Torreya Nucifera Research Articles

Extracellular Signal-Regulated Kinase Is a Direct Target of the Anti-Inflammatory Compound Amentoflavone Derived fromTorreya nucifera

Amentoflavone is a biflavonoid compound with antioxidant, anticancer, antibacterial, antiviral, anti-inflammatory, and UV-blocking activities that can be isolated from Torreya nucifera, Biophytum sensitivum, and Selaginella tamariscina. In this study, the molecular mechanism underlying amentoflavone's anti-inflammatory activity was investigated. Amentoflavone dose dependently suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW264.7 cells stimulated with the TLR4 ligand lipopolysaccharide (LPS; derived from Gram-negative bacteria). Amentoflavone suppressed the nuclear translocation of c-Fos, a subunit of activator protein (AP)-1, at 60 min after LPS stimulation and inhibited the activity of purified and immunoprecipitated extracellular signal-regulated kinase (ERK), which mediates c-Fos translocation. In agreement with these results, amentoflavone also suppressed the formation of a molecular complex including ERK and c-Fos. Therefore, our data strongly suggest that amentoflavone's immunopharmacological activities are due to its direct effect on ERK.

Tracheary elements that resemble secondary xylem in calli derived from the conifers, Torreya nucifera and Cryptomeria japonica

Differentiated cells were recognized in calli derived from needles of Torreya nucifera and in calli derived from immature zygotic embryos of Cryptomeria japonica. Some differentiated cells resembled tracheary elements of primary xylem with spiral or reticulate thickening of cell walls. Other cells resembled tracheary elements with thick cell walls and bordered pits, which are features of secondary xylem. These tracheary elements were formed in cell clusters. Tracheary elements in calli of T. nucifera formed more highly developed structures, such as bordered pits and spiral thickening, than those of C. japonica. Cultured cells derived from conifers might provide a good model for studies of the differentiation of secondary xylem in vitro.

Sex ratios and spatial structure of the dioecious tree Torreya nucifera in Jeju Island, Korea

The sex ratio and spatial structure of different sexes are major components that affect the reproductive success and population persistence of dioecious plants. The differential reproductive costs between male and female plants are often believed to cause a biased sex ratio and spatial segregation of the sexes through slower growth and/or lower female survivorship. In this study, we examined the sex ratio and spatial structure of one population of <TEX>$Torreya$</TEX> <TEX>$nucifera$</TEX> trees in Jeju Island, Korea. We also tested the effects of the current tending actions in relation to tree vitality. At the population level, the sex ratio of the 2,861 trees was significantly biased toward males; however, it also showed considerable variation among different diameter at breast height classes and across habitats according to terrain level (from upper to lower). In 1999, before tree management (tending) began, among the ecological traits examined, only climber coverage correlated with tree vitality. Intensive tending such as climber removal since 1999 clearly enhanced the vitality of the majority of trees, but its effects were more conspicuous in medium-sized trees than in small ones, in upper terrain trees than those in other terrains, and in females than in males. Both male tree domination in small and large trees and tending effects on females are likely to reflect the effects of female reproductive costs regarding growth and/or survivorship. Spatial segregation between males and females was not observed in <TEX>$T.$</TEX> <TEX>$nucifera$</TEX>. Habitat heterogeneity created by the forest's rocky ground and its implications regarding sex ratios and spatial structure require further studies.

Interannual Variation in Nut Abundance Is Related to Agonistic Interactions of Foraging Female Japanese Macaques (Macaca fuscata)

The importance of dominance status to foraging and ultimately survival or reproductive success in wild primates is known; however, few studies have addressed these variables simultaneously. We investigated foraging and social behavior among 17 adult female Japanese macaques (Macaca fuscata) on Kinkazan Island, northern Japan, from September to November in 2 consecutive years (2004 and 2005) to determine whether interannual variation in food availability was related to variation in agonistic interactions over food resources and the feeding behavior of individuals of different dominance rank. We compared energy obtained with daily energy requirements and also examined the effect of variation in feeding behavior on female survival and reproductive success. Fruiting conditions differed considerably between the 2 yr: of four nut-producing species, the nuts of only Torreya nucifera fruited in 2004, whereas all four species, particularly Fagus crenata, produced nuts in abundance in 2005. The abundance and average crown size of trees of Torreya nucifera were smaller than those of Fagus crenata, and there was a higher frequency of agonistic interactions during 2004, when dominant, but not subordinate, individuals were able to satisfy daily energy requirements from nut feeding alone through longer nut feeding bouts. In contrast, all macaques, regardless of their dominance rank, were able to satisfy their energy requirements by feeding on nuts in 2005. Subordinate macaques appeared to counter their disadvantage in 2004 by moving and searching for food more and maintaining larger interindividual distances. Several lower-ranking females died during the food-scarce season of 2004, and only one dominant female gave birth the following birth season. In contrast, none of the adult females died during the food-scarce season of 2005, and 12 females gave birth the following birth season. These findings suggest that an interaction between dominance rank and interannual variation in food availability are related to macaque behavior, survival, and reproduction.

한국산 깍지벌레과 Pupillarial종에 대한 보고 (노린재목, 깍지벌레과)

Two pupillarial species, Cryptoparlatorea leucaspis Lindinger and Fiorinia japonica Kuwana, are recorded for the first time in Korea. Both species were found on coniferous trees, Cryptomeria japonica and Torreya nucifera. Also an identification key, brief diagnoses, and photographs of Korean pupillarial species are provided to assist in their identification.

Tree-ring study of a Late Holocene forest buried in the Ubuka Basin, southwestern Japan

Buried trees in the Ubuka Basin, southwestern Japan, were investigated to identify the wood species and to build tree-ring chronologies. A total of 65 trees were collected belonging to three species: Japanese cedar (Cryptomeria japonica, 63 trees), Japanese nutmeg tree (Torreya nucifera, one tree) and Hinoki cypress (Chamaecyparis obtusa, one tree). Crossdating of the individual ring-width series resulted in four independent tree-ring chronologies covering 605, 576, 507 and 395 years. The absolute ages of these chronologies were determined to be 3676, 4166, 4576 and 4876 cal BP by definite-gap sequence modeling of radiocarbon dates measured at a constant gap of 30 years. An extremely long, continuous chronology can probably be developed in the future by increasing the number of samples at this site.

Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway

Arctigenin is a dibenzylbutyrolactone lignan isolated from Bardanae fructus, Arctium lappa L, Saussureamedusa, Torreya nucifera, and Ipomea cairica. It has been reported to exhibit anti-inflammatory activities, which is mainly mediated through its inhibitory effect on nuclear transcription factor-kappaB (NF-κB). But the role of arctigenin in JAK-STAT3 signaling pathways is still unclear. In present study, we investigated the effect of arctigenin on signal transducer and activator of transcription 3 (STAT3) pathway and evaluated whether suppression of STAT3 activity by arctigenin could sensitize cancer cells to a chemotherapeutic drug cisplatin. Our results show that arctigenin significantly suppressed both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Inhibition of STAT3 tyrosine phosphorylation was found to be achieved through suppression of Src, JAK1, and JAK2, while suppression of STAT3 serine phosphorylation was mediated by inhibition of ERK activation. Pervanadate reversed the arctigenin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, arctigenin can obviously induce the expression of the PTP SHP-2. Furthermore, the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to cisplatin-induced apoptosis. Arctigenin dramatically promoted cisplatin-induced cell death in cancer cells, indicating that arctigenin enhanced the sensitivity of cancer cells to cisplatin mainly via STAT3 suppression. These observations suggest a novel anticancer function of arctigenin and a potential therapeutic strategy of using arctigenin in combination with chemotherapeutic agents for cancer treatment.

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL pro inhibition

As part of our search for botanical sources of SARS-CoV 3CL pro inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL pro inhibitory activity (62% at 100 μg/mL). Following bioactivity-guided fractionation, eight diterpenoids ( 1– 8) and four biflavonoids ( 9– 12) were isolated and evaluated for SARS-CoV 3CL pro inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone ( 9) (IC 50 = 8.3 μM) showed most potent 3CL pro inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure–activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL pro activity with IC 50 values of 280.8, 20.2, and 23.8 μM, respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3′ of flavones, as biflavone had an effect on 3CL pro inhibitory activity.

Chemical constituents from needles of Torreya nucifera

Torreya nucifera (Linn.) Sieb. et Zucc. (Taxaceae), a species mainly distributed in Japan, Korea and China and it has long been extensively used in traditional medicines for the treatment of various diseases relating to oxidation and inflammation [1–3]. In this study, needles of T. nucifera were extracted with acetone-H2O (7:3, v/v), fractionated with hexane, chloroform and ethylacetate (EtOAc), and freeze dried to give some dark brown powders. The resulting EtOAc soluble fraction mixture was then repeatedly chromatographed on a Sephadex LH-20 open column using a series of aqueous methanol and ethanol-hexane mixtures as eluents. Most of the needle extractives were flavan and their methyl ether derivatives, such as (+)-catechin (1), (–)-epicatechin (2), (+)-gallocatechin (3), (–)-epigallocatechin (4), 3-O-methyl-(+)-catechin (5) and 3-O-methyl-(–)-epicatechin (6), as well as protocatechuic acid (7), one of benzoid acid. The structure elucidation and determination of the isolated compounds were based on physiochemical and spectroscopic methods, including 1H and 13C NMR, NOE and EI-MS analysis. Compounds 1, 3, 4, 5 and 7 were isolated from the needles of this species for the first time. Antioxidant potential of the isolates was assessed by DPPH free radical scavenging assay. Results indicated that EtOAc soluble fraction (IC50 13.8g/ml), compounds 1–4 and 7 (IC50 9.6, 9.4, 12.7, 9.5, and 12.9g/ml, respectively) exhibited significant scavenging effects, compared with positive control of curcumin (IC50 13.8g/ml. This indicates that T. nucifera needles could be a promising source for antioxidant-related-medicine exploitation.

Anti-proliferative and apoptosis-inducible activity of labdane and abietane diterpenoids from the pulp of Torreya nucifera in HeLa cells

Two abietane and one labdane type diterpenoids were isolated from the methanol extracts of Torreya nucifera pulp and investigated for their ability to inhibit the growth of human cancer cells. Among the three compounds, the labdane compound kayadiol was found to have the most effective inhibitory effect against a wide variety of human cancer cells. Using the MTT assay, kayadiol was determined to have an IC50 (50% inhibition concentration) of 30 µM in HeLa cells, and also to exhibit anti-proliferative effects towards six other human cancer cell lines, with IC50 values of 30-50 µM. Kayadiol treatment of HeLa cells resulted in a dose-dependent generation of apoptotic events, including DNA laddering (≤100 µM). Moreover, kayadiol-treated HeLa cells showed activation of caspases-3 and -9, as well as an increase in the depolarization of mitochondrial membrane potential and the Bax/Bcl-2 ratio. These results indicate that a mitochondria-related apoptotic pathway is involved in the kayadiol-induced death of HeLa cells. Kayadiol is therefore a promising novel anti-proliferative agent and merits further investigation.

Effects of Non-Methylene-Interrupted Polyunsaturated Fatty Acid, Sciadonic (All-cis-5,11,14-eicosatrienoic Acid) on Lipid Metabolism in Rats

We investigated effects of the non-methylene-interrupted polyunsaturated fatty acid, sciadonic acid (all-cis-5,11,14-eicosatrienoic acid), on the lipid metabolism in rats, to identify the mechanism for the plasma and hepatic triacylglycerol-lowering effects of Japanese torreya (Torreya nucifera) seed oil. Sciadonic acid was isolated from torreya seed oil by the combination of urea-adduct with lipase-esterification. Sprague-Dawley (SD) male rats were fed with experimental diets containing 5% and 10% sciadonic acid based on corn oil for 2 weeks. The serum and liver triacylglycerol levels were lower in the rats fed with sciadonic acid. Considerable amounts of sciadonic acid were detected in the triacylglycerol and phospholipid in both the serum and liver of the rats fed with sciadonic acid. These observations demonstrate that sciadonic acid could modify the lipid metabolism in rats.

비자(Torreya nucifera) 추출물의 생리활성

식용 및 한방생약재로 사용되고 있는 비자나무(T. nucifera)종자인 비자(榧子)를 천연 항산화 소재로 활용하기 위한 연구의 일환으로 물 추출물(WE)과 에탄올 추출물(EE) 그리고 열수 추출물(HWE)에 함유된 플라보노이드와 폴리페놀 화합물의 함량과 tyrosinase 저해, 아질산염 소거, 전자공여, xanthine oxidase 저해활성 등 항산화적 생리활성을 측정하였다. 추출방법과 용매를 달리한 비자의 수율은 100oC 이상의 고온과 압력이 가해진 HWE가 7.08 g/100 g으로 가장 높은 수율을 나타내었다. 또한 비자의 HWE는 35.47㎎/g의 플라보노이드와 112.95 ㎎/g의 폴리페놀 화합물을 함유하였다. 비자의 각 추출물에 대한 tyrosinase 저해율은 2.0 ㎎/mL의 농도에서 5.62∼28.71%로 WE에서 가장 높은 저해효과를 나타내었다. 아질산염의 소거능을 측정한 결과에서는 pH 1.2의 2.0 ㎎/mL의 조건에서 HWE가 94.30 %로 가장 높은 소거효과를 보였으며, pH 1.2와 3.0에서 비자의 세 가지 추출물 모두 약 90% 이상의 아질산염 소거능을 나타내었다. SOD 유사활성능은 HWE에서 33.58%로 WE(17.70%)와 EE(24.16%)보다 약 1.4～2배 높은 활성을 보였다. 전자공여능은 0.1 ㎎/mL의 농도에서 66.46∼89.72%로 WE에서 높았으며, 추출물의 농도가 증가함에 따라 유의적으로 전자공여능이 감소하였다(p＜0.05). Xanthine oxidase 저해는 HWE에서 89.29%로 가장 높은 저해효과를 나타내었으며, 0.5 ㎎/mL의 농도에서도 WE와 HWE는 75% 이상의 저해율을 나타내었다. 이상의 실험 결과 비자는 물을 용매로 추출하는 것이 다량의 플라보노이드와 폴리페놀 화합물 추출에 효과적인 것으로 판단되며 또한 우수한 생리활성 효과를 나타내므로 이를 이용하여 기능성식품이나 제품의 첨가물 또는 의약품 재료로 개발, 활용할 수 있는 유용한 한방생약자원인 것으로 판단된다.

Effects of Yearly Change in Nut Fruiting on Autumn Home-range Use by Macaca fuscata on Kinkazan Island, Northern Japan

Temporal changes in food availability affect foraging success and ultimately reproductive success of animals. They include both seasonal and annual changes. Although many researchers have investigated food availability and the corresponding ranging behavior of primates, studies of yearly changes have been limited. We studied the effects of the fruiting of nuts of Fagus crenata, Zelkova serrata, and Torreya nucifera on the ranging behavior of Japanese macaques (Macaca fuscata) in autumn on Kinkazan Island, northern Japan, for 5 yr between 2000 and 2005 (excluding 2003). We divided the study area (3.3 km2) into 100 × 100-m quadrats to assess the distribution of nut-rich quadrats that contained high densities of nut-producing species. The home ranges of the monkeys contained more nut-rich quadrats than expected, and the difference was significant for 3 of 5 yr. Autumn home-range use was also affected by the distribution of nut production, although the effect differed by year: monkeys frequently used nut-rich quadrats in a Zelkova-year (2000) and in 1 Torreya-year (2001), whereas they nonselectively used nut-rich quadrats in 2 Fagus-years (2002 and 2005) and in another Torreya-year (2004). Thus, Japanese macaques flexibly change their autumn ranging behavior according to yearly changes in distribution of nut production.

Torreya nucifera Essential Oil Inhibits Skin Pathogen Growth and Lipopolysaccharide-Induced Inflammatory Effects

Torreya nucifera Essential Oil Inhibits Skin Pathogen Growth and Lipopolysaccharide-Induced Inflammatory Effects

Effects of a Typhoon on Foraging Behavior and Foraging Success of Macaca fuscata on Kinkazan Island, Northern Japan

We studied the effects of typhoon damage on the food habits, time budgets, and moving distances of Japanese macaques (Macaca fuscata) on Kinkazan Island, northern Japan. Before the typhoon (pre-typhoon phase), the macaques fed on various food items, including fruit in trees (Swida macrophylla) and nuts (Torreya nucifera) on the ground. After the typhoon passed (post-typhoon phase), the macaques fed intensively on the seeds of Perilla frutescens (a forb) and the nuts of Quercus serrata on the ground. One may attribute the changes to decreased food availability or foraging efficiency of fruits and nuts on the ground, due to their concealment by leaf litter and mud and their consumption by other animals, such as sika deer (Cervus nippon) and field mice (Apodemus argenteus). In the post-typhoon phase, the macaques fed more quickly on seeds of Perilla frutescens, spent less time traveling, and moved over shorter distances. The differences may be due to changes in the distribution of staple foods between the 2 phases. We also evaluated the energy intake and energy balance of the macaques in both phases, based on observations of foraging and nutritional analyses of the food items. There is no significant difference in either parameter between the 2 phases. The changes in food habits and movement behavior may have compensated for the reduced food availability or foraging efficiency caused by the typhoon. Both behavioral changes and nutritional issues are important when investigating the effects of storms on animal ecology.

Nematocidal Screening of Essential Oils and Herbal Extracts against Bursaphelenchus xylophilus

Five essential oils and 15 herbal extracts were evaluated to control Bursaphelenchus xylophillus in laboratory. The essential oils from clove plant (Syzygium aromaticum), mustard (Brassica integrefolia), thyme (Thymus vulgaris), and Pelargonium inquinans were found to be highly promising and gave excellent control of the nematodes at all the time of exposure. Among them, the least one gave 91.3% mean mortality rate at 24 hours of exposure time, which is highly significant from the control. While in the second study, most of the methanol (Desmodium caudatum, Paulownia coreana, Auckulandia lappa, Sophota flavescens, Aloe sp., Rheum palmatum, Zingiber officinale, Magnolia officinalis, and Eugenia caryophyllata), hexane (Torreya nucifera, Pharbitis nil, Prunus mume, Melia azedarach, and Xanthium strumarium), and hot water (Cinnamomum cassia) herbal extracts killed the nematodes, but in varying degrees compared to the control. Only one extract was found to be promising viz Magnolia officinalis which found to be statistically different from the control and gave mean mortality of 72, 82.3, and 85.3 % for 24, 48, and 72 hours exposure, respectively. Further screening was conducted for M. officinalis with concentrations of 1,000, 100, and 10 ppm against the same species of nematode with the same time of exposure. However, it gave an excellent result for 1,000 ppm for all time of exposure, whereas for the 100 and 10 ppm it gave mean mortality of 39.5 and 25.8% for the time 72hrs, respectively that were statistically different from the control.

Inhibitory effects of natural plants of Jeju Island on elastase and MMP‐1 expression

In order to search for new active cosmetic ingredients of natural origin, we screened about 60 plants collected from Jeju Island, which is located in the southernmost part of the Republic of Korea. We investigated their free radical scavenging activity, elastase inhibition activity, and reduction of MMP-1 mRNA expression for the development of anti-aging ingredients as raw materials for use in cosmetics. In the free radical scavenging capacity assay, 12 extracts, including Typha orientalis (seed) and Torreya nucifera (leaf), showed significant free radical scavenging activity (up to SC(50)<30 microg/ml). Among these extracts, Nymphaea tetragona (rhizome) extract showed the highest free radical scavenging activity (SC(50)=4.7 microg/ml). In the anti-elastase inhibition assay, seven extracts, including Typha orientalis (seed) and Persicaria hydropiper (whole plant), showed high inhibitory activity (>50% at 100 mug/ml). Among these extracts, Persicaria hydropiper (whole plant) extract showed the highest elastase inhibition activity (IC(50) = 46.7 mug/ml). In the MMP-1 expression assay using RT-PCR, Typha orientalis (seed), Pyrrosia hastata (root), and Capsicum annum (whole plant) showed slightly lower inhibition activity than EGCG, which was used as a control. Furthermore, four extracts, including Persicaria hydropiper (whole plant), Filipendula glaberrima (root), Nymphaea tetragona (root), and Camellia japonica (leaf), completely inhibited the expression of MMP-1 in human fibroblast cells. The results showed that four of the 60 plant extracts may hold potential for use as natural active ingredients for anti-aging cosmetics.

Effects of Japanese Torreya (Torreya nucifera) Seed Oil on the Activities and mRNA Expression of Lipid Metabolism-Related Enzymes in Rats

The mechanism for the plasma and liver triacylglycerol-reducing effects of Japanese torreya (Torreya nucifera) seed oil containing sciadonic acid (all-cis-5, 11, 14-eicosatrienoic acid) is reported. Male SD rats were fed experimental diets containing 10% torreya, corn, or soybean oil for 4 weeks, and the activities and mRNA expression of the enzymes involved in lipid metabolism were measured in the liver. The activities of some hepatic enzymes involved in fatty acid synthesis were lower in the rats fed torreya oil.

Population genetic studies on indigenous conifers in Korea

Studies on the genetic variation in tree populations have been greatly facilitated by the development of molecular markers. Allozyme and DNA markers have been employed to estimate the level of genetic diversity and characterize the distribution patterns of genetic variation within and among populations. They have also been applied to examine the dispersal patterns and mating systems influencing the degree of population differentiation. We reviewed 18 population genetic studies on 8 native Korean conifers (Pinus densiflora, P. thunbergii, P. koraiensis, P. pumila, Abies holophylla, A. koreana, Taxus cuspidata, and Torreya nucifera), which appeared in the literature between 1992 and 2005. The level and distribution patterns of genetic variation observed in the 8 conifers were comparable with those reported in studies on other conifers. From studies on allozyme markers, the expected heterozygosity ranged from 0.168 to 0.262 for Taxus cuspidata and Pinus densiflora, and the degree of genetic differentiation between populations (GST) ranged from 0.031 to 0.067 for Pinus densiflora and Taxus cuspidata, respectively. From studies on the DNA markers of I‐SSR, the genetic diversity (Shannon's Index) ranged from 0.306 to 0.478 for Pinus koraiensis and Taxus cuspidata, respectively, and most of genetic variations were allocated within populations with FST values (i.e., degree of population differentiation) ranging from 0.066 to 0.198 for Pinus densiflora and Abies holophylla, respectively. Although the multilocus outcrossing rate (t m) of Pinus densiflora was 0.860, based on allozymes, suggesting a high level of outcrossing, highly correlated outcrossed paternity (r p=0.874) and evidence of biparental inbreeding (t m‐t s=0.115) were also verified. A study on the spatial autocorrelation of a scattered Korean pine (Pinus koraiensis) stand with relatively low density revealed a significant spatial genetic structure at distances between 24 and 32 m. At the allozyme level, the species with limited distributions tended to show less genetic diversity, but a higher level of genetic differentiation (G ST). For DNA markers, this tendency was not marked.

Antioxidant Activities of Abietane Diterpenoids Isolated from Torreya nucifera Leaves

Investigation on antioxidant compounds from the ethanolic extracts of Torreya nucifera leaves resulted in the isolation of abietane diterpenoids, a known 18-methylesterferruginol (1) and a new 18-dimethoxyferruginol (2). The structures of compounds 1 and 2 were elucidated on the basis of their spectroscopic analyses. Compounds 1 and 2 inhibited the Cu2+-mediated, 2,2'-azobis(2-amidinopropane)hydrochloride-mediated and 3-morpholinosydnonimine-1-mediated low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substances assay as well as the macrophage-mediated LDL oxidation. Compounds 1 and 2 exhibited the potent antioxidant activities in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Compound 1 also suppressed nitric oxide production and inducible nitric oxide synthase expression in lipopolysaccharide-stimulated RAW264.7 cells.