Production of hispidin polyphenols from medicinal mushroom Sanghuangporus vaninii in submerged cultures.

Objective: The medicinal mushroom Sanghuangporus vaninii produces pharmaceutically valuable hispidin polyphenols in natural habitats. However, due to the slow growth in nature, S. vaninii grown in the field (sclerotia) is not reliable for pharmaceutical purposes. Although higher biomass of fungal mycelia can be obtained in submerged cultures, the accumulation of hispidin polyphenols is rare. Methods: In this study, the polyunsaturated fatty acids (PUFAs), linoleic acid (LA), linolenic acid (ALA), and methyl jasmonate (MeJa) were employed as the stimulant agents to coordinate the accumulation of biomass and hispidin polyphenols in its submerged cultures. Results: The addition of LA and ALA promoted the mycelial accumulation, while the addition of MeJa inhibited the growth of S. vaninii concomitant with reduced total polyphenols. UPLC-Triple-TOF-MS analysis revealed an increased production of hispidin, phellinstatin, pinnilidine, and its derivatives upon the addition of LA and ALA, and hypholomine B and its isomer, 3,14'-bihispidinyl, and phelligridin E upon the addition of MeJa on day 13. Intriguingly, total polyphenols from the MeJa-supplementing cultures harbored a high capacity in scavenging free radicals. Chemical structural analysis showed that hispidin polyphenols had higher antioxidant activity due to more hispidin moieties induced by MeJa. Conclusion: The supplement of PUFAs affects the synthesis and composition of hispidin polyphenols in S. vaninii. Our results provide a possibility to coordinate the production of hispidin polyphenols via submerged cultures of S. vaninii.

Ultraviolet radiation promotes the production of hispidin polyphenols by medicinal mushroom Inonotus obliquus.

Production of hispidin polyphenols in Inonotus obliquus is a stress-induced response triggered by environmental factors. As one of the important environmental factors, ultraviolet (UV) radiation plays regulatory roles in fungal growth and development. However, whether UV radiation regulates the formation of hispidin polyphenols remains to be established. In this study, we cultivated I. obliquus on solid medium and imposed intermittent UV radiation. We showed that UV exposure inhibited the growth of mycelia but increased the production of polyphenols. Further bioassays revealed that UV radiation also increased the catalytic activities of phenylalanine ammonia-lyase (PAL) and chalcone isomerase (CHI), up-regulated expression of genes related to redox, transcriptional regulation, and metabolism. In addition, the total extracts from the UV-irradiated group were more capable of scavenging DPPH and ABTS+ free radicals, especially at the later stage of culture. Thus, UV radiation, acting as one of the environmental factors, stimulated the accumulation of polyphenols in I. obliquus by regulating the activities of enzymes and the expression of genes related to growth and metabolism, and can be tentatively used as a feasible strategy to enhance the production of polyphenols in I. obliquus.

Deciphering the antitumoral potential of the bioactive metabolites from medicinal mushroom Inonotus obliquus.

ETHNOPHARMACOLOGICAL RELEVANCE: The crude extracts of the medicinal mushroom Inonotus obliquus have been used as an effective traditional medicine to treat malicious tumors, gastritis, gastric ulcers, and other inflammatory conditions in Russia and most Baltic countries. AIM OF THIS REVIEW: Deciphering the antitumoral potential of the bioactive metabolites from I. obliquus and addressing its possibility to be used as effective agents for tumor treatment, restoration of compromised immunity and protection of gastrointestinal damage caused by chemotherapy. MATERIALS AND METHODS: We analysed the current achievements and dilemma in tumor chemo- or immunotherapy. In this context, we searched the published literatures on I. obliquus covering from 1990 to 2020, and summarized the activities of antitumor, antioxidation, and immunomodulation by the polysaccharides, triterpenoids, small phenolic compounds, and hispidin polyphenols. By comparing the merits and shortcomings of current and traditional methodology for tumor treatment, we further addressed feasibility for the use of I. obliquus as an effective natural drug for tumor treatment and prevention. RESULTS: The diverse bioactive metabolites confer I. obliquus great potential to inhibit tumor growth and metastasis. Its antitumor activities are achieved either through suppressing multiple oncogenic signals including but not limited to the activation of NF-κB and FAK, and the expression of RhoA/MMP-9 via ERK1/2 and PI3K/Akt signaling pathway. The antitumor activities can also be achieved by inhibiting tyrosinase activity via PAK1-dependent signaling pathway or altering lysosomal membrane permeabilization through blocking tubulin polymerization and/or disturbing energy metabolism through LKB1/AMPK pathway. In addition, the metabolites from I. obliquus also harbour the potentials to reverse MDR either through selective inhibition on P-gp/ABCB1 or MRP1/ABCC1 proteins or the induction of G2/M checkpoint arrest in tumor cells of chemoresistant phenotypes mediated by Nox/ROS/NF-kB/STAT3 signaling pathway. In addition to the eminent effects in tumor inhibition, the metabolites in I. obliquus also exhibit immunomodulatory potential to restore the compromised immunity and protect against ulcerative damage of GI tract caused by chemotherapy. CONCLUSIONS: I. obliquus possesses the potential to reduce incidence of tumorigenesis in healthy people. For those whose complete remission has been achieved by chemotherapy, administration of the fungus will inhibit the activation of upstream oncogenic signals and thereby prevent metastasis; for those who are in the process of chemotherapy administration of the fungus will not only chemosensitize the tumor cells and thereby increasing the chemotherapeutic effects, but also help to restore the compromised immunity and protect against ulcerative GI tract damage and other side-effects induced by chemotherapy.

Disturbance in biosynthesis of arachidonic acid impairs the sexual development of the onion blight pathogen Stemphylium eturmiunum.

The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.

Regulation of Anticancer Styrylpyrone Biosynthesis in the Medicinal Mushroom Inonotus obliquus Requires Thioredoxin Mediated Transnitrosylation of S-nitrosoglutathione Reductase.

The medicinal macrofungus Inonotus obliquus widely utilized as folk medicine in Russia and Baltic countries is a source of phenylpropanoid-derived styrylpyrone polyphenols that can inhibit tumor proliferation. Insights into the regulatory machinery that controls I. obliquus styrylpyrone polyphenol biosynthesis will enable strategies to increase the production of these molecules. Here we show that Thioredoxin (Trx) mediated transnitrosylation of S-nitrosoglutathione reductase (GSNOR) underpins the regulation of styrylpyrone production, driven by nitric oxide (NO) synthesis triggered by P. morii coculture. NO accumulation results in the S-nitrosylation of PAL and 4CL required for the synthesis of precursor phenylpropanoids and styrylpyrone synthase (SPS), integral to the production of styrylpyrone, inhibiting their activities. These enzymes are targeted for denitrosylation by Trx proteins, which restore their activity. Further, this Trx S-nitrosothiol (SNO) reductase activity was potentiated following S-nitrosylation of Trx proteins at a non-catalytic cysteine (Cys) residue. Intriguingly, this process was counterbalanced by Trx denitrosylation, mediated by Trx-dependent transnitrosylation of GSNOR. Thus, unprecedented interplay between Trx and GSNOR oxidoreductases regulates the biosynthesis of styrylpyrone polyphenols in I. obliquus.

Reversible S-nitrosylation limits over synthesis of fungal styrylpyrone upon nitric oxide burst.

Nitric oxide (NO) is known to be involved in modulating production of styrylpyrone polyphenols in the basidiomycete Inonotus obliquus. However, it remains unknown how NO orchestrates fungal styrylpyrone biosynthesis. Here, we show that a transient NO burst correlated with an enhanced expression of phenylalanine ammonia lyase (PAL), 4-coumarate CoA ligase (4CL), and styrylpyrone synthase (SPS), the key enzymes involved in styrylpyrone biosynthesis, and subsequently an increased production of styrylpyrone polyphenols. In parallel, the NO burst also resulted in S-nitrosylation of PAL, 4CL, and SPS, which compromised their enzymatic activities mediating a post-translational feedback mechanism that keeps NO-dependent transcriptional activation in check. Moreover, dysfunction of thioredoxin reductase (TrxR) further increased the formation of S-nitrosylated proteins, implicating the significance of the Trx system in maintaining a low level of protein-nitrosothiols. Three thioredoxin-like proteins (TrxLs) from I. obliquus show in vitro denitrosylation potential toward S-nitrosylated proteins via trans-denitrosylation or mixed disulfide intermediates. Thus, S-nitrosylation triggered by the NO burst limits over production of fungal styrylpyrone polyphenols, and denitrosylation by TrxLs that act in concert with TrxR play a key role in maintaining redox balance and orchestrating catalytic activities of the enzymes engaged in styrylpyrone synthetic metabolism.

Correlation of nitric oxide produced by an inducible nitric oxide synthase-like protein with enhanced expression of the phenylpropanoid pathway in Inonotus obliquus cocultured with Phellinus morii.

Fungal interspecific interactions enhance biosynthesis of phenylpropanoid metabolites (PM), and production of nitric oxide (NO) is known to be involved in this process. However, it remains unknown which signaling pathway(s) or regulator(s) mediate fungal PM biosynthesis. In this study, we cocultured two white-rot fungi, Inonotus obliquus and Phellinus morii, to examine NO production, expression of the genes involved in phenylpropanoid metabolism and accumulation of phenylpropanoid-derived polyphenols by I. obliquus. Coculture of the two fungi caused an enhanced NO biosynthesis followed by increased transcription of the genes encoding phenylalanine ammonia lyase (PAL) and 4-coumarate CoA ligase (4CL), as well as an upregulated biosynthesis of styrylpyrone polyphenols in I. obliquus. Addition of the NO synthase (NOS) selective inhibitor aminoguanidine (AG) inhibited NO production by more than 90% followed by cease in transcription of PAL and 4Cl. Treatment of guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one did not affect NO production but suppressed transcription of PAL and 4CL and reduced accumulation of total phenolic constituents. Genome-wide analysis of I. obliquus revealed two genes encoding a constitutive and an inducible NOS-like protein, respectively (cNOSL and iNOSL). Coculture of the two fungi did not increase the expression of the cNOSL gene but triggered expression of the iNOSL gene. Cloned iNOSL from Escherichia coli shows higher activity in transferring L-arginine to NO, and this activity is lost upon AG addition. Thus, iNOSL is more responsible for NO production in I. obliquus and may act as an important regulator governing PM production during fungal interspecific interactions.

Halogenated metabolites isolated from Penicillium citreonigrum.

Three chromone analogs, 1-3, a chlorinated alkaloid sclerotioramine (4), together with two 11-noreremophilane-type sesquiterpenes with a conjugated enolic OH group and a brominated one, 5 and 6, respectively, were isolated from Penicillium citreonigrum (HQ738282). Compounds 1, 5, and 6 were new. Biological tests revealed that 4 exhibited a significant activity (IC50 7.32 µg/ml), and 6 showed a moderate activity (IC50 16.31 µg/ml) in vitro against HepG2 cell line, and 4 also displayed an activity comparable to that of acarbose against α-glucosidase.

Design and diversity-oriented synthesis of novel 1,4-thiazepan-3-ones fused with bioactive heterocyclic skeletons and evaluation of their antioxidant and cytotoxic activities.

This study has achieved the design and diversity-oriented synthesis of novel 1,4-thiazepine derivatives embedded with carbazole, pyrazole or isoxazole motif via microwave-assisted multicomponent reactions under solvent-free condition, thus providing a green and facile access to 1,4-thiazepine derivatives with prominent features of high structural diversity, short reaction time, high yields and environmental friendliness. More importantly, these novel compounds have been subjected to the test of in vitro antioxidant and cytotoxic activities, resulting in the finding that these 1,4-thiazepine derivatives not only have significant antioxidant activity, but also exhibit remarkably selective cytotoxicity to carcinoma cell line HCT 116.

Involvements of S-nitrosylation and denitrosylation in the production of polyphenols by Inonotus obliquus.

Nitric oxide (NO) has been evidenced to mediate biosynthesis of polyphenols in Inonotus obliquus. However, it remains unknown how NO regulates their biosynthesis. Here we show that higher cellular NO levels coincided with higher accumulation of S-nitrosothiols (SNO; the products of NO combined with a specific residue in glutathione or proteins) and polyphenols, and higher activity of denitrosylated S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductase (TrxR). This homeostasis was breached by GSNOR or TrxR inhibitors. Inhibiting GSNOR boosted TrxR activity, but reduced SNO formation, coinciding with an enhanced production of polyphenols. Likewise, inhibiting TrxR increased GSNOR activity and SNO production, but downregulated accumulation of polyphenols. Inhibiting GSNOR or TrxR also modified the polyphenolic profiles of I. obliquus. Suppressing GSNOR-enhanced biosynthesis of phelligridins C and H, inoscavin C and methyl inoscavin B, but reduced that of phelligridin D, methyl inoscavin A, davallialactone and methyl davallialactone, the typical polyphenols in I. obliquus. Similarly, downregulating TrxR increased production of phelligridin D, methyl inoscavin A, davallialactone, and methyl davallialactone, but shrinking that of phelligridins C and H, methyl inoscavin B and inoscavin C. Thus, in I. obliquus, the state of S-nitrosylation and denitrosylation affects not only the accumulation of polyphenols, but also their metabolic profiles.

Analysis of antioxidant metabolites by solvent extraction from sclerotia of Inonotus obliquus (Chaga).

INTRODUCTION: The sclerotia of Inonotus obliquus (Chaga) are effective therapeutic agents to treat several human malignant tumours and other diseases without unacceptable toxic side-effects. OBJECTIVE: To investigate solvent effects on metabolic profiles and antioxidant activities of extracts of Chaga. METHODOLOGY: Chaga was extracted by petroleum ether, chloroform, ethyl acetate, acetone, ethanol and water. Solvent effects on metabolites in the extracts were assayed by NMR-based metabolomic analysis. Antioxidant activities were indicated as capacities for scavenging superoxide anion, DPPH and hydroxyl radicals. RESULTS: Petroleum ether and chloroform extracts contained primarily lanostane-type triterpenoids (LT), whereas the extracts of ethyl acetate, acetone and ethanol were characterised by the predominant presence of hispidin analogues and LT, and water extracts by polysaccharides and phenolic compounds. The ethyl acetate, acetone, ethanol and water extracts revealed remarkable potential for scavenging the tested radicals, while those of petroleum ether and chloroform did not. Polyphenols are the major contributors for quenching the tested free radicals, while in LT only compounds 16, 17 and 22 participated in scavenging hydroxyl radicals. CONCLUSION: Polyphenols in Chaga are the principles for quenching free radicals while polysaccharides and a few LT compounds contribute partially in scavenging DPPH and hydroxyl radicals, respectively. NMR-based metabolomic analysis is a useful method by which to correlate ¹H-NMR spectra of Chaga extracts with their antioxidant activities, and this allows the prediction of potentials for scavenging free radicals by ¹H-NMR spectroscopy.

Production of antioxidant and antitumor metabolites by submerged cultures of Inonotus obliquus cocultured with Phellinus punctatus.

While Inonotus obliquus produces a diverse range of bioactive metabolites in its natural habitats, it accumulates less in its submerged cultures. We show here that coculture of I. obliquus with Phellinus punctatus resulted in less production of mycelial biomass but an increased accumulation of phenolic compounds, melanins, and lanostane-type triterpenoids. Metabolites increased in production by coculture include phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, inoscavin B, davallialactone, methyl davallialactone, foscoparianol D, 21,24-cyclopentalanosta-3ß,21,25-triol-8-en, lanosta-7,9(11),23-triene-3ß,22,25-triol, and inotodisaccharide and melanins. Metabolites from coculture also showed an increased potential for scavenging free radicals and inhibiting the proliferation of HeLa 229 cells. Davallialactone, methyl davallialactone, and minor phenolic components are the major contributors for scavenging DPPH and hydroxyl radical in monoculture, and phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, methyl davallialactone, foscoparianol D, and inotodisaccharide are those for scavenging the tested radicals in coculture. Lanostane-type triterpenoids indicated limited roles in scavenging free radicals. Nearly all the detected metabolites correlate positively with inhibiting proliferation of HeLa 229 cells. Thus, coculture of I. obliquus with other fungi seems to be a cost-effective strategy for upregulating biosynthesis of bioactive metabolites.

Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production.

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called 'Chaga'. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.

Green chemoselective synthesis of thiazolo[3,2-a]pyridine derivatives and evaluation of their antioxidant and cytotoxic activities.

The green chemoselective synthesis of thiazolo[3,2-a]pyridine derivatives was achieved in water via microwave-assisted three-component reactions of malononitrile, aromatic aldehydes and 2-mercaptoacetic acid with molar ratios of 2:1:1.5 and 2:2.2:1, respectively. These compounds were subject to the experiments of antioxidant activity and cytotoxicity to carcinoma HCT-116 cells and mice lymphocytes. Nearly all of the tested compounds possessed potent capacities for scavenging free radicals. In addition, most of these compounds showed cytotoxicity to HCT-116 cells and mice lymphocytes with no selectivity. Of these, only thiazolo[3,2-a]pyridine derivative 5d suggested selective cytotoxicity to tumor cell line HCT-116 cells.

Nitric oxide mediates the fungal-elicitor-enhanced biosynthesis of antioxidant polyphenols in submerged cultures of Inonotus obliquus.

A fungal elicitor prepared from the cell debris of the plant-pathogenic ascomycete Alternaria alternata induces multiple responses by Inonotus obliquus cells, including an increase in generation of nitric oxide (NO), activity of phenylalanine ammonia lyase (PAL) and accumulation of total mycelial phenolic compounds (TMP), but does not trigger production of oxylipins or jasmonic acid (JA). The role of NO in TMP production was investigated via the effects of the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO) and the nitric oxide synthase (NOS) inhibitor aminoguanidine (AG). TMP profiles were assayed using (1)H NMR spectroscopy combining multivariate pattern recognition strategies. Pretreatment of I. obliquus mycelia with cPITO or AG suppressed not only elicitor-enhanced NO generation and PAL activity, but also the elicitor-induced increase in TMP production. This TMP reduction by either a NO scavenger or a NOS inhibitor was reversed by exogenous addition of either a NO donor, sodium nitroprusside, or JA separately. NMR-based metabonomic analysis of TMP profiles showed that the induced TMP were hispidin analogues including inoscavins, phelligridins, davallialactone and methyldavallialactone, which possess high antioxidant activities. Thus, NO mediates an elicitor-induced increase in production of antioxidant polyphenols in I. obliquus via a signalling pathway independent of oxylipins or JA, a mechanism which differs from those in some higher plants.

NMR-based metabonomic analysis on effect of light on production of antioxidant phenolic compounds in submerged cultures of Inonotus obliquus.

This study was designed to investigate the light effect on biosynthesis of antioxidant phenolic compounds by Inonotus obliquus grown in submerged cultures using (1)H NMR spectroscopy combining multivariate pattern recognition strategies. I. obliquus were exposed to a range of light conditions and resultant data were compared to those from field-grown sclerotia and the mycelia grown in daylight. Daylight illumination inhibited biosynthesis of davallialactone and phelligridins and other hispidin analogs. Continuous darkness enhanced the formation of phelligridins, davallialactone and inoscavins. Phelligridins and davallialactone also occurred in the mycelia grown in blue and red light with levels lower than those found in darkness. In addition, polyphenols synthesized under daylight conditions showed less potential antioxidant activity than those determined with other light regimes. These findings demonstrate that light regulates biosynthesis of polyphenols in I. obliquus and their subsequent antioxidant activities, and (1)H NMR-based metabolic profiling is a cost-effective approach for evaluating light effects on fungal metabolisms.

Oxidative stress response of Inonotus obliquus induced by hydrogen peroxide.

While the medicinal fungus Inonotus obliquus produces polyphenols as one of its main metabolites in natural habitats, it accumulates less polyphenols under laboratory conditions. In this study we found that the continuous addition of 1 mM H(2)O(2) at a rate of 1.6 ml/h into a submerged culture of the fungus enhanced its production of mycelia, melanins, flavonoids and hispidin analogs (HA). Simultaneous exposure of the fungus to both H(2)O(2) and arbutin resulted in reduced production of mycelia, glycosylated flavonoids (GF) and HA, and inhibition of melanogenesis. However, superoxide dismutases (SOD) and catalase (CAT) activity were enhanced following the addition of H(2)O(2) or H(2)O(2) plus arbutin. The maximum levels of SOD and CAT activities reached 355.2 U/mg protein and 39.8 U/mg protein respectively in H(2)O(2)-added medium, and 264 U/mg protein and 35.9 U/mg protein respectively in H(2)O(2) plus arbutin medium. Thus, detoxification of H(2)O(2) is conducted mainly by polyphenols under normal physiological conditions, and by both polyphenols and antioxidant enzymes under oxidative stress when melanogenesis is inhibited. Although enhanced HA production occurred after melanogenesis inactivation, total extracellular polyphenol levels were reduced. These findings suggest that enzymatic activities convert superoxide to H(2)O(2), and non-enzymatic mechanisms are largely responsible for detoxifying H(2)O(2). Enhanced production of melanins is the most important non-enzymatic response of this fungus against oxidative stress.

Accumulation of antioxidant phenolic constituents in submerged cultures of Inonotus obliquus.

Phenolic compounds produced by sclerotia of Inonotus obliquus are the active constituents responsible for antioxidant activities. In this study, I. obliquus was grown in a continuously stirred tank reactor (CSTR) to explore how it accumulates phenolic compounds in different culture media and whether these compounds possess antioxidant activities. Phenolic compounds produced by I. obliquus in the control medium consisted of melanins, flavonoids, polyphenols and small phenolics. Their accumulation was affected by adding H(2)O(2) to the medium, where increased levels of total intracellular phenols (TIP) and melanins, but less total extracellular phenol (TEP) occurred. Simultaneous exposure to H(2)O(2) and arbutin resulted in a further increase in TIP production and reduced accumulation of TEP. Both TIP and TEP obtained at different culture ages and media were active in scavenging superoxide anion and DPPH radicals. Therefore, production of phenolic compounds by I. obliquus is enhanced by imposing oxidative stress, which might allow it to be exploited as a reliable source of pharmaceutically important phenolic compounds.

Total flavonoids of Daphne genkwa root significantly inhibit the growth and metastasis of Lewis lung carcinoma in C57BL6 mice.

Daphne genkwa root has been traditionally used as an effective remedy to treat various tumors. However, the active constituents for its antitumor potency have not been well documented. During the screening for antitumor constituents, it was found that the total flavonoids of D. genkwa root (TFDR) were responsible for the inhibition of tumor growth and metastasis. In this study, TFDR was investigated for its chemical composition and activities against tumor growth and metastasis. HPLC indicates that daphnodorin B, containing 42.79% of the total, represents the predominant constituent in TFDR. Treatment of LLC-bearing mice with TFDR evidently protected peripheral lymphocytes from tumor-induced reduction, and increased lymphocyte proliferation potential and cytolytic activity of NK, and inhibited tumor progression and metastasis either 7 days before, or simultaneous with, or 7 days after LLC transplantation. TFDR also suggested higher cytotoxicity to a number of tumor cell lines than that to normal human kidney cell K293. TFDR also induced an enhancement on peripheral release of TNF-alpha at doses between 25 and 75 mg/kg. These results indicated that TFDR inhibited tumor growth and metastasis by protecting host immunocyte viability and its proliferation potential, and selectively inhibiting tumor cell proliferation, and improving cytolytic activity of NK cells, and enhancing TNF release in LLC-bearing mice. Daphnodorin B and its analogues in TFDR are the active constituents in the roots of D. genkwa, contributing to the inhibition of tumor growth and metastasis.

Antitumor activity of daphnodorins from Daphne genkwa roots.

Daphne genkwa root has been traditionally used as an effective remedy to treat various tumors. However, the active constituents for its antitumor potency have not been well documented. During the screening for antitumor constituents, it was found that daphnodorins were responsible for the inhibition of tumor growth and metastasis. In this study, six daphnodorins including daphnodorins B (1), G (2), H (3), H-3''-methylether (4), H-3-methylether (5) and G-3''-methylether (6) were investigated for the protection against LLC-induced reduction of lymphoid organs and peripheral lymphocytes, and for the activities against tumor growth and metastasis. The six daphnodorins showed selective cytotoxicity to a number of tumor cell lines. Treatment of LLC-bearing mice with daphnodorin B and/or daphnodorin complex evidently protected peripheral lymphocytes from tumor-induced reduction, increased lymphocyte proliferation potential and inhibited tumor progression and metastasis at doses of 40 and 80 mg/kg. These results indicated that daphnodorin B or daphnodorin complex inhibited tumor growth and metastasis by protecting host immunocyte viability and proliferation potential, and selectively inhibiting tumor cell proliferation.