Genome and Transcriptome Analysis of the Torreya grandis WRKY Gene Family during Seed Development.

Torreya grandis, an economically significant evergreen tree species exclusive to subtropical China, is highly valued for its seeds. However, the seed development process of T. grandis remains relatively unexplored. Given the pivotal role WRKY transcription factors (TFs) play in coordinating diverse cellular and biological activities, as well as crucial signaling pathways essential for plant growth and development, and the lack of comprehensive investigation into their specific functions in T. grandis, our study investigated its genome and successfully isolated 78 WRKY genes and categorized them into three distinct clades. A conserved motif analysis unveiled the presence of the characteristic WRKY domain in each identified TgWRKY protein. The examination of gene structures revealed variable numbers of introns (ranging from zero to eight) and exons (ranging from one to nine) among TgWRKY genes. A chromosomal distribution analysis demonstrated the presence of TgWRKY across eight chromosomes in T. grandis. Tissue-specific expression profiling unveiled distinctive patterns of these 78 TgWRKY genes across various tissues. Remarkably, a co-expression analysis integrating RNA-seq data and morphological assessments pinpointed the pronounced expression of TgWRKY25 during the developmental stages of T. grandis seeds. Moreover, a KEGG enrichment analysis, focusing on genes correlated with TgWRKY25 expression, suggested its potential involvement in processes such as protein processing in the endoplasmic reticulum, starch, and sucrose metabolism, thereby modulating seed development in T. grandis. These findings not only underscore the pivotal role of WRKY genes in T. grandis seed development but also pave the way for innovative breeding strategies.

Diterpenoids from Torreya grandis and their cytotoxic activities.

Eight previously undescribed diterpenoids, along with eleven previously reported analogues, were obtained from the supercritical CO2 extracts of Torreya grandis aril. The structures of these compounds were elucidated based on HRESIMS, NMR, ECD, and single-crystal X-ray diffraction data. In the MTT assay, compound 18 exhibited significant inhibitory effects on two human colon cancer cell lines, HT-29 and HCT 116 cells, with IC50 values of 7.37 µM and 6.55 µM, respectively. It was found that compound 18 induced apoptosis and significantly inhibited the migration of HCT 116 colon cancer cells in a concentration-dependent manner.

Identification of novel bioactive proteins and their produced oligopeptides from Torreya grandis nuts using proteomic based prediction.

Torreya grandis nut is a chief functional food in China consumed for centuries. Besides its rich protein composition, increasing studies are now focusing on T. grandis functional proteins that have not yet identified. In this study, liquid chromatography coupled with mass spectrometry detection of smaller and major proteins, revealed that the major peptide was 36935.00 Da. Proteome sequencing annotated 142 proteins in total. Bioactive proteins such as defensin 4 was annotated and its anti-microbial function was verified. Finally, functional oligopeptides were predicted by searching sequences of digested peptides in databases. Ten group of oligopeptides were suggested to exhibit antioxidant, Angiotensin-converting enzyme inhibition, anti-inflammatory. The predicted antioxidant activity was experimentally validated. It is interesting that a peptide GYCVSDNN digested from defensin 4 showed antioxidant activity. This study reports novel functional peptides from T. grandis nuts that have not been isolated and/or included as functional ingredients in nutraceuticals and in food industry.

In Vitro Antioxidant Properties and Phenolic Profile of Acid Aqueous Ethanol Extracts from Torreya grandis Seed Coat.

Torreya grandis is an important economic forestry product in China, whose seeds are often consumed as edible nuts, or used as raw materials for oil processing. To date, as an important by-product of Torreya grandis, comprehensive studies regarding the Torreya grandis seed coat phenolic composition are lacking, which greatly limits its in-depth use. Therefore, in the present study, the Torreya grandis seed coat was extracted by acid aqueous ethanol (TE), and NMR and UHPLC-MS were used to identify the major phenolics. Together with the already known phenolics including protocatechuic acid, catechin, epigallocatechin gallate, and epicatechin gallate, the unreported new compound 2-hydroxy-2-(4-hydroxyphenylethyl) malonic acid was discovered. The results of the antioxidant properties showed that both TE and 2-hydroxy-2-(4-hydroxyphenylethyl) malonic acid exhibited strong ABTS, DPPH, and hydroxyl radical-scavenging activity, and significantly improved the O/W emulsion's oxidation stability. These results indicate that the TE and 2-hydroxy-2-(4-hydroxyphenylethyl) malonic acid could possibly be used in the future to manufacture functional foods or bioactive ingredients. Moreover, further studies are also needed to evaluate the biological activity of TE and 2-hydroxy-2-(4-hydroxyphenylethyl) malonic acid to increase the added value of Torreya grandis by-products.

Multi-omics analysis reveals the molecular responses of Torreya grandis shoots to nanoplastic pollutant.

Micro/nanoplastic has become an emerging pollutant of global concern. At present, ecotoxic researches on micro/nanoplastics mostly focus on marine aquatic organisms and freshwater algae. Research on the ecological impacts of plastics on higher terrestrial plants, especially on forest plants, is relatively limited. Torreya grandis cv. Merrillii, a species of conifer in the family Taxaceae, is a unique and economically valuable tree species in China. The physiological and biochemical responses of T. grandis seedlings to polystyrene nanoplastics (PSNPs) with a diameter of 100 nm were systematically studied inthe present study. The results showed that nanoplastics enhanced the accumulation of the thiobarbituric acid reactive substance and the activities of catalase and peroxidase. The concentrations of iron, sulfur, and zinc were reduced after nanoplastic exposure. PSNP treatment had an important effect on a series of chemical and genetic indicators of T. grandis, includingantioxidants, small RNA, gene transcription, protein expressions, and metabolite accumulation. Multi-omic analysis revealed that PSNPs modulate terpenoid- and flavonoid-biosynthesis pathways by regulating small RNA transcription and protein expression. Our study provided novelty insights into the responses of forest plants to nanoplastic treatment.

Identification of key genes and enzymes contributing to nutrition conversion of Torreya grandis nuts during post-ripening process.

The seeds of Torreya grandis are necessary to go through a ripening process, which eventually leads to nutrition conversion and the production of edible nuts. However, the molecular basis of nutrition conversion remains unclear. Here, transcriptome sequencing was performed on seeds treated with different temperature and humidity. A total of 881 unigenes related to nutrition conversion were identified. The correlations between nutrient content and gene expression suggested that sucrose phosphate synthase (SPS), dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex (DLST), glycerol-3-phosphate acyltransferase (GPAT) and Pyruvate kinase (PK) may play key roles in nutrition conversion. Transient over-expression of TgDLST, TgPK and TgGPAT in tobacco leaves promoted nutritional conversion. Moreover, enzyme activity analysis indicated that diacylglycerol acyltransferase (DGAT) and pyruvate dehydrogenase (PDH) activities may also accelerate the nutritional conversion. This study uncovers the molecular basis of nutrition conversion in T. grandis seeds, which critical for shortening the time of nutrition conversion.

Effects of Harvest Time on the Yield, Quality and Active Substance of Torreya grandis Nut and Its Oil.

Torreya grandis is an important economic tree species in China. It provides nutritional value and is important to the health care industry. There are ongoing issues with product quality which are primarily related to improper management and early harvest. This study was carried out during the fruit ripening processes to evaluate the influence of harvesting date on T. grandis quality, and to determine the optimal harvest period. The effects of harvest time on the variation of quality and nutritional parameters of T. grandis nuts and its oil were evaluated, and the optimal harvest period was determined. The results showed that harvest timing had a strong effect on both oil yield and quality. Prolonged ripening could induce higher levels of kernel rate, fruit inclusions, oil and nutritional quality. When the sample harvested in the mid-September, the kernel rate and oil content were increased by 1.88±0.31% and 6.65±0.47%, respectively, compared to samples harvested in the beginning of late-August. Similarly, the mid-September harvest resulted in total unsaturated fatty acids content of the oil being increased by 5.3±0.34%, the FFA and peroxide value being decreased by 40.7±0.15% and 76±0.08%, respectively, and total tocopherols and free amino acids were increased 7.5±0.24% and 47.3±0.15%, respectively, compared to the samples harvested on Aug. 25. The results indicated that the optimal harvest time of T. grandis fruits was mid-September as it was beneficial for improving the quality of T. grandis nut and its oil. It was suggested that T. grandis fruit should be harvested later.

Anti-melanogenic effects of hot-water extracts from Torreya nucifera via MAPKs and cAMP signaling pathway on B16F10 cells.

Torreya nucifera is an evergreen tree in the family Taxaceae, the seeds, leaves, and stems of which have long been used as edible products and herbal medicines in Korea. Previous studies of biological activity have shown that T. nucifera has antioxidant and anti-inflammatory effects. However, the effect of T. nucifera leaves on melanogenesis are yet to be studied. In this investigation, we used B16F10 melanoma cells to test the efficacy of T. nucifera leaf hot water extract (TLWE). α-melanocyte stimulating hormone (α-MSH) stimulated B16F10 melanoma cells were treated with various concentrations of TLWE (50, 100, and 200 µg/mL). The results showed that TLWE reduced the melanin content and cellular tyrosinase activity in a concentration-dependent manner. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) signaling pathway. The compounds catechin and ρ-coumaric acid, which are known to have a whitening effect on skin, were detected by HPLC analysis. These results suggest that TLWE has an anti-melanogenic effect. In addition, the safety of TLWE was tested. The results of the skin irritation test showed that TLWE is harmless to the human skin, even at higher concentrations than those used in the experiment. Therefore, we suggest that the water extract of T. nucifera leaves has potential for use as a skin-whitening agent.

Amentotaxins C-V, Structurally Diverse Diterpenoids from the Leaves and Twigs of the Vulnerable Conifer Amentotaxus argotaenia and Their Cytotoxic Effects.

A phytochemical investigation of the MeOH extract of the leaves and twigs of Amentotaxus argotaenia, a relict vulnerable coniferous species endemic to China, led to the isolation and characterization of 35 diterpenoids/norditerpenoids. Twenty of these are new, including 11 ent-kaurane-type (amentotaxins C-M, 1-11, respectively), three icetexane-type [= 9(10→20)abeo-abietane-type (amentotaxins N-P, 12-14, respectively)], four ent-labdane-type (amentotaxins Q-T, 15-18, respectively), and two isopimarane-type [amentotaxins U (19) and V (20)] compounds. Their structures were elucidated on the basis of spectroscopic data, single-crystal X-ray diffraction, the modified Mosher's method, and electronic circular dichroism data analyses. Compounds 1-9 are rare 18-nor-ent-kaurane-type diterpenoids featuring a 4ß,19-epoxy ring. All the isolates were evaluated for their cytotoxic effects against a small panel of cultured human cancer cell lines (HeLa, A-549, MDA-MB-231, SKOV3, Huh-7, and HCT-116), and some of them exhibited cytotoxicities with IC50 values ranging from 1.5 to 10.0 µM.

Antioxidant, metabolic enzymes inhibitory ability of Torreya grandis kernels, and phytochemical profiling identified by HPLC-QTOF-MS/MS.

In this study, the antioxidant activities, α-glucosidase and tyrosinase inhibitory ability of Torreya grandis kernels (TGK) were performed. Samples were extracted with various polarity of ethanol, and the major phytochemical profile was characterized. The results showed that 70% of ethanol extract gave the richest phenolics and flavonoids. The strongest DPPH· and ABTS·+ scavenging ability, as well as the best inhibition on tyrosinase and α-glucosidase was also detected on 70% of ethanol extract. Among the fractions of 70% of ethanol extract, the ethyl acetate fraction (EAF) owned the highest phenolics, flavonoids, and the best DPPH· and ABTS·+ scavenging ability, and tyrosinase inhibition. Unexpectedly, the dichloromethane fraction possessed the strongest inhibition on α-glucosidase, which was much greater than that of acarbose. HPLC-QTOF-MS/MS analysis result to the characterization of 19 compounds from EAF. The results implied that TGK can be a potential source of natural antioxidants, α-glucosidase and tyrosinase inhibitors. Practical applications The kernels of T. grandis are one of the precious nuts in the world, and the extracts were advertised to show a variety of biological activities and pharmacological effects. However, researches on the phytochemical constituents and bioactivities are fewer. In this study, TGK was found to show good potency in antioxidant, α-glucosidase and tyrosinase inhibitory activities. The 70% ethanol is the best solvent for extracting above mentioned active components, and ethyl acetate can be the suitable enriching solvent. In addition, the predominant phytochemical compounds in EAF were characterized. Therefore, this research can help to the performance of further research and application of TGK in functional products.

Fractionated Antioxidant and Anti-inflammatory Kernel Oil from Torreya fargesii.

Polymethylene-interrupted polyunsaturated fatty acids (PMI-PUFAs) are emerging functional lipids with proven antioxidant and anti-inflammatory effects. In this study, a typical PMI-PUFA, sciadonic acid (C20:3, 5c 11c 14c), was enriched in the kernel oil of Torreya fargesii (T. fargesii) by fractionation. Fractionated kernel oil of T. fargesii (containing 25% sciadonic acid) showed equal stability and similar radical scavenging ability compared with the non-fractionated oil. In anti-inflammatory tests, fractionated kernel oil was shown to inhibit the activity of phosphodiesterase (PDE-5, efficiency 80% at 133.7 µg/mL) and lipoxygenase-5 (LOX-5, efficiency 65% at 66.7 µg/mL) more effectively than the non-fractionated oil. This shows that increasing the amount of sciadonic acid can enhance the anti-inflammatory effect of the kernel oil. This research also indicates that fractionation is a feasible way to obtain sciadonic acid-rich functional oil with potential pharmacological effects.

Sunlight induced biosynthesis of silver nanoparticle from the bark extract of Amentotaxus assamica D.K. Ferguson and its antibacterial activity against Escherichia coli and Staphylococcus aureus.

Green synthesis of nanoparticles has gained importance due to its eco-friendly, low toxicity and cost effective nature. This study deals with the biosynthesis of silver nanoparticles (AgNPs) from the bark extract of Amentotaxus assamica. The AgNPs have been synthesised by reducing the silver ions into stable AgNPs using the bark extract of Amentotaxus assamica under the influence of sunlight irradiation. The characterisation of the biosynthesised AgNPs was carried out by UV-vis spectroscopy, X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray analysis. The UV-vis spectrum showed a broad peak at 472 nm. Also, the XRD confirmed the crystalline structure of the AgNPs. Moreover, the SEM analysis revealed that the biosynthesised AgNPs were spherical in shape. Also, dynamic light scattering techniques were used to evaluate the size distribution profile of the biosynthesised AgNPs. Furthermore, the biosynthesised AgNPs showed a prominent inhibitory effect against both Escherichia coli (MTCC 111) and Staphylococcus aureus (MTCC 97). Thus the biosynthesis of AgNPs from the bark extract of Amentotaxus assamica is found to eco-friendly way of producing AgNPs compared to chemical method.

Full-Length Transcriptome Analysis of the Genes Involved in Tocopherol Biosynthesis in Torreya grandis.

The seeds of Torreya grandis (Cephalotaxaceae) are rich in tocopherols, which are essential components of the human diet as a result of their function in scavenging reactive oxygen and free radicals. Different T. grandis cultivars (10 cultivars selected in this study were researched, and their information is shown in Table S1 of the Supporting Information) vary enormously in their tocopherol contents (0.28-11.98 mg/100 g). However, little is known about the molecular basis and regulatory mechanisms of tocopherol biosynthesis in T. grandis kernels. Here, we applied single-molecule real-time (SMRT) sequencing to T. grandis (X08 cultivar) for the first time and obtained a total of 97 211 full-length transcripts. We proposed the biosynthetic pathway of tocopherol and identified eight full-length transcripts encoding enzymes potentially involved in tocopherol biosynthesis in T. grandis. The results of the correlation analysis between the tocopherol content and gene expression level in the 10 selected cultivars and different kernel developmental stages of the X08 cultivar suggested that homogentisate phytyltransferase coding gene ( TgVTE2b) and γ-tocopherol methyltransferase coding gene ( TgVTE4) may be key players in tocopherol accumulation in the kernels of T. grandis. Subcellular localization assays showed that both TgVTE2b and TgVTE4 were localized to the chloroplast. We also identified candidate regulatory genes similar to WRI1 and DGAT1 in Arabidopsis that may be involved in the regulation of tocopherol biosynthesis. Our findings provide valuable genetic information for T. grandis using full-length transcriptomic analysis, elucidating the candidate genes and key regulatory genes involved in tocopherol biosynthesis. This information will be critical for further molecular-assisted screening and breeding of T. grandis genotypes with high tocopherol contents.

New sights into lipid metabolism regulation by low temperature in harvested Torreya grandis nuts.

BACKGROUND: Torreya grandis, a large evergreen coniferous tree with oil-rich nuts, undergoes a crucial ripening stage after harvest that results in oil accumulation, finally giving rise to the nut that is edible in roasted form. To understand lipid metabolism in T. grandis nuts during the post-harvest ripening period, the effects of low temperature on lipid content, fatty acid composition, lipid biosynthesis and degradation were investigated. RESULTS: The lipid content increased during ripening at room temperature and a low temperature slowed down this increase. Linoleic acid content increased at low temperature, which was accompanied by an increase in the microsomal oleate desaturase (FAD2) activity and FAD2 expression. Furthermore, a low temperature attenuated lipid peroxidation as indicated by lower contents of malondialdehyde, hydroperoxide and total free fatty acid in T. grandis nuts during the ripening stage, as well as the down-regulation of gene expression of lipid degradation-related enzymes such as phospholipase D and lipoxygenases. CONCLUSION: The findings of the present study indicate that a low temperature increased polyunsaturated fatty acid contents by increasing FAD2 biosynthesis and decreasing lipid peroxidation, thereby improving the oil yield in T. grandis nuts during the post-harvest ripening period. © 2019 Society of Chemical Industry.

Antioxidant and Tyrosinase Inhibitory Activities of Seed Oils from Torreya grandis Fort. ex Lindl.

Torreya grandis Fort. ex Lindl. is a plant belonging to the Taxaceae family and Torreya grandis cv. Merrillii is the only grafted and thoroughbred species belonging to this species. In this study, we extracted five different seed oils, including T. grandis seed oil (TGSO), T. grandis "Xiangyafei" seed oil (XYSO), T. grandis "Zhimafei" seed oil (ZMSO), T. grandis "Majus"seed oil (TGMSO), and T. grandis "cunguangfei" seed oil (CGSO) using physical pressure. The resulting extracts were analyzed to determine their fatty acid composition, antioxidant activity, and inhibitory activity towards tyrosinase. The results of the antioxidant activity assays revealed that XYSO and ZMSO exhibited much greater DPPH radical scavenging activity and ferric reducing power than TGSO. Notably, all five of the seed oils showed dose-dependent inhibitory activity towards tyrosinase. XYSO and TGSO gave the highest activities of all of the seed oils tested in the current study against monophenolase and diphenolase, with IC50 values of 227.0 and 817.5µg/mL, respectively. The results of this study show that wild TGSOs exhibit strong antioxidant and tyrosinase inhibition activities. These results therefore suggest that wild TGSOs could be used as a potential source of natural antioxidant agents and tyrosinase inhibitors.

Fast and accurate separation of the paclitaxel from yew extracum by a pseudo simulated moving bed with solvent gradient.

A pseudo simulated moving bed (SMB) with solvent gradient was used to trap and separate paclitaxel from yew extracum. This SMB process consisted of three steps: feeding, purification and recovery. In comparison with methanol/water as an eluent, acetonitrile/water could give a better separation but had a poor dissolubility of the yew extracum, and therefore methanol/water was used in the feeding followed by acetonitrile/water in the purification. In the first two steps, water was deliberately added into zone III to modulate the eluotropic strength of the liquid entering zone III, so as to make paclitaxel separation from impurities be more efficient. Once most of impurities discarded, the columns were in turn eluted to recover the trapped paclitaxel of 98% yield with a purity of 78% from the yew extracum containing 1.5% paclitaxel. Afterward, an additional operation of crystallization improved the purity further to 97.8% with the yield of 95%.

Preparation, Characterization, and Antibacterial Effects of Chitosan Nanoparticles Embedded with Essential Oils Synthesized in an Ionic Liquid Containing System.

Several chitosan sodium tripolyphosphate (TPP) nanoparticles embedded with Torreya grandis aril essential oils (TEOs) were synthesized using an emulsion-ionic gelation technique. Mannosylerythritol lipid A (MEL-A), a type of biosurfactant, was selected as the emulsifier. In order to replace acetic acid, an ionic liquid (IL) was employed to dissolve chitosan. The physical properties, diameters, morphology, embedding rate, and antibacterial effects of those essential oil loaded chitosan (CS) nanoparticles were characterized. The results demonstrated that chitosan nanoparticles can be successfully prepared in an ionic liquid containing system and the diameters for nanoparticles in acetic acid and ionic liquid solutions are 144.1 ± 1.457 and 219.0 ± 4.045 nm. After loading with essential oils, the size increased to 349.6 ± 10.55 and 542.9 ± 16.74 nm, respectively. Antibacterial properties were investigated by the observation of the inhibition zone against S. aureus. The results revealed that TEO loaded nanoparticles synthesized in acid and IL aqueous systems have stronger antibacterial activities than CS nanoparticles.

Bioassay-guided isolation of active substances from Semen Torreyae identifies two new anthelmintic compounds with novel mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Torreyae, the seeds of Torreya grandis Fortune ex Lindley (Cephalotaxaceae) is a well-known traditional Chinese medicinal plant recorded in the Chinese Pharmacopeia (2010 version). It is widely used for treating intestinal parasites in China, owing to its desirable efficacy and safety. However, the anthelmintic compounds in Semen Torreyae have not yet been identified. AIM OF THE STUDY: This study aims to identify the compounds active against helminths from Semen Torreyae. In addition, we tested whether C. elegans strains resistant to currently-used anthelmintic drugs showed cross-resistance to these compounds. METHODS: A bioassay-guided isolation of anthelmintic compounds from Semen Torreyae was performed using a Caenorhabditis elegans (C. elegans) testing model. The structures of active compounds were elucidated by a combination of GC-MS, high resolution MS, and NMR. The median-effect method was employed to generate a combination index (CI) to evaluate the synergistic effect of the anthelmintic compounds. A panel of C. elegans mutant strains resistant against the major anthelmintic drug classes was used to study the cross-resistance to currently-used anthelmintic drugs. A panel of transient receptor potential (TRP) channel mutant strains was also tested to explore the possible mechanisms of action of the anthelmintic compounds. RESULTS: The bioassay-guided isolation led to two active compounds, i.e. galangal acetate (IC50: 58.5 ±â€¯8.9 µM) and miogadial (IC50: 25.1 ±â€¯5.4 µM). The combination of galangal acetate and miogadial resulted in a synergistic effect at IC50, IC70, and IC90 levels (CIs < 1). Galangal acetate and miogadial demonstrated similar activity against drug-resistant C. elegans strains compared to the wild-type strain. In addition, none of the TRP mutants was significantly resistant to galangal acetate or miogadial compared to wild type worms. CONCLUSIONS: We identified the bioactive compounds from Semen Torreyae responsible for its anthelmintic activity: galangal acetate and miogadial. The two anthelmintic compounds demonstrated a synergistic effect against C. elegans. Galangal acetate and miogadial are unlikely to act on the targets of currently-used anthelmintics (ivermectin, levamisole, benomyl and aldicarb), and an action on TRP channels appears to be ruled out as well. In summary, galangal acetate and miogadial are promising anthelmintic hits worth further investigation.

Physicochemical and functional properties of protein extracts from Torreya grandis seeds.

Proteins extracted from Torreya grandis seeds were investigated for their physicochemical and functional properties. The results showed protein extracts from two cultivars of T. grandis, Shengzhou I (SZPI) and Dazinaiyou (DNPI), had similar protein contents and appropriate amino acid balances with about 41% of the essential amino acid. The molecular weights of seed protein fractions were mostly about 31-37kDa and 20-21kDa. SZPI and DNPI had similar denaturation temperature of around 93.7°C while free sulfhydryl group and disulfide bond contents were found to differ slightly. The surface hydrophobicity of DNPI was 982, significantly (p<0.05) greater than that of SZPI (649). Both proteins exhibited high solubilities and favourable emulsifying abilities, foaming and fat absorption capacities, although their in-vitro digestibilities were rather low. Therefore, T. grandis seed proteins have potential as valuable nutrition sources and functional ingredients in food industry.

Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. MATERIALS AND METHODS: For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. RESULTS: Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. CONCLUSION: Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.