Japan.

Pinellia Tuber (the dried tuber of Pinellia ternata (Thunb.) Makino [Araceae]) (PT) is a crude drug used in traditional Chinese medicine (TCM) and Japanese Kampo medicine. PT is subjected to additional processing before use in TCM because of its toxic, while the processing has not been used in Kampo medicine. The aim of this study is to clarify the reason why the differences about the processing of PT between TCM and Kampo medicine have been appeared. We investigated successive literatures published in China and in Japan from the Han dynasty to the modern age. The descriptions about the processing of PT in China had appeared since the Later Han dynasty as washing, and after that, various processing methods have been recorded, such as boiling, steaming, making cakes, and fermenting to prepare PT malt (PTM) with various drug additives. The objective of the processing for PT was not only to remove its toxicity but to change drug properties, and several kinds of processed PT had been developed to treat different types of "phlegm" in the Ming dynasty. The current Chinese Pharmacopoeia recommends the use of processed PT to avoid the toxicity, and registers unprocessed PT as well as three kinds of processed PT except for PTM which had been deleted in 2015 edition. These processing methods for PT have been established in the Qing dynasty. The oldest description in Japan was appeared in 1363, and the processing methods had been influenced by the literatures in the Song dynasty. After that, the processed PT in Japan had mainly been PTM until the 18th century. In 1738, Shuan Kagawa wrote that PT should not be processed because its pharmacological effects disappeared and the toxicity of PT disappeared by preparing its decoction without processing. Then, the processing of PT has been unpopular, and the Japanese Pharmacopoeia has registered PT since 1939 without any processing. Compared to TCM, Japanese Kampo medicine has tended to avoid ideologism based on traditional knowledge and to adopt positivism. This policy has reflected the differences in the processing of PT between Kampo medicine and TCM.

History and the immunostimulatory effects of heat-processed licorice root products with or without honey.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, the dried root of Glycyrrhiza uralensis Fisch. (licorice root) is usually used after stir-baked with honey. However, in Japanese traditional Kampo medicine, processed licorice root is prepared by roasting without honey. AIM OF THE STUDY: We summarized our previous studies on the processed licorice root products to review the effectiveness of the processing for licorice root. MATERIALS AND METHODS: We summarized our previous studies about processed licorice root. The first report was about investigating the successive literatures of traditional medicine in China and Japan about the processing of licorice root. Next was the report about chemically analyzing for prepared various kinds of processed licorice root samples. The last reports were evaluating in vitro effects of the extracts of these samples and heated honey on granulocyte colony-stimulating factor (G-CSF) secretion in cultured intestinal epithelial cells. RESULTS: Before the Song dynasty in mainland China, the processing of licorice root for the internal usage had been roasted without any drug adjuvants. Then, clinicians had also used honey-roasted licorice to treat throat pain since the Song dynasty, and honey-roasted licorice has been used as the substitute to roasted licorice since the end of the Qing dynasty. While the descriptions using honey have been disappeared in 18th century in Japan. We found that the conversion between liquiritigenin and isoliquiritigenin or between liquiritin and isoliquiritin in licorice root by heating was accelerated by using honey as drug adjuvant. The inducible effect of G-CSF of licorice root was not augmented by roasting, but significantly augmented by stir-baked with honey. Heated honey also had this activity, and isomaltose contributed the appearance of this activity among the constituents in honey. The best activity was appeared when isomaltose was heated at 180 °C for 60 min or at 200 °C for 15-30 min, and the average molecular weight of the active product was 790 kDa. CONCLUSIONS: By our previous studies, we believe that the processing method in China is better than that in Japan for licorice root, since the immunostimulatory effects are appeared in honey used as drug adjuvant when honey is heated. Among the ingredients of honey, isomaltose can be used as the marker compound to choose a conforming honey product for the processing of licorice root.

Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice - A comparison with that of V. officinalis root.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. AIM OF THE STUDY: We compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. MATERIALS AND METHODS: We prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. RESULTS: The ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. CONCLUSIONS: Based on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating.

We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15-30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.

Historical and pharmacological studies on rehmannia root processing- Trends in usage and comparison of the immunostimulatory effects of its products with or without steam processing and pretreatment with liquor.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Rehmannia glutinosa (RR) is a crude drug used in traditional Japanese Kampo medicine and traditional Chinese medicine (TCM). Sometimes, the crude drug is subjected to additional processing before use. AIM OF THE STUDY: To determine the effects of steam processing and pretreatment with liquor of RR through historical investigation, analytical chemistry, and pharmacological experiments. MATERIALS AND METHODS: We inspected TCM literature from the Later Han Dynasty to the present day. Dried RR steamed for 3, 6, 9, or 12 h (steamed RRs, SRRs), dried RR soaked in yellow rice wine (liquor) (liquor-RR), and dried RR steamed for 6 h pretreated with liquor (liquor-SRR) were prepared. These samples were extracted using boiling water, and the inducible effects of the extracts on granulocyte colony-stimulating factor (G-CSF) secretion in cultured enterocytes and the content of their marker compounds were evaluated by using HPLC. RESULTS: The effect of processing using both steaming and the pretreatment using liquor described in TCM literature over different eras was to enhance the warming effect and tonifying qi (energy) of RR. We found that SRR, processed by pretreatment with liquor, became mainstream since the Qing Dynasty. In SRR, stachyose content was decreased and fructose and manninotriose contents were increased with steaming time. However, the content of these compounds was not altered by pretreatment with liquor. RR extract induced G-CSF secretion in cultured enterocytes; moreover, the SRR extract steamed for more than 6 h had significantly stronger effects than that RR. Pretreatment with liquor did not cause any significant differences in the effects of RR or SRR. CONCLUSIONS: The aim of processing for RR by both steaming and pretreatment with liquor in TCM literature over different eras was to enhance the tonifying effect on qi and its immunostimulatory effect. Although the effect of RR on the induction of G-CSF secretion in intestinal epithelial cells was enhanced by steaming, this enhancement was not enhanced by the pretreatment with liquor. These results provide scientific support for steaming, but could not elucidate a reason for pretreatment with liquor in TCM theory.

The immunostimulatory effects and chemical characteristics of heated honey.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), honey has been used as an additive in the heat-processing of herbal medicines to enhance their immunostimulatory activities. AIM OF THE STUDY: We investigated the immunostimulatory activity of heated honey in vitro and in vivo. MATERIALS AND METHODS: For the in vitro study, we compared the differences among the inducible effects of honey subjected to various heating conditions on granulocyte colony-stimulating factor (G-CSF) secretion from the cultured enterocytes and investigated the active ingredient. For the in vivo study, we conducted a survival test of mice infected by Streptococcus pyogenes with and without oral administration of heated honey. RESULTS: We found that heating the honey induced the appearance of G-CSF secretions from the cultured enterocytes, and that this appearance depended on the heating temperature and time. No G-CSF secretions appeared when honey was not heated. Mice infected with Streptococcus pyogenes that were fed heated honey revealed prolonged survival. The active ingredient in heated honey was a high-molecular compound with about 730 kDa. When this compound was hydrolyzed, galactose, glucose, rhamnose, α-ribofuranose ß-ribofuranose 1,5':1',5-dianhydride, and 5-hydroxymethylfurfural were generated. CONCLUSIONS: Heated honey reveals immunostimulatory activity both in vitro and in vivo. These results support the scientific evidences of the TCM theory.

Comparison of the inducible effects of licorice products with or without heat-processing and pre-treatment with honey on granulocyte colony-stimulating factor secretion in cultured enterocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice (the roots and rhizomes of Glycyrrhiza uralensis Fisch.) is occasionally used as crude drug following processing including roasting or honey-roasting (soaking with honey before roasting) in traditional Japanese Kampo medicine and traditional Chinese medicine (TCM). AIM OF THE STUDY: We investigated the differences in the inducible effect of processed licorice products on granulocyte colony-stimulating factor (G-CSF) secretion in cultured intestinal epithelial cells and elucidated the active ingredients in both unprocessed and processed licorice products. MATERIALS AND METHODS: We prepared heat-processed licorice with or without pretreatment with honey, and fractionated the extracts by Sephadex G-100. Enterocyte-like differentiated MCE301 cells were incubated in media comprising a hot water extract of licorice products for 24h, and the concentrations of G-CSF in the media were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Licorice extract induced G-CSF secretion in MCE301 cells, and the active ingredients of licorice were high molecular compounds. Although the roasted licorice extract exhibited the activity similar to that of the unprocessed licorice extract, honey-roasted licorice extracts exhibited a significantly higher inducible effect on G-CSF secretion in the cells than that of unprocessed or roasted licorice extracts without pretreatment with honey. This enhanced activity was dependent on the temperature and heating time. CONCLUSIONS: The enhanced inducible effect of honey-roasted licorice on G-CSF secretion might be attributed to the combined effect of licorice-derived high molecular compounds and heated-honey-derived compounds. The results of this study can scientifically explain the objective of processing via honey-roasting in TCM theory.

Comparison of byakujutsu (Atractylodes rhizome) and sojutsu (Atractylodes lancea rhizome) on anti-inflammatory and immunostimulative effects in vitro.

The Japanese Pharmacopoeia defines byakujutsu (Atractylodes rhizome) as the rhizome of Atractylodes japonica or A. macrocephala and sojutsu (Atractylodes lancea rhizome) as the rhizome of A. lancea, A. chinensis, or their interspecific hybrids. Because their pharmaceutical uses differ in traditional Japanese Kampo medicine and traditional Chinese medicine, with less apparent scientific evidence, we compared the pharmacological properties between byakujutsu and sojutsu. Crude drug specimens of byakujutsu (n = 40) and sojutsu (n = 49) obtained in markets were identified by their species using DNA profiling. Their pharmacological properties were evaluated by the inhibitory effect of a MeOH extract of the samples on nitric oxide (NO) production by lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells and by the inducing effect of boiling water extract of the samples on granulocyte-colony stimulating factor (G-CSF) secretion from murine normal colonic epithelial MCE301 cells. We authenticated A. macrocephala (n = 8), A. japonica (n = 35), and the hybrid between A. macrocephala and A. japonica (n = 1), and they were used as byakujutsu. We authenticated A. chinensis (n = 25), A. lancea (n = 14), and the hybrid between A. chinensis and A. lancea (n = 6), and they were used as sojutsu. The inhibitory effects of byakujutsu on NO production were significantly higher than those of sojutsu (P < 0.05). This activity of A. japonica rhizome was significantly higher than that of A. macrocephala rhizome and A. lancea rhizome (P < 0.01). The activity of A. chinensis rhizome was significantly higher than that of A. lancea rhizome (P < 0.05). The extract of A. japonica rhizome significantly induced G-CSF secretion from MCE301 cells in a concentration-dependent manner. These effects of byakujutsu samples were not significantly different from those of sojutsu samples. A. japonica rhizome had significantly higher activity than A. macrocephala rhizome; however, there were no statistically significant differences among A. japonica, A. chinensis, and A. lancea. The pharmacological differences of byakujutsu and sojutsu may not be large among highly variated crude drug samples with average values, and quality control with the identification of the original plant species of byakujutsu and sojutsu may guarantee their pharmacological properties.

Comparison of chemical constituents among licorice, roasted licorice, and roasted licorice with honey.

Licorice (root and rhizome of Glycyrrhiza uralensis Fisch.) is sometimes used as crude drug after processing. In this report, we prepared roasted licorice with or without honey using 3 lots of crude drug samples derived from wild G. uralensis, and analyzed the constituents in unprocessed, roasted, and honey-roasted licorice samples by high performance liquid chromatography-electrospray ionization-ion trap-time of flight mass spectrometry (HPLC-ESI-IT-TOF-MSn) with principal component analysis. We found that the areas of 41 peaks were noticeably changed by processing. Among them, the areas of 12 peaks, viz. isoliquiritin, isoliquiritigenin, glucoisoliquiritin, 6″-O-acetylisoliquiritin, 6″-O-acetylisoliquiritin apioside, glycyrrhetinic acid 3-O-glucuronide, 5 kinds of sugar-derivatives and one compound whose molecular weight was 386 Da were increased by roasting in all 3 lots, and those peak areas were increased by higher heating temperatures. Among the increased peaks, 3 kinds of sugar-derivatives had larger areas, and 6″-O-acetylisoliquiritin had lower areas than those in honey-roasted licorice. Those sugar-derivatives were the only characteristics differing between honey-roasted licorice and roasted licorice. Meanwhile, the areas of 9 peaks, four of them identified as 6″-O-acetylliquiritin, 6″-O-acetylliquiritin apioside, formononetin and gancaonin L, were decreased by roasting in all 3 lots, but there were no differences between roasted licorice with or without honey. Those compounds whose amounts were changed by processing could be used as markers for the quality control of roasted and honey-roasted licorice.

[Herbological Study on the Medicinal Effects of Roasted Licorice and Honey-roasted Licorice].

In China, the crude drug licorice ("kanzo" in Japanese, "gancao" in Chinese) has been used both dried and roasted as the situation demands from ancient times. The meaning of "roasted licorice" is simply roasted and honey-roasted in ancient and modern times, respectively. However, it is not clear medicinal purposes of processed licorice or why licorice processed with honey began to be used. We researched ancient literature and found that the main objective of roasting was to change the property of licorice from cool to warm (i.e., dried licorice had the effect of draining fire), while roasted licorice was used as an energy supplement, having a digestive effect and thus warming the body. Meanwhile, doctors began using honey-roasted licorice to treat throat pain from the Song dynasty, and then at the end of the Qing dynasty, honey-roasted licorice was expected to have the same effects of roasted licorice (i.e., supplementing energy and having a digestive effect).