Antinociceptive effects of flower extracts and the active fraction from Styrax japonicus.

ETHNOPHARMACOLOGICAL RELEVANCE: Flowers from Styrax japonicus sieb. et Zucc. have been used as a Chinese folk medicine to alleviate pain such as toothache and sore throat. AIM OF THE STUDY: To testify the analgesic effect of flowers from Styrax japonicus, analyze components of the active fraction, and investigate the mechanism of analgesia. MATERIALS AND METHODS: Flower extracts were obtained by ethanol, petroleum ether and hydrodistillation extraction. Different fractions of ethanol extracts (EE) were isolated by silica gel column chromatography and preparative liquid chromatography. Analgesic effects of EE, petroleum ether extracts (PEE), hydrodistillation extracts (HDE), and fractions of EE were evaluated using hot plate, acetic acid-induced writhing and formalin tests on mice. Components of the active fraction 1 (F1) were determined by the ultrahigh-performance liquid chromatography Q extractive mass spectrometry (UHPLC-QE-MS). Anti-inflammatory and sedative effects involving analgesic mechanisms were evaluated by carrageenan induced hind paw oedema and pentobarbital sodium sleep tests, respectively. In addition, antagonists including naloxone hydrochloride (NXH), flumazenil (FM), SCH23390 (SCH) and WAY100635 (WAY) were used to investigate the possible mechanism of analgesia. Contents of neurotransmitters and relevant metabolites in different brain regions of mice were also quantified by the ultraperformance liquid chromatography with a fluorescence detector (UPLC-FLD). RESULTS: EE rather than PEE and HDE at medium and high doses (150 mg/kg and 300 mg/kg) significantly prolonged the latency time of the response of mice to the thermal stimulation in the hot plate test. Moreover, EE significantly decreased number of writhes in the acetic acid-induced writhing test, and reduced licking time in both two phases of the formalin test in a dose-dependent manner. The F1 (50 mg/kg) showed effective antinociceptive responses in all mice models. However, fraction 2 (F2) and fraction 3 (F3) at 50 mg/kg performed no analgesic action. Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, pinoresinol-4-O-glucoside, forsythin and arctiin were identified from components of the F1. Furthermore, F1 (50 mg/kg) did not significantly affect hind paw oedema of mice induced by carrageenan but significantly shortened sleep latency and increased sleep duration in the pentobarbital sodium sleep test. In addition, the antinociceptive response of F1 was not affected by NXH in two mice models, but significantly blocked by FM and WAY in the hot plate test. In the formalin test, FM avoided the effect of F1 only in the first phase, while the analgesic activity of F1 was totally suppressed by WAY in both two phases. Otherwise, contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased significantly in hippocampus and striatum of mice in the F1 group. CONCLUSION: EE from flowers of Styrax japonicus, and F1, the active part isolated from EE, showed significant antinociceptive activities. The analgesic effect of F1 appeared to be related to the sedative effect, partially mediated by the GABAergic system, and highly involved in the serotonergic system. This was the first study confirming the analgesic effect of Styrax japonicus flower, which provided a candidate for the development of non-opioid analgesics.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Styrax camporum, a typical species of the Brazilian cerrado, attenuates DNA damage, preneoplastic lesions and oxidative stress in experimental rat colon carcinogenesis.

Styrax camporum Pohl, a typical species from the Brazilian cerrado, commonly known as "benjoeiro", is used to treat gastroduodenal diseases. In previous studies carried out by our research group, hydroalcoholic extract of S. camporum stems (SCHE) exhibited antigenotoxic and antiproliferative effects. For a comparative analysis of the chemopreventive effect of SCHE, the aim of this study was to investigate the influence of SCHE against carcinogen 1,2-dimethylhydrazine (DMH)-induced DNA damage and pre-neoplastic lesions in Wistar rat colon. Animals were treated orally with SCHE at 250, 500 or 1000 mg/kg body weight in conjunction with a subcutaneous injection of DMH. DNA damage was assessed using the comet assay while tpre-neoplastic lesions by aberrant crypt foci (ACF) assay. The following hepatic oxidative stress markers were determined including activities of catalase (CAT) and glutathione S-transferase (GST) as well as levels of reduced glutathione (GSH) and malondialdehyde (MDA). Treatment with SCHE was not genotoxic or carcinogenic at the highest dose tested (1000 mg/kg b.w.). The extract effectively inhibited DNA damage and pre-neoplastic lesions induced by DMH administration at all concentrations tested. Measurement of CAT, and GST activities and levels of GSH showed that SCHE did not reduce oxidative processes. In contrast, treatment with SCHE (1000 mg/kg b.w.) decreased liver MDA levels. Taken together, these findings suggested the chemopreventive effect attributed to SCHE in colon carcinogenesis, may be related to its capacity to inhibit DNA damage as well as an antioxidant action associated with its chemical constituents egonol and homoegonol.

Benzoinum from Styrax tonkinensis (Pierre) Craib ex Hart exerts a NVU protective effect by inhibiting cell apoptosis in cerebral ischaemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Benzoinum (Styraceae) is a traditional Chinese medicine used to treat stroke and other cardio-cerebrovascular diseases for thousands of years. Benzoinum has also proven to have diverse pharmacological activity, but the neuroprotection mechanism of apoptosis in ischaemic stroke was not determined. AIM OF THIS STUDY: To investigate the protective effect of a neurovascular unit (NVU) and the mechanisms of benzoinum on cerebral ischaemic rats. MATERIALS AND METHODS: The neuroprotective activity of benzoinum against middle cerebral artery occlusion (MCAO)-induced cerebral ischaemic injury. Neurological scores, 2,3,5-Triphenyltetrazolium chloride (TTC) staining, and hematoxylin-eosin staining (HE) staining were conducted to evaluate the neurological damage. Infarction rate and denatured cell index (DCI) were also calculated. The ultrastructure of neuron and blood-brain-barrier (BBB) was observed by transmission electron microscopy (TEM). Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect Bax, Bcl-2 and Caspase 3 expression. Furthermore, Claudin 5 also was detected through immunohistochemistry. RESULTS: Benzoinum could significantly improve neurological function score and reduce cerebral infarction rate and DCI. In addition, benzoinum alleviated pathomorphological change and apoptosis in the brain tissue of MCAO rats. The results of TEM and claudin 5 expression of immunohistochemistry showed that benzoinum could play a neuroprotective effect in NVU. Also, benzoinum-enhanced Bcl2, and reduced Bax and Bax/Bcl-2 and Caspase 3, suggest that benzoinum provided a neuroprotective effect by inhibited cell apoptosis. CONCLUSION: Benzoinum could play a neuroprotective role and regulate apoptosis for repair and stabilisation of NVU. This anti-apoptosis activity might be associated with the downregulation of Bax and Caspase 3, and the upregulation of Bcl2. Our present findings provide a promising medication for the treatment of ischaemic stroke.

Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor.

Styrax Japonica Sieb. et Zucc. has been used as traditional medicine in inflammatory diseases, and isolated compounds have shown pharmacological activities. Pinoresinol glucoside (PIN) belonging to lignins was isolated from the stem bark of S. Japonica. This study aimed to investigate the biological function and mechanisms of PIN on cell migration, osteoblast differentiation, and matrix mineralization. Herein, we investigated the effects of PIN in MC3T3-E1 pre-osteoblasts, which are widely used for studying osteoblast behavior in in vitro cell systems. At concentrations ranging from 0.1 to 100 µM, PIN had no cell toxicity in pre-osteoblasts. Pre-osteoblasts induced osteoblast differentiation, and the treatment of PIN (10 and 30 µM) promoted the cell migration rate in a dose-dependent manner. At concentrations of 10 and 30 µM, PIN elevated early osteoblast differentiation in a dose-dependent manner, as indicated by increases in alkaline phosphatase (ALP) staining and activity. Subsequently, PIN also increased the formation of mineralized nodules in a dose-dependent manner, as indicated by alizarin red S (ARS) staining, demonstrating positive effects of PIN on late osteoblast differentiation. In addition, PIN induced the mRNA level of BMP2, ALP, and osteocalcin (OCN). PIN also upregulated the protein level of BMP2 and increased canonical BMP2 signaling molecules, the phosphorylation of Smad1/5/8, and the protein level of Runt-related transcription factor 2 (RUNX2). Furthermore, PIN activated non-canonical BMP2 signaling molecules, activated MAP kinases, and increased ß-catenin signaling. The findings of this study indicate that PIN has biological roles in osteoblast differentiation and matrix mineralization, and suggest that PIN might have anabolic effects in bone diseases such as osteoporosis and periodontitis.

Pentacyclic triterpenoid acids in Styrax as potent and highly specific inhibitors against human carboxylesterase 1A.

Human carboxylesterase 1A1 (hCES1A) is a promising target for the treatment of hyperlipidemia and obesity-associated metabolic diseases. To date, the highly specific and efficacious hCES1A inhibitors are rarely reported. This study aims to find potent and highly specific hCES1A inhibitors from herbs, and to investigate their inhibitory mechanisms. Following large-scale screening of herbal products, Styrax was found to have the most potent hCES1A inhibition activity. After that, a practical bioactivity-guided fractionation coupling with a chemical profiling strategy was used to identify the fractions from Styrax with strong hCES1A inhibition activity and the major constituents in these bioactive fractions were characterized by LC-TOF-MS/MS. The results demonstrated that seven pentacyclic triterpenoid acids (PTAs) in two bioactive fractions from Styrax potently inhibit hCES1A, with IC50 values ranging from 41 nM to 478 nM. Among all the identified PTAs, epibetulinic acid showed the most potent inhibition activity and excellent specificity towards hCES1A. Both inhibition kinetic analyses and in silico analysis suggested that epibetulinic acid potently inhibited hCES1A in a mixed inhibition manner. Collectively, our findings demonstrate that some PTAs in Styrax are potent and highly specific inhibitors of hCES1A and these constituents can be used as promising lead compounds for the development of more efficacious hCES1A inhibitors.

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism.

BACKGROUND: Styrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions. PURPOSE: This study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered. STUDY DESIGN: The inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated. METHODS: The inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis. RESULTS: In vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6ß-hydroxylation. CONCLUSION: Styrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.

Two new phenylpropanoids from the resin of Styrax tonkinensis (Pierre) Craib ex Hartw.

Two new phenylpropanoids, named stytonkinol A (1) and stytonkinol B (2), have been isolated from the resin of Styrax tonkinensis (Pierre) Craib ex Hartw. Their structures were determined by spectroscopic analysis, including 1D and 2D NMR, and HR-ESI-MS. Two isolated compounds were assayed for cytotoxic activities against five tumor cell lines (HepG-2, A549, Hela, MCF-7, and PC-3) by Cell Counting Kit-8 (CCK-8) test in vitro. The cytotoxic effectiveness observed against Hela, and MCF-7 cell lines of compound 1 were superior or similar to the positive control cisplatin (IC50 values of 40.95 and 47.36 µM), with IC50 values of 26.75 and 45.16 µM, respectively, while it showed moderate cytotoxic activities against the HepG-2 and PC-3 cell lines. Compound 2 showed moderate cytotoxic activities on cells MCF-7 with IC50 values of 57.1 µM.

A new epicatechin glucopyranoside derivative from Styrax suberifolius.

A new derivative of epicatechin glucopyranoside, (2R,3R)-3,7,4'-trihydroxy-5,3'-dimethoxyflavan 7-O-ß-d-glucopyranoside (1), together with three mononuclear phenolic acid esters, methyl orsellinate (2), ethyl orsellinate (3) and methyl ß-orcinolcarboxylate (4) were isolated from the bark of Styrax suberifolius. The structures of 1-4 were determined on the basis of extensive analysis of NMR and MS spectra combined with chemical hydrolysis. The antifungal activities of the isolated compounds against three plant pathogenic fungi, Alternaria solani, Fusarium oxysporum and Phomopsis cytospore were evaluated using radial growth inhibition assay. Compounds 2, 3 and 4 exerted selective inhibitory activities against the tested fungi. Among of them, methyl ß-orcinolcarboxylate (4) exhibited obvious inhibitory effect against P. cytospore, with an inhibition rate of 86.72% at 100 µg/ml.

Oral Administration of Incense Resin (Styrax benzoin) Extract Enhances Spatial Learning, Memory, and Dendrite Complexity of Mice

Abstract Hippocampus is a part of the brain that has a major role in spatial learning and memory which can be affected by herbal extracts. Incense resin (Styrax benzoin) has been used by local communities to improve intelligence. However, there is no scientific evidence of the functions of Styrax benzoin for regulating hippocampal function. The aim of this study was intended to analyze and investigate the effect of incense resin on learning, memory, and dendrite complexity of mice. Three months old male Deutch Democratic Yokohama (DDY) mice were injected orally with graded doses of 100, 150, and 200 mg/kg of incense resin aqueous extract daily for 30 days. Spatial learning and memory performance levels were tested with Y-maze alternation, novel object recognition, and Morris water maze. The branches and maximum dendritic span in the dentate gyrus were observed by the Golgi-Cox staining. Overall, our results showed that incense resin extract increased learning and memory ability, and the number of dendrite branching in the dentate gyrus.

[Quality evaluation of Styrax based on quantitative determination of 7 chemical components using HPLC wavelength switching method and chemometrics].

In order to evaluate the quality of Styrax more comprehensively,this study attempted to establish an HPLC wavelength switching method to simultaneously determine the content of seven compounds in Styrax,and chemometrics were used to analyze the quality difference between different sources of Styrax,and finally establish a characteristic chromatogram of Styrax. The column was Agilent ZORBAX Extend C18( 4. 6 mm×250 mm,5 µm) with phase a mixture of acetonitrile-0. 1% phosphoric acid aqueous solution as the mobile phase in a gradient elution procedure; the detection wavelength was set as follows: 0-13. 5 min,194 nm( benzoic acid);13. 5-20. 5 min,278 nm( cinnamic acid); 20. 5-32 min,194 nm( benzyl benzoate,benzyl cinnamate,cinnamyl cinnamate,dehydroabietic acid); 32-55 min,241 nm( abietic acid). The methodological verification results showed that when the injection masses of benzoic acid,cinnamic acid,benzyl benzoate,benzyl cinnamate,cinnamyl cinnamate,dehydroabietic acid and abietic acid were0. 006 948-0. 694 8,0. 001 426-0. 142 6,0. 013 16-0. 658 0,0. 006 148-0. 614 8,0. 008 035-0. 803 5,0. 002 121-0. 212 1,and0. 010 172-1. 017 2 µg,respectively,there were good linear relationship between injection mass and peak area. The average recovery rates of seven compounds were in the range from 94. 34% to 105. 8%,and all RSD were less than 3. 0%( n = 6). The methodological verification results showed that the developed HPLC wavelength switching method has good accuracy and repeatability. The results of the sample analysis showed that the quality of Styrax from different sources was quite different. The chromatogram of Styrax reference material( S1) was used as the reference chromatogram to calculate the fingerprint similarity of each batch of samples. The results showed that the similarities of samples S2-S10 were 0. 952,0. 949,0. 981,0. 351,0. 751,0. 969,0. 979,0. 992 and 0. 971,respectively.The similarity values of other batches samples were satisfactory,except for sample S5 and S6,indicating that the quality difference among these samples is small. The similarity values of S11-S20 were 0. 060,0. 055,0. 054,0. 285,0. 092,0. 002,0. 044,0. 044,0. 044,and 0. 040,respectively. The results showed that compared with the sample S1,there was a large quality difference among S11-S20. Based on the chromatograms of S1-S10,the HPLC characteristic chromatograms of Styrax was established and the purpose is to give reference to other pharmaceutical researchers. The newly developed HPLC wavelength switching method have the advantages of simplicity,reproducibility and specificity,and the developed HPLC characteristic chromatograms provided a reference method for the overall quality evaluation of Styrax.

Fast and Green - CO2 Based Extraction, Isolation, and Quantification of Phenolic Styrax Constituents.

In this study the first supercritical fluid based protocol for the extraction, analysis, and isolation of six polar compounds, i.e., o-vanillin (1), styracin (2), vanillin (3), trans-cinnamic acid (4), vanillic acid (5), and shikimic acid (6), was developed. First, eight styrax resin products (R1-R8) obtained from various Liquidambar tree species, which are known to contain compounds 2-6, were extracted with a 1 : 1 mixture of supercritical CO2 and EtOH. Within 4 minutes, the compounds were successfully baseline separated on an Acquity UPC2 BEH 2-EP (3.0 × 100 mm, 1.7 µm) column using a mobile phase of supercritical CO2 and MeOH with 0.1 % phosphoric acid. The compounds were quantified and the method was validated according to current ICH guidelines. Scaling up to preparative supercritical fluid chromatography using a Viridis BEH 2-EP (10 × 250 mm, 5 µm) column allowed for a fast separation and isolation of the selected constituents 2 and 4 from R6 within 7 minutes. This supercritical fluid protocol is easily adaptable to compounds of similar polarity. The increase in speed and its environmental friendliness underline its superiority over conventional set-ups.

Cytotoxic Activity of Compounds from Styrax obassia.

Cancer is a major public health burden in both developed and developing countries. Plant-derived compounds have played an important role in the development of useful anti-cancer agents. The current study was designed to evaluate the cytotoxic activity of chemical compounds from the stem bark of Styrax obassia. Seven known compounds (1-7) were isolated and identified. Compound 2 exhibited cytotoxic activity against the breast cancer cell line MCF-7 with an IC550 of 27.9 µM, followed by the human cervical cancer cell line Hela with an IC50 of 23.3 µM, and the human promyelocytic leukemia cell line HL-60 with an IC50 of 47.8 RM. Compound 7 exhibited cytotoxicity against Hela cells with an ICso of 16.8 pM, followed by MCF-7 cells with an IC50 of 53.5 µM. This is the first study to investigate the significant anti-tumor properties of isolated compounds from the stem bark of S. obassia.

Antibacterial, anti-biofilm and anticancer potentials of green synthesized silver nanoparticles using benzoin gum (Styrax benzoin) extract.

This study described a simple and green approach for the synthesis of silver nanoparticles (AgNPs) employing benzoin gum water extract as a reducing and capping agent and their applications. The AgNPs were characterized by ultraviolet-visible spectrophotometer, X-ray diffraction pattern, field emission transmission electron microscopy, dynamic light scattering, zeta potential and fourier transform infrared spectroscopy. The AgNPs showed promising antimicrobial activity against various pathogens (Gram-negative, Gram-positive and fungus) and possessed high free radical scavenging activity (104.5 ± 7.21 % at 1 mg/ml). In addition, the AgNPs exhibited strong cytotoxicity towards human cervical cancer and human lung cancer cells as compared to the normal mouse macrophage cells. Moreover, the AgNPs possessed anti-biofilm activity against Escherichia coli, and compatibility to human keratinocyte HaCaT cells, which suggests the use of dressing with the AgNPs in chronic wound treatment. Therefore, AgNPs synthesized by benzoin gum extract are comparatively green and may have broad spectrum potential application in biomedicine.

Characterization of Phenolic Compounds and Antioxidant and Anti-inflammatory Activities from Mamuyo (Styrax ramirezii Greenm.) Fruit.

Extracts of Styrax ramirezii Greenm., a fruit traditionally valued for health and wellness in Mexico, were analyzed phytochemically and evaluated for antioxidant and anti-inflammatory activity. Six norneolignans were identified by HPLC-TOF-MS, and the two major compounds were isolated for further evaluation. The effects of the isolated norneolignans, egonol and homoegonol, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, reactive oxygen species (ROS) production, and biomarkers of inflammation were evaluated. Of the tested compounds, egonol potently inhibited the production of NO and also significantly reduced the release of ROS. Consistent with these observations, the mRNA expression levels of inducible nitric oxide synthase (iNOS) (0.668 ± 0.108), cyclooxygenase-2 (COX-2) (0.553 ± 0.007), interleukin-1ß (IL-1ß) (0.093 ± 0.005), and interleukin-6 (IL-6) (0.298 ± 0.076) were reduced by egonol. The activity for both egonol and homoegonol increased in a concentration-dependent manner. These results suggest the potential of S. ramirezii Greenm. fruit to contribute to a healthy diet, rich in antioxidant and anti-inflammatory compounds.

Two new triterpenoids from the resin of Styrax tonkinensis.

Two new triterpenoids, 3ß,6ß-dihydroxy-12-oxo-13Hα-olean-28,19ß-olide (1) and 3-oxo-olean-11,13(18)-dien-28,19ß-olide (2) were isolated from the resin of Styrax tonkinensis (Pier.) Craib. The structures of both triterpenoids were determined by physicochemical and spectroscopic methods. Compound 1 is the second triterpene found with cis-fused C/D ring from the resin, which is rarely observed in oleanane-type triterpenes.

[Lignans from Stem Bark of Styrax perkinsiae].

OBJECTIVE: To study the chemical constituents in the stem bark of Styrax perkinsiae. METHODS: The chemical constituents were separated and purified by chromatographic methods after solvent extraction and identified by spectroscopic analyses. RESULTS: Ten lignans were isolated from the stem bark of Styrax perkinsiae and identified as following: pinoresinol 4-O-ß-D-glucopyranoside (1), matairesinoside (2), styraxlignolide B (3), 3- (ß-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl )-7-methoxy-(2R , 3S) -dihydrobenzofuran (4), burselignan (5), (+) -neo-olivil (6), threo-1-(4-hydroxy-3-methoxyphenyl )-2-[ 4-(3-hydroxypropyl)-2-methoxyphenoxy]-1, 3-propanediol (7), erythro-1-(4-hydroxy-3-methoxyphenyl )-2-[ 4-(3-hydroxypropyl )-2-methoxyphenoxy ] -1 ,3-propanediol (8), isolariciresinol(9) and (+) -lariciresinol (10). CONCLUSION: Compounds 5 - 10 are isolated from the plants of Styrax genus for the first time.

SJSZ glycoprotein (38 kDa) modulates macrophage type 1/2-related factors at hepatocarcinogenic stage in N-nitrosodiethylamine-treated Balb/c.

Macrophage plays critical role for tumor progression: Type 1 (M1) for tumor prevention and type 2 (M2) for promotion in hepatocellular carcinoma. In order to study the chemopreventive effects of the SJSZ glycoprotein (38 kDa) on M1- or M2-related factors, Balb/c was injected intraperitoneally with N-nitrosodiethylamine (DEN; 50 mg/kg, BW) for 7 weeks. After 7 weeks, the mice were sacrificed. After that, peritoneal macrophages were isolated. We evaluated the production of reactive oxygen species (ROS) and nitric oxide (NO), hepatocarcinogenic signals [activities of mitogen-activated associated kinase (MAPKs), inducible nitric oxide synthase (iNOS), nuclear factor (NF)-κB, and signal transducer and activator of transcription (STAT) 6,], cytokines [interleukin (IL)-10, IL-4, IL-12, and interferon (IFN)-γ], and CD163-positive macrophages (M2 polarization) using biochemical methods, immunoblot analysis, qRT-PCR, ELISA, and flow cytometry. The results revealed that the SJSZ glycoprotein (10 mg/kg, BW) inhibits the phosphorylation of MAPKs and expression of NF-κB, pSTAT6, IL-10, and IL-4; and normalizes production of ROS and NO, and expression of iNOS, IL-12, and IFN-γ. Especially, it inhibited CD163-positive macrophages. In conclusion, these results indicated that SJSZ glycoprotein modulates polarization of macrophage type 1 and type 2 at hepatocarcinogenic initial stage in DEN-treated Balb/c. Thus, SJSZ glycoprotein may be useful as one of immunomodulating agents which have to regulate M1- and M2-related factors to prevent tumor progression.

In vivo protective activity of Styrax camporum hydroalcoholic extract against genotoxicity induced by doxorubicin and methyl methanesulfonate in the micronucleus and comet assays.

Styrax camporum Pohl is a tall shrub or a tree with small white flowers, which grows in the states of São Paulo and Minas Gerais and is popularly used for the treatment of gastroduodenal diseases. Considering this last fact, the aim of this study was to evaluate the genotoxic potential of S. camporum hydroalcoholic extract and its influence on genotoxicity induced by doxorubicin and methyl methanesulfonate in Swiss mice using the micronucleus and comet assays, respectively. The animals were treated by gavage with different doses of the extract (250, 500, and 1000 mg/kg body weight). For antigenotoxicity assessment, different doses of the S. camporum extract were administered simultaneously with doxorubicin (micronucleus test; 15 mg/kg) and methanesulfonate (comet assay; 40 mg/kg). The results showed that the S. camporum extract itself was not genotoxic in the mouse micronucleus or comet assay. The number of micronucleated polychromatic erythrocytes was significantly lower in animals treated with the S. camporum extract and doxorubicin when compared to animals treated only with doxorubicin. In the comet assay, the S. camporum extract, at the doses tested, significantly reduced the extent of DNA damage in liver cells induced by methanesulfonate. The putative activity of the active compounds of S. camporum extract may explain the effect of this plant on genotoxicity induced by doxorubicin and methanesulfonate.

Egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae promote the biosynthesis of estrogen by aromatase.

Estrogen deficiency is associated with a variety of diseases, including osteoporosis, atherosclerosis, and Alzheimer's disease. Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyze the biosynthesis of estrogens from androgens. Inhibitors of aromatase have been developed for the treatment of estrogen-dependent breast cancer. However, small molecular agonists of aromatase, which would be useful to locally promote estrogen biosynthesis for the prevention of estrogen deficiency-induced diseases, are rarely reported. In this study, we established a nonradioactive assay for measuring aromatase activity by using human ovarian granulosa KGN cells and identified two estrogen biosynthesis-promoting compounds, egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae. The compounds also promoted estrogen biosynthesis in 3T3-L1 preadipocyte cells. Further study showed that neither compound affected the transcriptional and translational expression of aromatase in KGN cells, but that both significantly promoted the in vitro enzyme activity of recombinant expressed aromatase. Egonol gentiotrioside was also found to increase the serum estrogen level in ovariectomized rats. These results suggest that these two compounds may promote estrogen biosynthesis through the allosterical regulation of aromatase activity. Egonol gentiobioside and egonol gentiotrioside are, therefore, valuable targets for structural modification and warrant further investigation for their potential as novel pharmaceutical tools for the prevention of estrogen deficiency-induced diseases.