[Development of an analytical system for dried blood spots for forensic toxicology: a case study of five common drugs and poisons].

Dried blood spot (DBS) technology is a simple and convenient method for collecting, transporting, and storing blood samples on filter paper, and has numerous applications in the clinical, research, and public health settings. This technique is gaining popularity in the field of forensic science because it facilitates the rapid analysis of prohibited drugs in blood samples and offers significant advantages in toxicology scenarios such as drinking-driving screening, drug abuse detection, and doping detection. However, the lack of a standardized system and the fact that its stability and reliability have not been thoroughly researched and demonstrated limit its application in judicial practice in China. DBS samples can be prepared, stored, and analyzed in various ways, all of which may significantly affect the results. In this study, we developed a method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that focuses on the preparation, pretreatment, analysis, and storage of DBS samples. A thorough investigation was conducted to examine the optimal preparation conditions, including the blood spot matrix, drying technique, and preprocessing parameters, such as the solvent and extraction method. Moreover, the analytical conditions, such as the mobile phase system and elution gradient, were established to facilitate the quantitative detection of methamphetamine, lidocaine, ketamine, fentanyl, and diazepam in both DBS and whole-blood samples. The impact of storage conditions, such as the temperature, humidity, and sealing, on the analytical results of the DBS and whole-blood samples was also examined. The results showed a strong linear relationship for lidocaine and fentanyl within the range of 0.5-100 ng/mL. Similarly, methamphetamine, ketamine, and diazepam exhibited good linearity within the range of 2-100 ng/mL. The coefficients of determination (r2) ranged from 0.9983 to 0.9997, and the limits of detection ranged from 0.2 to 0.5 ng/mL, indicating a high degree of correlation and sensitivity. Stability tests demonstrated that the five target substances remained stable in the DBS for 60 days, with the measured contents deviating from the nominal values by 15%. Moreover, the measurement results of the DBS samples were highly similar to those of the whole-blood samples, with mean percentage differences of 4.44%, 3.50%, 7.66%, 5.10%, and 5.25% for fentanyl, diazepam, ketamine, lidocaine, and methamphetamine, respectively. Throughout the 60-day storage period, the maintenance of temperatures of -20 and 4 ℃, as well as sealing and dry storage, was not necessary. Room temperature was the most practical storage environment for the DBS samples. The results for each target showed very small concentration differences between the whole-blood and DBS samples, indicating that the DBS samples were suitable for drug and poison analysis in blood. Furthermore, the DBSs exhibited high quantitative consistency with the whole-blood samples, rendering them suitable matrices for preserving blood samples. Because DBS samples are easy to handle and store, they can realize the lightweight preservation of blood samples and provide a novel solution for the analysis and preservation of blood samples in public security practice. We recommend conducting comprehensive validations before utilizing DBS for analysis, particularly in terms of quantification, to ensure the judicial reliability of the results.

Green tea extract synergistically enhances the effectiveness of an antiresorptive drug on management of osteoporosis induced by ovariectomy in a rat model.

Antiresorptive drugs are effective for reducing bone loss in postmenopausal women, but their long-term application may be associated with adverse effects. The present study aimed to investigate the potential in vivo synergistic effects of green tea extract (GTE) and alendronate or raloxifene on the management of osteoporosis. Ovariectomized rats were fed orally with GTE, alendronate and raloxifene at different concentrations and various combinations for 4 weeks. Bone mineral density (BMD) at the lumbar spine, femur and tibia was monitored weekly using peripheral quantitative computed tomography. Bone microarchitecture in the left distal femur was analyzed using micro-CT, while serum biochemical levels were measured using ELISA kits at the end of the study. GTE alone effectively mitigated BMD loss and improved bone microarchitecture in rats. The co-administration of GTE and alendronate increased total BMD in the lumbar spine, femur and tibia. Particularly, GTE synergistically enhanced the effect of alendronate at a low dose on bone microarchitecture and decreased serum tartrate-resistant acid phosphatase. These findings imply that the dosage of certain antiresorptive agents could be reduced when they are administrated simultaneously with GTE, so that their adverse effects are minimized. The findings may be used to support the development of a new synergistic intervention between food therapy and pharmacotherapy on the management of osteoporosis in a long-term basis.

GSP-2, a polysaccharide extracted from Ganoderma sinense, is a novel toll-like receptor 4 agonist.

Ganoderma sinense is a Chinese unique medicinal fungus that has been used in folk medicine for thousands of years. Polysaccharides are considered to be biologically active ingredients due to their immune-modulating functions. Previously we found that GSP-2, a new polysaccharide isolated from Ganoderma sinense, exerts an immunomodulatory effect in human peripheral blood mononuclear cells but the underlying mechanism is unclear. The present study aimed to investigate how GSP-2 triggers immunologic responses and the implicated signaling pathways. GSP-2 effects were investigated both in a macrophagic cell line, RAW264.7, and in primary macrophages. Moreover, the molecular basis of GSP-2 recognition by immune cells, and the consequent activation of signaling cascades, were explored by employing recombinant human HEK293-TLR-Blue clones, individually overexpressing various Toll-like receptors. GSP-2 dose-dependently induced the overexpression of Toll-like receptor 4 (TLR4) but did not affect the expression of other TLRs. Moreover, GSP-2 induced TNFα secretion in primary macrophages from wild-type, but not TLR4-knockout mice. In addition, GSP-2 upregulated TLR4 protein expression and activated the MAPK pathway in RAW246.7 macrophages. Finally, GSP-2 induced the production of the cytokines TNFα, IL1ß, and IL6. Our data demonstrated that GSP-2 was specifically recognized by TLR4, promoting cytokine secretion and immune modulation in macrophages.

In situ Study on the Diffusion Kinetics of Seal Ink by Microinfrared Spectroscopy.

In document examination, it is of great importance to determine the composition of seal ink with different imprint times, and spectroscopic methods are widely used today. In this research, the diffusion of seal inks from three different brands on the same type of paper is monitored in situ by microinfrared spectroscopy and microinfrared imaging technology. The area of the absorption peak at 1743 cm-1 gradually decreases with increasing diffusion time. The diffusion kinetics of seal ink on paper are also studied by analyzing the infrared spectra of seal inks at the same measuring point with different diffusion times. The research provides a basic study in understanding the diffusion behavior of seal ink on paper over short time spans.

Inhibitory effect of different Dendrobium species on LPS-induced inflammation in macrophages via suppression of MAPK pathways.

Dendrobii Caulis (DC), named 'Shihu' in Chinese, is a precious herb in traditional Chinese medicine. It is widely used to nourish stomach, enhance body fluid production, tonify "Yin" and reduce heat. More than thirty Dendrobium species are used as folk medicine. Some compounds from DC exhibit inhibitory effects on macrophage inflammation. In the present study, we compared the anti-inflammatory effects among eight Dendrobium species. The results provided evidences to support Dendrobium as folk medicine, which exerted its medicinal function partially by its inhibitory effects on inflammation. To investigate the anti-inflammatory effect of Dendrobium species, mouse macrophage cell line RAW264.7 was activated by lipopolysaccharide. The nitric oxide (NO) level was measured using Griess reagent while the pro-inflammatory cytokines were tested by ELISA. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) phosphorylation were evaluated by Western blotting analysis. Among the eight Dendrobium species, both water extracts of D. thyrsiflorum B.S.Williams (DTW) and D. chrysotoxum Lindl (DCHW) showed most significant inhibitory effects on NO production in a concentration-dependent manner. DTW also significantly reduced TNF-α, MCP-1, and IL-6 production. Further investigations showed that DTW suppressed iNOS and COX-2 expression as well as ERK and JNK phosphorylation, suggesting that the inhibitory effects of DTW on LPS-induced macrophage inflammation was through the suppression of MAPK pathways. In conclusion, D. thyrsiflorum B.S.Williams was demonstrated to have potential to be used as alternative or adjuvant therapy for inflammation.

[Short-Term Effects of Different Fertilization Treatments on Greenhouse Gas Emissions from Northeast Black Soil].

A 7-day incubation experiment was conducted at 25℃ with 60% water holding capacity (WHC) to study the short-term effects of different fertilization treatments on the regularity of greenhouse gas emissions from northeast black soil. The results showed that application of chemical N fertilizer had no effect on CO2 emission, as compared with the non-fertilizer control treatment; however, a combined application of N fertilizer with pig manure or straw increased CO2 emissions by one magnitude compared to that of the chemical N fertilizer treatment, with the effect of chemical N fertilizer and straw being more prominent. Nitrification was the main process resulting in N2O emission for the non-fertilizer control and chemical N fertilizer treatments, and the application of chemical N fertilizer had no significant effect on N2O emission, as compared with the non-fertilizer control. The combined application of N fertilizer with pig manure or straw promoted the occurrence of denitrification and increased N2O emissions by two magnitudes compared to that of the chemical N fertilizer treatment, with the effect of chemical N fertilizer with straw being more remarkable. Compared with the non-fertilizer control, the application of chemical N fertilizer inhibited CH4 emissions and promoted the slightly absorption of CH4, while the combined application of chemical N fertilizer with pig manure or straw increased significantly the emission of CH4.

Dihydrofisetin exerts its anti-inflammatory effects associated with suppressing ERK/p38 MAPK and Heme Oxygenase-1 activation in lipopolysaccharide-stimulated RAW 264.7 macrophages and carrageenan-induced mice paw edema.

Dihydrofisetin is a flavanonol derived from some edible wild herbs and traditional Chinese medicines. It has been found to possess many biological activities. However, the anti-inflammatory potential of Dihydrofisetin remains uncharacterized. The aim of the present study was to investigate the anti-inflammatory activity of Dihydrofisetin and its underlying mechanisms. We found that Dihydrofisetin dose-dependently inhibited lipopolysaccharide-induced productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages, probably through suppressing the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The expressions of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and monocyte chemotactic protein (MCP-1) were also suppressed. We further demonstrated that Dihydrofisetin inhibited the activation of mitogen-activated protein kinases (MAPKs) pathway and phosphorylation of IκB-α whereas upregulated the expression of heme oxygenase-1 (HO-1). The in vivo carrageenan-induced mice paw edema study also indicated that treatment with 100 mg/kg of Dihydrofisetin could significantly inhibit carrageenan induced paw edema, decrease the levels of TNF-α, IL-6 and MDA, and increase the activity of GSH-Px in paw tissues. Taken together, Dihydrofisetin may act as a natural agent for treating inflammatory diseases by targeting MAPK, NF-κB and HO-1 pathways.

A traditional Chinese formula composed of Chuanxiong Rhizoma and Gastrodiae Rhizoma (Da Chuanxiong Formula) suppresses inflammatory response in LPS -induced RAW 264.7 cells through inhibition of NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuanxiong Formula (DCXF) which origins from Jin Dynasty is a famous classical 2-herb Chinese medicinal prescription. It is composed of dried rhizomes of Ligusticum chuanxiong (Chuanxiong Rhizoma, CR) and Gastrodia elata (Gastrodiae Rhizoma, GR) at the ratio of 4:1 (w/w). It has been used to treat headache which is caused by wind pathogen and blood stasis for thousands of years in China. AIM OF STUDY: The present study was performed to investigate the anti-inflammatory effect of DCXF and elucidate its underlying molecular mechanisms using LPS-stimulated RAW 264.7 cells. MATERIALS AND METHODS: The anti-inflammatory effect of DCXF was evaluated using LPS-stimulated RAW 264.7 cells. Generation of nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. The gene expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the effect of DCXF on NF-κB activation was measured by western blot assay. RESULTS: Treatment with DCXF significantly suppressed the productions of NO and PGE2 through inhibitions of iNOS and COX-2 expressions in LPS-stimulated RAW 264.7 cells. DCXF significantly decreased IκBα phosphorylation, inhibited p65 expression and reduced p-p65 level. These results suggested the anti-inflammatory effect of DCXF was associated with the reduction of inflammatory mediators through inhibition of NF-κB pathway. CONCLUSIONS: These results indicated that DCXF inhibited inflammation in LPS-stimulated RAW 264.7 cells through inactivation of NF-κB pathway.

In vivo and in vitro anti-inflammatory effects of Zao-Jiao-Ci (the spine of Gleditsia sinensis Lam.) aqueous extract and its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Zao-Jiao-Ci (ZJC), as the spine of Chinese Honey locust (Gleditsia sinensis Lam.), is traditionally used as Chinese medicine to reduce inflammation. AIM OF THE STUDY: The present study aimed to investigate an anti-inflammatory effect of ZJC aqueous extract both in vitro and in vivo, as well as its underlying mechanisms. MATERIALS AND METHODS: Anti-inflammatory effect of ZJC aqueous extract was evaluated by using carrageenan-induced paw edema in rats. In addition, the inhibitory effects of ZJC on nitric oxide production, intracellular reactive oxygen species production, pro-inflammatory mediator expression and prostaglandin E2 (PGE2) production were determined by using LPS-activated RAW 264.7 cells. The anti-oxidant activity of ZJC was assessed using 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid assay. RESULTS: ZJC aqueous extract showed significant suppressive effect on paw edema in rats at 100mg/kg. Moreover, ZJC aqueous extract decreased the expression of cyclooxygenase (COX)-2 and significantly decreased the PGE2, tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 production in LPS-activated macrophages in dose-dependent manners. ZJC aqueous extract inhibited the mRNA expression of these inflammatory cytokines as well. Furthermore, ZJC aqueous extract was found as an anti-oxidant and could inhibit ROS production in the LPS-induced cells. CONCLUSIONS: These findings show the potential of ZJC aqueous extract as a naturally occurring COX-2 inhibitor to reduce inflammation.

Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes.

Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea.

Green tea polyphenols decreases uric acid level through xanthine oxidase and renal urate transporters in hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea is a Chinese materia medica with the main functions of "inducing urination and quenching thirst". Green tea polyphenols (GTP) are generally acknowledged as the main active fraction with multiple pharmacological functions in green tea. However, the effect of GTP on hyperuricemia is not clear till now. AIM OF STUDY: The present study was carried out to investigate the effect of GTP on serum level of uric acid in potassium oxonate (PO)-induced hyperuricemic mice, and explore the underlying mechanisms from two aspects of production and excretion of uric acid. MATERIALS AND METHODS: PO and GTP were intragastricly administered to mice for consecutive 7 days. Serum level of uric acid, and xanthine oxidase (XOD) activity in serum and liver were examined. Simultaneously, expression of XOD protein in liver was analyzed by Western blot assay. Expressions of urate transporters including urate-anion transporter (URAT) 1, organic anion transporter (OAT) 1 and 3 in kidney were analyzed by immunohistochemistry staining method. RESULTS: 300 and 600 mg/kg GTP significantly decreased serum level of uric acid of hyperuricemic mice in a dose-dependent manner (p<0.05 or p<0.01). Besides, 300 and 600 mg/kg GTP markedly reduced XOD activity in serum and liver of hyperuricemic mice (both p<0.01). Furthermore, 300 and 600 mg/kg GTP clearly reduced XOD expression in liver, as well as reduced URAT1 expression and increased OAT1 and OAT3 expressions in kidney of hyperuricemic mice (p<0.05 or p<0.01). CONCLUSIONS: These results demonstrated that GTP had the effect of lowering uric acid through decreasing the uric acid production and increasing uric acid excretion. Our study suggested that GTP would be a promising candidate as a novel hypouricaemic agent for further investigation.

Er-Miao-San, a traditional herbal formula containing Rhizoma Atractylodis and Cortex Phellodendri inhibits inflammatory mediators in LPS-stimulated RAW264.7 macrophages through inhibition of NF-κB pathway and MAPKs activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Er-Miao-San (EMS) is a traditional Chinese herbal formulation that contains combinations of Rhizoma Atractylodis (RA) and Cortex Phellodendri (CP). It exhibits analgesic and anti-inflammatory activities and have been used for the treatment of various "Bi Zheng" for thousand years in China. The aims of the present study were to investigate the anti-inflammatory activities of EMS and elucidate the underlying mechanisms with regard to its molecular basis of action for the best combination. MATERIALS AND METHODS: The anti-inflammatory effects of EMS were studied by using lipopolysaccharide (LPS)-stimulated activation of nitric oxide (NO) and pro-inflammatory cytokine production in mouse RAW264.7 macrophages. Expression of inducible NO synthase (iNOS), mitogen-activated protein kinases (MAPKs) phosphorylation, p65 phosphorylation, inhibitor-κBα (IκBα) degradation, and NF-κB DNA-binding activity were further investigated. RESULTS: The present study demonstrated that EMS could suppress the production of NO in LPS-stimulated RAW264.7 macrophages. However, CP and RA did not have significant inhibitory effect on them. EMS also inhibited the production of tumor necrosis factor-alpha, interleukin-1 beta and macrophage chemotactic protein-1. Further investigations showed EMS could suppress iNOs expression and p38 phosphorylation. EMS significantly decreased the content of IκBα, reduced the level of phosphorylated p65 and suppressed the NF-κB DNA-binding activity. All these results suggested the inhibitory effects of EMS on the production of inflammatory mediators through the inhibition of the NF-κB pathway. CONCLUSIONS: Our results indicated that EMS inhibited inflammatory events and iNOS expression in LPS-stimulated RAW264.7 cells through the inactivation of the MAPK and NF-κB pathway. This study gives scientific evidence validating the use of EMS in treatment of patients with "Bi Zheng" in clinical practice in traditional Chinese medicine.

Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice.

Green tea (Camellia sinensis, CS), a kind of Chinese tea commonly consumed as a healthy beverage, has been demonstrated to have various biological activities, including antioxidation, antiobesity and anticancer. Our study aims to investigate the antitumor, antimetastasis and antiosteolytic effects of CS aqueous extract both in vitro and in vivo using metastasis-specific mouse mammary carcinoma 4T1 cells. Our results showed that treatment of 4T1 cells with CS aqueous extract resulted in significant inhibition of 4T1 cell proliferation. CS extract induced 4T1 apoptosis in a dose-dependent manner as assessed by annexin-V and propidium iodide staining and caspase-3 activity. Western blot analysis showed that CS increased the expression of Bax-to-Bcl-2 ratio and activated caspase-8 and caspase-3 to induce apoptosis. CS also inhibited 4T1 cell migration and invasion at 0.06-0.125 mg/ml. In addition, CS extract (0.6 g/kg, orally fed daily for 4 weeks) was effective in decreasing the tumor weight by 34.8% in female BALB/c mice against water treatment control (100%). Apart from the antitumor effect, CS extract significantly decreased lung and liver metastasis in BALB/c mice bearing 4T1 tumors by 54.5% and 72.6%, respectively. Furthermore, micro-computed tomography and in vitro osteoclast staining analysis suggested that CS extract was effective in bone protection against breast cancer-induced bone destruction. In conclusion, the present study demonstrated that the CS aqueous extract, which closely mimics green tea beverage, has potent antitumor and antimetastasis effects in breast cancer and could protect the bone from breast cancer-induced bone destruction.

In vitro and in vivo mechanistic study of a novel proanthocyanidin, GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis.

Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (P<.001) and tubule formation (P<.001-.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC-(4→8)-GCG in a dose-dependent manner after 3 days of treatment (P<.001-.05). In conclusion, these results revealed that our novel proanthocyanidin, GC-(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent.

Probing the time scale for bulk equilibration and mass transport of water in amorphous inorganic aerosol.

New techniques are required to explore directly the kinetics of water transport in aerosol between the gas and condensed phases, both at high relative humidity (RH) close to saturation and at low RH where the role of amorphous states must be considered. Here, we present micro-Raman measurements of the kinetics of water transport between the bulk of a particle and the surrounding gas phase by examining the rate of exchange of D(2)O by H(2)O in droplets initially composed of MgSO(4)/D(2)O. The formation of an amorphous gel inhibits the response of the droplet composition to changes in the ambient RH and leads to a substantial reduction of the mass transfer rate of water in the droplet bulk with an apparent diffusion constant of 10(-15) to 10(-14) m(2) s(-1). These measurements are consistent with the imposition of a kinetic limitation on the time response of the aerosol particle size to changes in RH.

In-situ confocal Raman observation of structural changes of insulin crystals in sequential dehydration process.

In-situ confocal Raman spectroscopy combined with relative humidity (RH) control technique was used to study the sequential dehydration process of insulin crystals. By gradually decreasing the ambient RH of the insulin crystal, the content of the hydration water in the crystal was quantitatively controlled. Tyrosine (Tyr) residues were very sensitive to the micro-environmental changes, and four Raman features 828cm(-1), 852cm(-1), 1174cm(-1) and 1206cm(-1) of Tyr were employed to monitor the dehydration process. Taking advantage of the ratios I(852)/I(828) at different RH values, the mole fractions of the 'exposed' and 'buried' Tyr residues were estimated. Moreover, using the ratio I(1174)/I(1206) as an indicator of the dehydration process, three RH regions were discriminated. This is believed to imply that different types of the hydration water were lost step by step, i.e. firstly the 'second-layer' and 'first-layer' classes, then the 'contact' class, and finally, the 'inside' class. In addition, the profile of the amide I band was observed to gradually change with RH. By band fitting of the amide I region, changes in secondary structure were quantitatively determined. And the results showed that nearly 17% of α-helix converted into ß-sheet with RH decreasing from 92% to 2%.

The metabolites of mangrove endophytic fungus Zh6-B1 from the South China Sea.

Two new metabolites, 3R,5R-Sonnerlactone (1) and 3R,5S-Sonnerlactone (2), were isolated from the mangrove endophytic fungus Zh6-B1 obtained from the South China Sea. Their structures were elucidated by MS and NMR. The absolute configuration of compound 1 was determined by single-crystal X-ray analysis using Cu Kalpha radiation. The absolute configuration of compound 2 was determined by NOESY analysis and comparing circular dichroism spectroscopy with compound 1. The antiproliferative activity of compound 1 and 2 against the multi-drug resistant human oral floor carcinoma cells (KV) was evaluated.