Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.     

Production and secretion of resveratrol in hairy root cultures of peanut

Resveratrol and its derivatives are natural stilbenes associated with many health benefits that include those conferred by their antioxidant and anticancer properties. While stilbenes can be recovered as an extract from a selected number of plants, these products are not suitable for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its derivatives in the extract. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut (Arachis hypogaea) were established and tested as a bioproduction system for resveratrol and associated derivatives. Analyses by HPTLC and GC-MS of ethyl acetate extracts showed that a single 24 h sodium acetate elicitation resulted in a 60-fold induction and secretion of trans-resveratrol into the medium of peanut hairy root cultures. trans-Resveratrol accumulated to levels of 98 microg/mg of the dried extract from the medium representing 99% of the total resveratrol produced. Other stilbenes, including trans-pterostilbene, were also detected in the medium. Our results demonstrate the capacity of hairy root cultures as an effective bioprocessing system for valued nutraceuticals like resveratrol and resveratrol derivatives. In being able to effectively induce and recover high levels of resveratrol and associated derivatives from the media fraction, hairy roots may offer a scalable and continuous product recovery platform for naturally-derived, high quality, enriched nutraceuticals.     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

Effects of indole‐3‐butyric acid on growth,pigments and UV‐screening compounds in Nostoc linckia

In the present research, the effect of indole‐3‐butyric acid (IBA) on the growth, and the production of some primary and secondary metabolites was studied in Nostoc linckia. In this respect, algae cultures were supplied with 0, 0.01, 0.1, 1, 10, and 100 μM IBA for 14 days. IBA at concentrations of 10 and 100 μM induced algal growth expressed as fresh weight in N. linckia. Treatment with IBA at all concentrations stimulated heterocyst formation. In addition, low concentrations of IBA (0.01, 0.1, and 1 μM) had a stimulatory effect on chlorophyll a and carotenoids accumulation. In contrast, higher concentrations of IBA induced the accumulation of phycocyanin, allophycocyanin, and phycoerythrin in the treated algae. In this case, IBA at the concentration of 10 μM was more effective. A significant decrease in protein content was observed in the algae treated by 0.01 μM IBA. All concentrations of IBA caused a decrease in sugar content, but lower concentrations were more effective. IBA application in all of the concentrations except 100 μM increased oligosaccharide‐linked mycosporine‐like amino acids (OS‐MAAs) content. Lower concentrations had a more significant effect on increasing OS‐MAAs content. However the concentrations of 10 and 100 μM IBA decreased scytonemin content. These results indicated the stimulatory impact of IBA on weight, heterocyst formation, and photosynthetic pigments in N. linckia.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Liver microsomal cytochromes P450‐dependent alkoxyphenoxazone O‐dealkylation in vitro by alligator and rat: Activities,inhibition, substrate preference,and discrimination factors

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450‐dependent mixed‐function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3‐methylcholanthrene (3MC), and PB–3MC co‐pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O‐dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7‐ethoxy, 7‐methoxy, 2‐phenylbenzyloxy, 7‐pentoxy, or 7‐benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O‐dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A‐type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2′,4,4′ tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, α‐napthaflavone, 1‐ethynylpyrene, SKF 525A, and 9‐ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 μM α‐napthaflavone inhibited liver microsomal EROD catalyzed by 3MC‐inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 μM SKF 525A inhibited PROD catalyzed by PB‐inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 17–27, 1999     

Synthesis of Novel Chiral Sulfonamide‐Bearing 1,2,4‐Triazole‐3‐thione Analogs Derived from D‐ and L‐Phenylalanine Esters as Potential Anti‐Influenza Agents

Novel enantiopure 1,2,4‐trizole‐3‐thiones containing a benzensulfonamide moiety were synthesized via multistep reaction sequence starting with D‐phenylalanine methyl ester and L‐phenylalanine ethyl ester as a source of chirality. The chemical structures of all compounds were characterized by elemental analysis, UV, IR, ¹H NMR, ¹³C NMR, 2D NMR (HETCOR), and mass spectral data. All compounds were tested in vitro antiviral activity against a broad variety of DNA and RNA viruses and in vitro cytostatic activity against murine leukemia (L1210), human T‐lymphocyte (CEM) and human cervix carcinoma (HeLa) cell lines. Although enantiopure 1,2,4‐triazole‐3‐thione analogs in (R) configuration emerged as promising anti‐influenza A H1N1 subtype in Madin Darby canine kidney cell cultures (MDCK), their enantiomers exhibited no activity. Especially compounds 18a , 21a , 22a , 23a , and 24a (EC₅₀: 6.5, 6.1, 2.4, 1.6, 1.7 μM, respectively) had excellent activity against influenza A H1N1 subtype compared to the reference drug ribavirin (EC₅₀: 8.0 μM). Several compounds have been found to inhibit proliferation of L1210, CEM and HeLa cell cultures with IC₅₀ in the 12–53 μM range. Compound 5a and 27a in (R) configuration were the most active compounds (IC₅₀: 12–22 μM for 5a and IC₅₀: 19–23 μM for 27a ). Chirality 28:495–513, 2016. © 2016 Wiley Periodicals, Inc.     

Cytotoxic ent‐Kaurane Diterpenoids from Isodon nervosus

Four new ent‐kaurane diterpenoids, rabdonervosins G–J ( 1 – 4 , resp.), were isolated from the leaves and stems of Isodon nervosus. Their structures were elucidated by extensive spectroscopic analyses, including 1D‐, 2D‐NMR and HR mass spectra. Compound 2 showed potent cytotoxicity against the HepG2 and PC‐9/ZD cell lines with IC₅₀ values of 2.36 and 6.07 μM , respectively, and compound 3 exhibited cytotoxicity against the HepG2 and CNE2 cell lines with IC₅₀ values of 8.64 and 9.77 μM , respectively.     

Stilbene derivatives from Gnetum gnemon Linn

Four stilbene derivatives, gnemonols K and L (resveratrol trimers), M (isorhapontigenin dimer), and gnemonoside K (glucoside of resveratrol trimer) together with eleven known stilbenoids and a lignan were isolated from the acetone, methanol and 70% methanol soluble parts of the root of Gnetum gnemon (Gnetaceae). The structures of the isolates were determined by spectral analysis. The antioxidant activity of the stilbenoids on lipid peroxide inhibition and super oxide scavenging activity were also investigated.     

Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular‐signal‐regulated kinases (ERK)1/2‐ and p38 kinase‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells, concomitant with an increase in the expression of COX‐2 and p53 phosphorylation. Blockade of cyclooxygenase‐2 (COX‐2) activity by siRNA or NS398 correspondingly inhibited ceramide‐induced p53 Ser‐15 phosphorylation and apoptosis; thus COX‐2 appears at the apex of the p38 kinase‐mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide‐treated cells. Ceramide‐treated cells underwent a dose‐dependent reduction in trans‐membrane potential. Although both ceramide and resveratrol induced the expressions of caspase‐3 and ‐7, the effect of inducible COX‐2 was different in caspase‐7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis‐requiring, ERK1/2‐dependent signal transduction pathway and induction of COX‐expression as an essential molecular antecedent for subsequent p53‐dependent apoptosis. In addition, expressions of caspase‐3 and ‐7 are observed. However, a p38 kinase‐dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide‐induced apoptosis. J. Cell. Biochem. 114: 1940–1954, 2013. © 2013 Wiley Periodicals, Inc.     

Lignan accumulation in two-phase cultures of <Emphasis Type="Italic">Taxus</Emphasis> x <Emphasis Type="Italic">media</Emphasis> hairy roots

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively.     

Secondary metabolites of Bagassa guianensis Aubl. wood: A study of the chemotaxonomy of the Moraceae family

In order to explain the durability of the Moraceae plant family, phytochemistry of Bagassa guianensis was performed. Ethyl acetate extract was obtained from the heartwood and 18 secondary metabolites were isolated, including 6 moracins [6-O-methyl-moracin M, 6-O-methyl-moracin N and moracin Z; previously identified: moracin M, moracin N and moracin P], 8 stilbenoids [presently identified: (?)-epialboctalol and arachidin 4; previously identified: alboctalol, trans-resveratrol, arachidin 2, trans-oxyresveratrol and artogomezianol], 3 previously identified flavonoids, steppogenin, katuranin and dihydromorin, β-sitosterol and resorcinol. Previous studies suggest that stilbenoids are responsible for the natural durability of wood. Our study has determined that B. guianensis is closely related to Morus sp. in phylogeny and should be included in the Moreae sensu stricto tribe of the Moraceae family.     

A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases.A substantial part of nonhost defense responses in many plants is the pathogen-induced production of secondary metabolites, generally termed phytoalexins, that locally restrict disease progression due to bioactivities toxic to the pathogen (for review, see Ahuja et al., 2012). Peanut or groundnut (Arachis hypogaea) tissues mount a defense against infection by the soil fungus Aspergillus flavus and other pathogens by overproducing stilbene derivatives around sites of wounding and elicitor perception (Sobolev, 2013). Resveratrol, one of the most studied phytoalexin stilbenoids, has attracted great attention because of its bioactive properties shown through in vitro and in vivo assays to benefit human health. These include antiinflammatory (Das and Das, 2007) and antioxidant properties as well as antitumor and favorable cardiovascular effects (Gambini et al., 2015). However, the limited oral bioavailability of resveratrol due to its rapid absorption and metabolism restricts the future of this potentially valuable drug in clinical trials (Tomé-Carneiro et al., 2013; Gambini et al., 2015).Prenylated stilbenoids naturally produced as phytoalexins in the peanut plant possess, for the most part, one isoprenyl moiety bound to the aromatic ring of the stilbene molecule (Fig. 1). When compared with resveratrol, these compounds exhibit similar or enhanced bioactivity in in vitro experiments. For instance, arachidin-1 and resveratrol showed similar antiinflammatory activity in lipid polysaccharide-treated RAW 264.7 macrophages, and this correlated with the inhibition of PG E₂ production (Chang et al., 2006; Djoko et al., 2007). Arachidin-1, arachidin-2, and arachidin-3, also applied to macrophages, were more effective than resveratrol in inhibiting inducible nitric oxide production (Sobolev et al., 2011). In other antioxidant activity assays, arachidin-1 inhibited lipid oxidation more effectively than resveratrol (Abbott et al., 2010), and arachidin-2 and arachidin-3 showed greater potency over resveratrol in inhibiting the production of intracellular reactive oxygen species (Sobolev et al., 2011). Arachidin-1 further showed higher cytotoxicity than resveratrol to leukemia HL-60 cells (Huang et al., 2010) and other cancer cells (SK-MEL, KB, BT-549, and SK-OV-3; Sobolev et al., 2011). Interestingly, arachidin-1 and arachidin-3 were shown to bind to human cannabinoid receptors 2 (hCBR2s), while the affinity of their nonprenylated analogs, piceatannol and resveratrol, for hCBR2s was 5- to 10-fold lower. Molecular modeling studies with hCBR2s indicated that the prenyl moiety of the arachidins improved the binding affinity to the receptors (Brents et al., 2012).Open in a separate windowFigure 1.A, Structure of the stilbene backbone and main prenylation patterns found on peanut prenylated stilbenoids. B, Chemical structures of stilbenoids identified in elicited peanut hairy roots: resveratrol (a), piceatannol (b), arachidin-2 (c), arachidin-5 (d), arachidin-3 (e), and arachidin-1 (f). All compounds are shown in their trans-isomers.In addition to the above-mentioned stilbenoids (arachidin-1, arachidin-2, and arachidin-3), more than 20 other prenylated stilbenoids have been described in peanut tissues (Sobolev et al., 2006, 2016; Wu et al., 2011; Sobolev, 2013). The biosynthesis of stilbenoids derives from both the phenylpropanoid and acetate pathways. These merge to produce resveratrol by the action of resveratrol synthase, which catalyzes the cyclization of 4-coumaroyl-CoA and malonyl-CoA (Schöppner and Kindl, 1984). The prenylation step, in which one of two prenyl patterns (3,3-dimethylallyl and 3-methyl-but-1-enyl) is introduced to various positions of the stilbene backbone (Fig. 1), along with the oxidation, methylation, and cyclization steps plays a major role in the diversification of peanut prenylated stilbenoids. Although the enzymes involved in resveratrol biosynthesis have been elucidated (Chong et al., 2009), the enzymes involved in the prenylation steps of resveratrol or any other stilbenoid have not been described.For other prenylated aromatic compounds, a prenyltransferase was found to be the critical activity for coupling the aromatic compound biosynthesis and terpenoid biosynthesis, the latter leading to the formation of the prenyl unit (Yazaki et al., 2009). Two pathways are known for the biosynthesis of prenylated compounds in plants, the mevalonic acid (MVA) pathway in the cytosol and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway in the plastid (Lohr et al., 2012). Many studies have shown that dimethylallyl pyrophosphate (DMAPP) derived from the MEP pathway is used as a prenyl donor to form prenylated flavonoids or prenylated isoflavonoids in the plastid (Yamamoto et al., 2000; Yazaki et al., 2009). To determine the biosynthetic origin of these terpenoids, distinct metabolic inhibitors were applied to inhibit the key rate-limiting enzymes involved in either the MVA or MEP pathway. For instance, mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase involved in the MVA pathway, was used in hairy root cultures of ginseng (Panax quinquefolius) to study the biosynthesis of ginsenosides (Zhao et al., 2014), while clomazone, an herbicide that inhibits 1-deoxy-d-xylulose-5-phosphate synthase during early steps of DMAPP biosynthesis in the plastid, was used to investigate the synthesis of monoterpenes in Catharanthus roseus (Han et al., 2013).In order to elucidate the biosynthesis of peanut prenylated stilbenoids, we established hairy root cultures of peanut (Condori et al., 2010) and recently demonstrated that a sustainable production of the prenylated stilbenoids arachidin-1 and arachidin-3 can be achieved upon cotreatment of these cultures with methyl jasmonate (MeJA) and cyclodextrin (CD; Yang et al., 2015a). In this study, we took advantage of this bioproduction system and identified arachidin-2 and a new prenylated stilbenoid, named arachidin-5, as the prenylated products of the hairy root microsomal fraction using resveratrol and piceatannol as substrates, respectively. To determine the biosynthetic origin of the prenyl moiety of these prenylated stilbenoids, two metabolic inhibitors, mevastatin and clomazone were selected and applied to peanut hairy root cultures cotreated with MeJA and CD as elicitors. In the process, we identified and characterized a resveratrol 4-dimethylallyl transferase from the microsomal fraction of elicited peanut hairy roots. To our knowledge, this enzyme is the first stilbenoid-specific prenyltransferase that prenylates resveratrol and other specific stilbenoids at the 4-C position of the aromatic ring (Fig. 1).     

In vitro production of metabolism-enhancing phytoecdysteroids from <Emphasis Type="Italic">Ajuga turkestanica</Emphasis>

In order to develop a sustainable source of metabolism-enhancing phytoecdysteroids, cell suspension and hairy root cultures were established from shoot cultures of wild-harvested Ajuga turkestanica, a medicinal plant indigenous to Uzbekistan. Precursors of phytoecdysteroids (acetate, mevalonic acid cholesterol) or methyl jasmonate (an elicitor) were added to subculture media to increase phytoecdysteroid accumulation. In cell suspension cultures, 20-hydroxyecdysone (20E) content increased 3- or 2-fold with the addition of 125 or 250 μM methyl jasmonate, respectively, compared to unelicited cultures. Precursor addition, however, did not provoke phytoecdysteroid accumulation. In hairy root cultures, addition of sodium acetate, mevalonic acid, and methyl jasmonate, but not cholesterol, increased phytoecdysteroid content compared to unelicited cultures. Hairy root cultures treated with 150 mg l⁻¹ sodium acetate, or 15 or 150 mg l⁻¹ mevalonic acid, increased 20E content approximately 2-fold to 19.9, 20.4 or 21.7 μg mg⁻¹, respectively, compared to control (10.5 μg mg⁻¹). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 μg mg⁻¹), turkesterone (0.9 μg mg⁻¹) and cyasterone (8.1 μg mg⁻¹) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 μg ml⁻¹ hairy root extract increased protein synthesis by 25.7% or 31.1%, respectively, in a C2C12 mouse skeletal cell line. These results suggest that sustainable production of metabolically active phytoecdysteroid can be achieved through hairy root culture systems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .     

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m^?2 s^?1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m^?2 s^?1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.     

Myrtucommulone‐A Induces both Extrinsic and Intrinsic Apoptotic Pathways in Cancer Cells

Myrtucommulone‐A is the active compound derived from Myrtus communis. The molecular targets of myrtucommulone‐A is widely unknown, which impedes its potential therapeutic use. In this study, we demonstrated the cytotoxicity of MC‐A and its potential to induce apoptosis in cancer cells. Myrtucommulone‐A was also found to be antiproliferative and strongly inhibited cancer cell migration. Eighty four apoptotic pathway genes were used to assess the effect of myrtucommulone‐A on cancer cells. Myrtucommulone‐A mediated an increase in apoptotic genes including Fas, FasL, Gadd45a, Tnf, Tnfsf12, Trp53, and caspase 4. The increase in myrtucommulone‐A dose (25 μM versus 6.25 μM) also upregulated the expression of genes, which are involved mainly in apoptosis, regulation of apoptosis, role of mitochondria in apoptotic signaling, cytokine activity, and tumor necrosis factor signaling. Our data indicate that myrtucommulone‐A could be utilized as a potential therapeutic compound with its molecular targets in apoptotic pathways.