Hydrophobic constituents of Polygonum multiflorum roots promote renal erythropoietin expression in healthy mice.

The roots of Polygonum multiflorum Thunberg (Polygonaceae) are used as a crude drug Kashu that is considered to improve blood deficiency based on a Kampo concept. Kashu has been included in Kampo formulas, such as Tokiinshi, which is used to treat eczema and dermatitis with itchiness by inhibiting inflammation and facilitating blood circulation in the skin. However, the effects of P. multiflorum roots on erythropoiesis are unclear. Previously, we isolated six phenolic constituents from an ethyl acetate (EtOAc)-soluble fraction of P. multiflorum root extract and identified them as (E)-2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside [(E)-THSG], emodin, emodin-8-O-ß-D-glucopyranoside, physcion, physcion-8-O-ß-D-glucopyranoside, and catechin. To examine whether P. multiflorum roots facilitate erythropoiesis, the EtOAc-soluble fraction was orally administered to healthy ICR mice. When compared with mice fed a standard diet alone (Controls), the mice fed a diet including the EtOAc-soluble fraction exhibited significantly higher serum erythropoietin (Epo) levels. The renal Epo mRNA levels in EtOAc-soluble fraction-administered mice were significantly higher than those in the control mice. Then, we administered roxadustat, which is a drug to treat the patient suffering with renal anemia by specifically inhibiting hypoxia-inducible factor prolyl hydroxylases. Roxadustat slightly increased renal Epo mRNA levels in healthy mice. Administration of (E)-THSG, a major constituent, significantly increased serum Epo levels. It is likely that (E)-THSG may facilitate the process to convert inactive renal Epo-producing cells to active Epo-producing cells. Collectively, it is implied that (E)-THSG in the EtOAc-soluble fraction of P. multiflorum roots may primarily improve blood deficiency of Kampo concept by promoting erythropoiesis.

Identification of Anti-Inflammatory Compounds from Peucedanum praeruptorum Roots by Using Nitric Oxide-Producing Rat Hepatocytes Stimulated by Interleukin 1ß.

The roots of Peucedanum praeruptorum Dunn and Angelica decursiva Franchet et Savatier are designated Zenko, which is a crude drug defined by the Japanese Pharmacopoeia. This crude drug is used as an antitussive and an expectorant and is included in the Kampo formula Jinsoin, which improves cough, fever, and headache. Although the anti-inflammatory effects of this crude drug have been determined, the constituents responsible for this effect remain unknown. To investigate biologically active compounds, rat hepatocytes were used, which produce proinflammatory mediator nitric oxide (NO) in response to proinflammatory cytokine interleukin 1ß (IL-1ß). A methanol extract of P. praeruptorum roots, which suppressed IL-1ß-induced NO production, was fractionated into three crude fractions (ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions) based on hydrophobicity. The EtOAc-soluble fraction markedly inhibited NO production. After this fraction was purified, three biologically active compounds were identified as praeruptorins A, B, and E, the contents of which were high. A comparison of their activities indicated that praeruptorin B exhibited the highest potency to inhibit NO production by decreasing inducible NO synthase expression and suppressed the expression of mRNAs encoding proinflammatory cytokines. Collectively, the three praeruptorins may primarily contribute to the anti-inflammatory effects of P. praeruptorum roots.

Detection of Decarboxylated Amino Acids after in Vitro Protease Digestion of the Hydrophilic Fraction of Crude Drug Extracts.

Many constituents of crude drugs in Japanese Kampo formulas are thought to function as pro-drugs, whose pharmacological activity is manifested after oral administration. Proteins and peptides in crude drugs may be digested and metabolized in the digestive tract and liver. However, few studies have reported the pharmacological activity of peptides in crude drugs. Here, we applied an analysis using LC-tandem mass spectrometry (LC-MS/MS) to identify the compounds derived from six crude drugs that are assumed to have anti-inflammatory effects. To simulate in vivo protease digestion, each water-soluble fraction of the crude drug extracts was treated with proteases, including endoproteinases and exopeptidases. Amines in the resultant digests were modified by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and analyzed using LC-MS/MS, which demonstrated the presence of four decarboxylated amino acids (primary amines). In the digest of the hydrophilic fraction of the fruit of Ziziphus jujuba Miller var. inermis Rehder (Taiso), isobutylamine, isoamylamine, and 2-methylbutylamine were identified, which may be derived from valinyl, leucinyl, and isoleucinyl residues, respectively. Additionally, tyramine possibly derived from tyrosyl residues was identified in the digests of all the crude drugs. In primary cultured rat hepatocytes treated with interleukin-1ß, all these decarboxylated amino acids suppressed the production of nitric oxide, a proinflammatory mediator. Our approach, i.e., in vitro protease digestion and LC-MS/MS analysis, suggests that decarboxylated amino acids may be formed in vivo from peptides and may be responsible for the anti-inflammatory effect of crude drugs included in Kampo medicine.

Bitter melon fruit extract enhances intracellular ATP production and insulin secretion from rat pancreatic ß-cells.

Bitter melon (Momordica charantia L.) has been shown to have various health-promoting activities, including antidiabetic and hypoglycaemic effects. Improvement in insulin sensitivity and increase in glucose utilisation in peripheral tissues have been reported, but the effect on insulin secretion from pancreatic ß-cells remains unclear. In this study, we investigated the effect of bitter melon fruit on insulin secretion from ß-cells and the underlying mechanism. The green fruit of bitter melon was freeze-dried and extracted with methanol. The bitter melon fruit extract (BMFE) was fractionated using ethyl acetate (fraction A), n-butanol (fraction B) and water (fraction C). Insulin secretory capacity from INS-1 rat insulinoma cell line and rat pancreatic islets, as well as glucose tolerance in rats by oral glucose tolerance test (OGTT), was measured using BMFE and fractions. ATP production in ß-cells was also examined. BMFE augmented insulin secretion from INS-1 cells in a dose-dependent manner. The significant augmentation of insulin secretion was independent of the glucose dose. Fraction A (i.e. hydrophobic fraction), but not fractions B and C, augmented insulin secretion significantly at the same level as that by BMFE. This finding was also observed in pancreatic islets. In OGTT, BMFE and fraction A decreased blood glucose levels and increased serum insulin levels after glucose loading. The decrease in blood glucose levels was also observed in streptozotocin-induced diabetic rats. In addition, BMFE and fraction A increased the ATP content in ß-cells. We concluded that hydrophobic components of BMFE increase ATP production and augment insulin secretion from ß-cells, consequently decreasing blood glucose levels.

Comparative Analysis of Anti-inflammatory Activity of the Constituents of the Rhizome of Cnidium officinale Using Rat Hepatocytes.

The rhizome of Cnidium officinale (Umbelliferae) (known as Senkyu in Japan; COR) has been used as a crude drug in Japanese Kampo formulas, such as Jumihaidokuto (to treat eczema and urticaria) and Kakkontokasenkyushin'i (to treat rhinitis). COR contains phthalides, which are thought to be potent principal constituents. Few studies have been reported about the comparison of anti-inflammatory activity of COR constituents. We aimed to identify the constituents in COR and compare their anti-inflammatory activity. COR was extracted with methanol and fractionated into ethyl acetate (EtOAc)-soluble, n-butanol-soluble, and water-soluble fractions. Primary cultured rat hepatocytes were used to assess anti-inflammatory activity by monitoring the interleukin (IL)-1ß-induced production of nitric oxide (NO), an inflammatory mediator. The EtOAc-soluble fraction significantly suppressed NO production without showing cytotoxicity in IL-1ß-treated hepatocytes, whereas the n-butanol-soluble fraction showed less potency, and the water-soluble fraction did not significantly affect the NO levels. Four constituents were isolated from the EtOAc-soluble fraction and identified as senkyunolide A, (3S)-butylphthalide, neocnidilide, and cnidilide. Among these phthalides and (Z)-ligustilide, senkyunolide A and (Z)-ligustilide efficiently suppressed NO production in hepatocytes, whereas the others showed less potency in the suppression of NO production. Furthermore, senkyunolide A decreased the levels of the inducible nitric oxide synthase (iNOS) protein and mRNA, as well as the levels of mRNAs encoding proinflammatory cytokines (e.g., tumor necrosis factor α) and chemokine C-C motif ligand 20. These results suggest that senkyunolide A may cause the anti-inflammatory and hepatoprotective effects of COR by suppressing the genes involved in inflammation.

Correction to: Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice.

The article Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice, written by Toshinari Ishii, Tetsuya Okuyama, Nao Noguchi, Yuto Nishidono, Tadayoshi Okumura, Masaki Kaibori, Ken Tanaka, Susumu Terabayashi, Yukinobu Ikeya and Mikio Nishizawa was originally published Online First without Open Access. After publication in volume 74 issue 1, page 51-64 the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2020 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice.

Qing Dai/Indigo Naturalis (QD) has been shown to ameliorate ulcerative colitis (UC) in clinical trials; however, its mechanism remains elusive. This study investigates the effects of QD on murine dextran sulfate sodium salt-induced colitis. Oral administration of QD protected the animals from colitis as manifested by weight loss, diarrhea, and rectal bleeding. QD was distinguishingly more effective than 5-aminosalicylate. Focused microarray analysis of genes expressed in the distal colon suggested that QD influences the inflammatory pathway. Anti-inflammatory activity of QD was confirmed by the suppression of nitric oxide (NO) production in response to interleukin-1ß in cultured hepatocytes. Some of the constituents in QD, such as tryptanthrin (TRYP) and indigo, suppressed NO production. TRYP maintained body weight but did not inhibit bleeding. Indigo, on the other hand, partially ameliorated bleeding, but did not maintain body weight. The combination of TRYP and indigo did not show additive ameliorating activity. The methanol extract of QD showed an anti-colitis activity like that of TRYP. In contrast, the methanol-insoluble QD fraction moderately ameliorated diarrhea and bleeding. Combining these two fractions resulted in full anti-colitis activity. Further clarification of the active constituents will help in the discovery of a safe and potent prescription for UC.

Antiinflammatory constituents of Atractylodes chinensis rhizome improve glomerular lesions in immunoglobulin A nephropathy model mice.

The crude drug Sojutsu, as defined by the Japanese Pharmacopoeia, is the rhizome of Atractylodes lancea De Candolle, Atractylodes chinensis Koidzumi, or their interspecific hybrids (Asteraceae). Sojutsu is one of the traditional Kampo formulas, which are administered to patients suffering from stomach disorders, edema, and nephrotic syndrome. Although antiinflammatory effects of Sojutsu have been reported, its effects on the liver and kidney have not been extensively investigated. Here, we used a Sojutsu sample identified as A. chinensis rhizome and isolated several constituents from its ethyl acetate (EtOAc)-soluble fraction that decreased production of the proinflammatory mediator nitric oxide (NO) in interleukin 1ß-treated rat hepatocytes. Among the constituents in this fraction, atractylodin showed the highest activity to suppress NO production, whereas hinesol, ß-eudesmol, and α-bisabolol showed low activity. Atractylodin decreased the levels of inducible nitric oxide synthase, tumor necrosis factor α, and lipocalin 2 messenger RNAs (mRNAs). The EtOAc-soluble fraction of the A. chinensis rhizome extract was administered daily for 20 weeks to high immunoglobulin A (HIGA) mice, whose pathological findings resemble human immunoglobulin A nephropathy. This fraction decreased the weight of white adipose tissue and decreased mesangial proliferation and immunoglobulin A deposition in glomeruli. These results indicate that the EtOAc-soluble fraction, which included antiinflammatory constituents, may be responsible for improvement of the mesangial lesions in HIGA mice.

Bitter melon fruit extract has a hypoglycemic effect and reduces hepatic lipid accumulation in ob/ob mice.

Bitter melon (Momordica charantia L.) is a vegetable and has been used as traditional medicine. Recently, we reported that bitter melon fruit extracts and its ethyl acetate (EtOAc)-soluble fraction markedly suppressed the expression of proinflammatory genes, including the inducible nitric oxide synthase gene. However, it is unclear whether bitter melon exhibits antidiabetic effects. In this study, we showed that cucurbitacin B, a cucurbitane-type triterpenoid, was present in an EtOAc-soluble fraction and suppressed nitric oxide production in hepatocytes. When the EtOAc-soluble fraction was administered for 7 days to ob/ob mice, a type 2 diabetes mellitus model, the mice fed with this fraction exhibited a significant decrease in body weight and blood glucose concentrations compared with the mice fed without the fraction. The administration of the fraction resulted in significant increases in serum insulin concentrations and the levels of both insulin receptor mRNA and protein in the ob/ob mouse liver. The EtOAc-soluble fraction decreased the interleukin-1ß mRNA expression, as well as hepatic lipid accumulation in hepatocytes. Taken together, these results indicate that administration of an EtOAc-soluble fraction improved hyperglycemia and hepatic steatosis, suggesting that this fraction may be responsible for both the antidiabetic and anti-inflammatory effects of bitter melon fruit.

Anti-inflammatory kavalactones from Alpinia zerumbet.

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. (Zingiberaceae) is a perennial plant native to the East Indies and is widely distributed in South America, Oceania, and Asia. The mature fruits of the plant have been used in traditional medicine in China. In this study, we compared the chemical constituents in the methanol extracts of the leaves, the placenta, the pericarps, and the seeds obtained from the same plant using LC-MS, and we examined the NO inhibitory activities of the respective extracts and the isolated compounds. As a result of LC-MS analyses, kavalactone derivatives (1-6) were detected in the methanol extracts of the leaves, placenta, and pericarps. Of these, compound 6 was identified as a new asymmetrical cyclobutane dimer of 5,6-dehydrokawain. Quantitative analysis showed that the total amounts of kavalactone derivatives were highest in the methanol extract of the pericarps. Moreover, the results of measurements of the anti-inflammatory activity revealed that the pericarps extract showed the strongest activity. The compounds responsible for the anti-inflammatory activity of the extracts from A. zerumbet were identified. Of these, five were known kavalactone derivatives and one was a new kavalactone derivative (aniba dimer C). The results showed that the pericarps of A. zerumbet are a rich source of kavalactone derivatives, and that the pericarps of A. zerumbet can be utilized as an important medicinal resource.

Effect of heat processing on the chemical constituents and NO-suppressing activity of Bletilla Tuber.

Bletilla Tuber (dried tuber of Bletilla striata) is used as an astringent hemostatic medicine for the treatment of ulcers, bleeding, and burns in traditional Chinese medicine (TCM). The Chinese Pharmacopoeia describes the heat processing methods used on raw tubers of Bletilla striata to produce the herbal medicine "Bletilla Tuber". In this study, we compared the chemical constituents of well-processed Bletilla Tuber (BT1) and normally processed Bletilla Tuber (BT2) derived from the same origin. In addition, as an indicator of the hemostatic activity of Bletilla Tuber, the NO inhibitory activities of extracts obtained from BT1 and BT2 and the isolated compounds were examined. As a result of LC-MS analysis, three types of compounds, glucosyloxybenzyl 2-isobutylmalates, bibenzyl derivatives and phenanthrene derivatives, were detected. Comparison of the chemical profiles of the extracts indicated that the relative contents of glucosyloxybenzyl 2-isobutylmalates had changed by heat processing, whereas the relative contents of bibenzyls and phenanthrenes had not changed. The extracts of BT1 and BT2 showed similar IC50 values on NO production suppressing activity. Furthermore, phenanthrenes and bibenzyls were identified as the compounds responsible for suppressing the NO activity. These results suggest that the biological activities, such as the anti-inflammatory and hemostatic activities, of Bletilla Tuber are not affected by heat processing.

Active Hexose Correlated Compound Has Protective Effects in Ischemia-Reperfusion Injury of the Rat Small Intestine.

BACKGROUND: Ischemia-reperfusion (IR) injury of the small intestine is a serious problem in abdominal aortic aneurysm surgery or small intestine transplantation. Active hexose correlated compound (AHCC) is a popular anti-inflammatory drug in complementary and alternative medicine. The aim of this study was to examine whether pretreatment with AHCC reduces intestinal IR injury. METHODS: Rats were given a normal diet (IR group) or normal diet supplemented with 2% AHCC (IR + AHCC group) ad libitum for 10 d. After 1 d of fasting, the superior mesenteric artery was occluded by clipping for 45 min. Intestinal and blood samples were collected for 1-6 h after reperfusion. The messenger RNA (mRNA) and protein levels of inflammatory factors were analyzed. RESULTS: The IR + AHCC group had reduced mucosal abrasion and significantly increased mucosal thickness of the intestinal tissues 6 h after reperfusion, compared with the IR group. AHCC decreased mRNA expression of inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant 1 and interleukin 6 in the mucosa of the small intestine. AHCC also decreased expression of iNOS protein. Serum levels of cytokine-induced neutrophil chemoattractant 1 and tumor necrosis factor α were decreased in the IR + AHCC group compared with the IR group. Electrophoretic mobility shift assay of mucosal nuclear extracts revealed that AHCC inhibited the activation of nuclear factor kappa B. AHCC also inhibited the expression of iNOS antisense transcript, which stabilizes iNOS mRNA. CONCLUSIONS: Our findings suggest that AHCC reduces expression of inflammatory mediators, in part, by inhibiting nuclear factor kappa B activation. AHCC may have anti-inflammatory effect in patients with intestinal IR injury.

Sakuranetin downregulates inducible nitric oxide synthase expression by affecting interleukin-1 receptor and CCAAT/enhancer-binding protein ß.

Pruni Cortex is a herbal drug from the bark of the Japanese flowering cherries, Prunus jamasakura or Prunus verecunda, and is included in the traditional Japanese herbal (Kampo) formula Jumihaidokuto, which is administered orally to patients suffering from inflammatory skin diseases. The flavanones contained in Pruni Cortex (e.g., sakuranetin and naringenin) have potent anti-inflammatory, anti-allergic, and anti-microbial activities. Although the effects of Pruni Cortex on skin disease have been well studied, reports regarding its pharmacological effects on the liver are limited. In this study, we extracted the bark of Prunus jamasakura and purified it to isolate the pharmacologically active constituents by monitoring nitric oxide (NO) production in rat hepatocytes that were treated with the pro-inflammatory cytokine, interleukin (IL)-1ß. Sakuranetin and (-)-naringenin, which were present in an ethyl acetate-soluble fraction of the bark extract, significantly inhibited NO induction and inducible nitric oxide synthase (iNOS) expression. These two flavanones decreased the expression of type 1 IL-1 receptor gene and phosphorylation of Akt, also known as protein kinase B, which is regulated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Furthermore, sakuranetin decreased the phosphorylation of the activator isoforms of CCAAT/enhancer-binding protein ß (C/EBPß), which synergistically activates the transcription of the iNOS gene with nuclear factor κB (NF-κB). Therefore, sakuranetin inhibited the co-activating activity of C/EBPß with NF-κB, leading to the suppression of iNOS gene expression in hepatocytes. Taken together, sakuranetin in Pruni Cortex downregulated the iNOS gene by inhibiting PI3K/Akt signal transduction and the phosphorylation of C/EBPß. These results imply that sakuranetin may be primarily responsible for the anti-inflammatory effects of Pruni Cortex in the liver.

Identification of PGC-1α activating constituents in Zingiberaceous crude drugs.

The activity of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) as an index of thermogenesis induced by four Indonesian Zingiberaceous crude drugs, Boesenbergia rotunda, Curcuma longa, Kaempferia galanga, Zingiber montanum, was examined, and GC-MS analyses of extracts of these drugs were performed. The results showed that activation of PGC-1α by K. galanga was high, whereas no activation was shown for the other drugs. Ethyl p-methoxycinnamate and ethyl cinnamate were identified as the PGC-1α activating compounds of K. galanga. Furthermore, study on the structure-activity relationship revealed that ethyl p-methoxycinnamate has the strongest activity among the cinnamic acid derivatives. This suggests that the ester structure and the methoxy group are important factors responsible for the PGC-1α activity.

Identification of anti-inflammatory constituents in Phellodendri Cortex and Coptidis Rhizoma by monitoring the suppression of nitric oxide production.

Phellodendri Cortex (Obaku in Japanese) and Coptidis Rhizoma (Oren), both of which contain berberine, have been used to prepare the kampo formula orengedokuto to treat inflammatory diseases, including dermatitis, gastric ulcers, and gastritis. These drugs are blended differently in other formulas, such as the use of Phellodendri Cortex in shichimotsukokato to treat hypertension and Coptidis Rhizoma in hangeshashinto to treat diarrhea and stomatitis. However, the differences in their medicinal properties are not well characterized. We prepared extracts from Phellodendron amurense bark (PAB) and Coptis chinensis rhizome (CCR) and separated them into alkaloid and non-alkaloid fractions. Anti-inflammatory effects were examined by monitoring the production of nitric oxide (NO), which is a pro-inflammatory mediator. A non-alkaloid fraction of the PAB extract suppressed NO production in hepatocytes more efficiently than that of the CCR extract. When each non-alkaloid fraction of the PAB and CCR extracts was administered to mice, the fractions of both extracts decreased the levels of mRNAs encoding inducible NO synthase and molecules in the interleukin-1ß signaling pathway. Limonin and obakunone identified in the PAB non-alkaloid fraction suppressed NO production, exhibiting IC50 values of 16 and 2.6 µM, respectively, whereas berberine and coptisine displayed IC50 values of 12 and 14 µM, respectively. Limonin and obakunone reduced the expression of the iNOS gene, probably through the transcription factor nuclear factor-κB. Therefore, both limonoids and alkaloids may be responsible for the anti-inflammatory effects of the PAB extract, whereas alkaloids may be primarily responsible for those of the CCR extract. The different composition of the constituents may modulate the anti-inflammatory effects of Phellodendri Cortex and Coptidis Rhizoma.

Genipin Inhibits the Induction of Inducible Nitric Oxide Synthase Through the Inhibition of NF-κB Activation in Rat Hepatocytes.

BACKGROUND/AIMS: Genipin is a component of Japanese traditional herbal medicine (Kampo), inchinkoto, and is used for the treatment of various liver injuries. However, there are few scientific evidence for its anti-inflammatory effects and mechanisms. In inflamed liver, proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß stimulate liver cells, followed by the expression of inducible nitric oxide synthase (iNOS). Excessive levels of NO produced by iNOS have been implicated as one of the factors in liver injury. Thus it is essential to inhibit iNOS induction for the prevention of liver injury. In this study, we examined IL-1ß-stimulated hepatocytes as a simple "in vitro liver injury model" to investigate liver protective effects of genipin. METHODS: Primary cultured rat hepatocytes were treated with IL-1ß in the presence or absence of genipin. The induction of NO production and iNOS, and its signaling pathway were analyzed. RESULTS: In IL-1ß-stimulated hepatocytes, genipin inhibited the production of NO dose- and timedependently, and reduced the levels of iNOS protein and its mRNA expression. Genipin also reduced mRNA expressions of TNF-α and IL-6. Genipin inhibited two essential signaling pathways for iNOS induction, IκB degradation/NF-κB activation and type I IL-1 receptor upregulation. Transfection experiments revealed that genipin decreased the expression of iNOS mRNA through both inhibitions of the promoter activation and mRNA stabilization. Delayed administration of genipin after IL-1ß addition also inhibited iNOS induction. CONCLUSION: Genipin influenced the induction of inflammatory mediators, iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. Genipin may have therapeutic potential for organ injuries including liver.

Lignans from guaiac resin decrease nitric oxide production in interleukin 1ß-treated hepatocytes.

Guaiac resin, extracted from the heartwood of Guaiacum officinale L. or G. sanctum L., is speculated to have anti-inflammatory effects. Lignans were purified from guaiac resin (also known as gum guaiacum) by monitoring the nitric oxide (NO) production in rat hepatocytes treated with an inflammatory cytokine interleukin-1ß (IL-1ß). Six lignans were purified from guaiac resin and identified as: dehydroguaiaretic acid (1), (+)-trans-1,2-dihydrodehydroguaiaretic acid (2), furoguaiaoxidin (3), meso-dihydroguaiaretic acid (4), furoguaiacin (i.e., α-guaiaconic acid) (5), and nectandrin B (6). To our knowledge, this is the first time that 1 has been isolated from guaiac resin as a non-derivative. Compounds 2 and 6 were first found in guaiac resin. Compound 3 was first isolated from a natural source as a non-derivative. Furthermore, 1-6 significantly suppressed NO production in IL-1ß-treated hepatocytes. Because anti-inflammatory compounds suppress NO production, this system is often used to measure the anti-inflammatory effects of Kampo drugs and herbal constituents. The NO-suppressing activity of the six lignans isolated in this study indicates that guaiac resin has anti-inflammatory effects and that these lignans may be responsible for the anti-inflammatory effects of guaiac resin.

Comparative analysis of the constituents in Saposhnikoviae Radix and Glehniae Radix cum Rhizoma by monitoring inhibitory activity of nitric oxide production.

During the development of natural herbal medicines in Japan, Glehniae Radix cum Rhizoma (Hamabofu in Japanese) has been used as a substitute for Saposhnikoviae Radix (Bofu). Bofu and Hamabofu are blended differently in several Kampo formulae. For example, Bofu is included in Jumihaidokuto by a manufacturer, whereas Hamabofu is included instead of Bofu in the same formula by other manufacturers. Although both Bofu and Hamabofu are used for their expected anti-inflammatory effects, differences in their medicinal properties are not well characterized. In addition, there have been very few reports comparing the pharmacological activities of the constituents in Bofu and Hamabofu. In the present study, we investigated the anti-inflammatory effects of the extracts of Bofu and Hamabofu by monitoring levels of the inflammatory mediator nitric oxide (NO) produced in rat hepatocytes. Moreover, the chemical constituents responsible for the activity were investigated. Our results showed that ethyl acetate fractions of Bofu and Hamabofu extracts contain different compounds, although both fractions suppressed NO production in rat hepatocytes. The linear dihydropyranochromones from the Bofu extract (i.e., 3'-O-angeloylhamaudol, ledebouriellol and hamaudol) suppressed NO production, whereas the coumarins from the Hamabofu extract (i.e., umbelliferone and scopoletin) also suppressed NO production. These results suggest that linear dihydropyranochromones and coumarins are responsible for the anti-inflammatory effects of Bofu and Hamabofu. It is plausible that Bofu and Hamabofu are blended differently in several Kampo formulae due to many constituents with as yet unidentified pharmacological activity.

Japanese Kampo Saireito Has a Liver-Protective Effect Through the Inhibition of Inducible Nitric Oxide Synthase Induction in Primary Cultured Rat Hepatocytes.

BACKGROUND: Japanese herbal medicine, Kampo saireito, is used for treatments in patients with digestive diseases, including chronic hepatitis and cirrhosis. However, few studies demonstrate scientific evidence for liver-protective effects of saireito. In inflamed liver, proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß stimulate the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. Excessive levels of NO synthesized by iNOS have been implicated as one of the factors in liver injury, so it is essential to reduce the induction of iNOS for the prevention of liver injury. In this study, we examined IL-1ß-stimulated hepatocytes as a simple "in vitro injury model" to investigate liver-protective effects of saireito. METHOD: Primary cultured rat hepatocytes were treated with IL-1ß in the presence or absence of saireito. The induction of NO production and iNOS and its signaling pathway were analyzed. RESULTS: Saireito inhibited the production of NO dose and time dependently and reduced the expression of iNOS messenger RNA (mRNA) and its protein. Saireito had no effect on IκB degradation but inhibited the translocation of nuclear factor (NF)-κB to the nucleus and its DNA binding. Saireito also inhibited the activation of Akt, resulting in the reduction of type I IL-1 receptor (IL-1RI) mRNA and protein expression. CONCLUSION: These findings demonstrate that saireito suppresses iNOS gene expression through the inhibition of NF-κB and IL-1RI-dependent pathways, leading to the reduction of NO production. Saireito may have therapeutic potential for organ injuries, including liver.

A new flavanone and other flavonoids from green perilla leaf extract inhibit nitric oxide production in interleukin 1ß-treated hepatocytes.

A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1ß-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.