Design and synthesis of forsythin derivatives as anti-inflammatory agents for acute lung injury.

Acute lung injury (ALI) is a clinically high mortality disease, which has not yet been effectively treated. The development of anti-ALI drugs is imminent. ALI can be effectively treated by inhibiting the inflammatory cascade and reducing the inflammatory response in the lung. Forsythia suspense is a common Chinese herbal medicine with significant anti-inflammatory activity. Using forsythin as the parent, 27 Forsythin derivatives were designed and synthesized, and the anti-AIL activity of these compounds was evaluated. Among them, compound B5 has the best activity to inhibit the release of IL-6, and the inhibition rate reaches 91.79% at 25 µM, which was 7.5 times that of the parent forsythin. In addition, most of the compounds have no significant cytotoxicity in vitro. Further studies showed that compound B5 had a concentration-dependent inhibitory effect on NO, IL-6 and TNF-α. And the IC50 values of compound B5 for NO and IL-6 are 10.88 µM and 4.93 µM, respectively. We also found that B5 could significantly inhibit the expression of some immune-related cytotoxic factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, B5 inhibits NF-κB/MAPK signaling pathway. In vivo experiments showed that B5 could alleviate lung inflammation in LPS-induced ALI mice and inhibit IL-6, TNF-α, COX-2 and iNOS. In summary, B5 has anti-inflammatory effects and alleviates ALI by regulating inflammatory mediators and inhibiting MAPK and NF-κB signaling pathways.

Safety, tolerability and pharmacokinetics of forsythin in healthy subjects: a double-blinded, placebo-controlled single-dose and multiple-dose escalation and food effect study.

BACKGROUND: Forsythin, an active compound from Forsythiae Fructus, has the potential to treat the common cold and influenza through its antipyretic-analgesic, anti-inflammatory and antiviral effects. The safety, tolerability and pharmacokinetic (PK) profile of forsythin were evaluated in healthy Chinese subjects. METHODS: This phase 1a study included three parts: double-blind, randomized, placebo-controlled single-ascending-dose (SAD) (50, 100, 200, 400, 600 or 800 mg), food effect investigation (100 mg) and multiple-ascending-dose (MAD) (50, 100 or 200 mg TID for 5 days). RESULTS: Forsythin is safe and tolerable in healthy Chinese subjects. The rates of adverse events (AEs) in the forsythin cohort were similar to those in the placebo cohort. Forsythin is well-absorbed after single or multiple doses and is extensively metabolized. The primary metabolites were aglycone M1, M1 sulphate (M2) and M1 glucuronide (M7). Exposure to forsythin (100 mg) was higher after food intake by approximately 1.4-fold, whereas M2 and M7 did not change. The steady state was reached around three days in the MAD study. Forsythin, M2 and M7 accumulation on day 5 was 1, 3 and 2, respectively. CONCLUSIONS: The safety and PK profiles of forsythin support further evaluation of its efficacy in individuals with the common cold or influenza.

Highly hygroscopicity and antioxidant nanofibrous dressing base on alginate for accelerating wound healing.

The removal of bacterium and free radicals is important for wound healing. Therefore, it is necessary to prepare biological dressings with antibacterial and antioxidant properties. In this study, high-performance calcium alginate/carbon polymer dots/forsythin composite nanofibrous membrane (CA/CPDs/FT) was explored under the influence of carbon polymer dots and forsythin. The addition of carbon polymer dots improved the nanofiber morphology and therefore enhanced the mechanical strength of the composite membrane. Moreover, CA/CPDs/FT membranes displayed satisfactory antibacterial and antioxidant properties because of the natural properties of forsythin. Meanwhile, outstanding hygroscopicity over 700% was also obtained for the composite membrane. In vitro and in vivo experiments showed that the CA/CPDs/FT nanofibrous membrane could prevent the invasion of bacteria, scavenge free radicals, and promote wound healing. Moreover, its good hygroscopicity and antioxidation characteristics were friendly for the clinical application of high-exudate wounds.

Forsythin inhibits ß-hydroxybutyrate-induced oxidative stress in bovine macrophages by regulating p38/ERK, PI3K/Akt, and Nrf2/HO-1 signaling pathways.

Ketosis is a metabolic disease of dairy cows in the perinatal period, ß-hydroxybutyrate (ß-HB) is the main component of ketosis. High levels of ß-HB can trigger oxidative stress and inflammatory response in dairy cows, leading to decreased milk yield and multiple postpartum diseases. Forsythin (FOR), the major constituent of the herbal medicine Forsythia, has anti-inflammatory, anti-oxidant, and antiviral effects. FOR was demonstrated to have an antioxidant effect on PC12 cells. However, the effects of FOR on ß-HB-stimulated bovine macrophages (BMs) has not been reported. Thus, the aim of the present study was to investigate the effects of FOR on ß-HB-stimulated BMs. Firstly, the CCK8 test confirmed that FOR (50, 100, 200 µg/mL) has no effect on BMs activity, and we selected these concentrations for subsequent experiments. Secondly, through detecting the oxidation indexes ROS, MDA and antioxidant indexes CAT and SOD, we confirmed the antioxidant effect of FOR on BMs. Next, qRT-PCR confirmed that FOR dramatically reduced the mRNA levels of IL-1ß and IL-6. Furthermore, the western blotting confirmed that FOR observably down-regulated ß-HB-stimulated phosphorylation of p38, ERK and Akt and up-regulated expression of Nrf2, and HO-1. Above results suggested that FOR plays antioxidant effects on ß-HB-induced BMs through p38, ERK and PI3K/Akt, Nrf2 and HO-1 signaling pathways. Therefore, we speculated that FOR may be a potential medicine to alleviate ß-HB-induced inflammatory response and provide a preliminary reference for the research and development of FOR.

Qualitative and quantitative determination of the primary active components and metabolites in human plasma after oral administration of Shuanghuanglian liquid.

Shuanghuanglian oral liquid is a common traditional Chinese medicine used to treat respiratory tract infections. Its major components are baicalin, chlorogenic acid, and forsythin. In this study, the main drug-related components in human plasma after oral administration of Shuanghuanglian were initially identified using ultra-performance liquid chromatography-ultraviolet detector/quadrupole time-of-flight mass spectrometry. Thirteen components from baicalin were identified, including the parent drug baicalin and aglycone baicalein. Only one metabolite related to chlorogenic acid, a sulfate conjugate formed after hydrolysis, and one metabolite related to forsythin, a sulfate conjugate of forsythin aglycone, were detected. Subsequently, a liquid chromatography-tandem mass spectrometry method was established and validated to simultaneously determine baicalin and baicalein, the primary active components. After simple protein precipitation, the analytes were separated on a BEH C18 column using a 5 min-gradient elution to avoid interference from baicalin isomers and their in-source dissociation. Excellent linearity was observed over the concentration ranges of 5.00-2000 ng/ml for baicalin and 1.00-100 ng/ml for baicalein. The validated method was successfully applied to a pharmacokinetic study of an oral administration of 60 ml Shuanghuanglian in healthy subjects. This study provided a foundation to investigate the clinical efficacy and safety of Shuanghuanglian further.

Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry.

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC-MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00-1000 ng/mL for forsythin, 2.50-2500 ng/mL for KD-2-Glc, and 5.00-5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC-MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.

Sulfation predominates the pharmacokinetics, metabolism, and excretion of forsythin in humans: major enzymes and transporters identified.

Forsythin extracted from Forsythiae Fructus is widely used to treat fever caused by the common cold or influenza in China, Japan and Korea. The present study aimed to analyze the pharmacokinetics, metabolism and excretion routes of forsythin in humans and determine the major enzymes and transporters involved in these processes. After a single oral administration, forsythin underwent extensive metabolism via hydrolysis and further sulfation. In total, 3 of the 13 metabolites were confirmed by comparison to reference substances, i.e., aglycone M1, M1 sulfate (M2), and M1 glucuronide (M7). Hydrolysis was the initial and main metabolic pathway of the parent compound, followed by extensive sulfation to form M2 and a reduced level of glucuronidation to form M7. In addition, the plasma exposure of M2 and M7 were 86- and 4.2-fold higher than that of forsythin. Within 48 h, ~75.1% of the administered dose was found in urine, with M2 accounting for 71.6%. Further phenotyping experiments revealed that sulfotransferase 1A1 and UDP-glucuronosyltransferase 1A8 were the most active hepatic enzymes involved in the formation of M2 and M7, respectively. The in vitro kinetic study provided direct evidence that M1 showed a preference for sulfation. Sulfated conjugate M2 was identified as a specific substrate of organic anion transporter 3, which could facilitate the renal excretion of M2. Altogether, our study demonstrated that sulfation dominated the metabolism and pharmacokinetics of forsythin, while the sulfate conjugate was excreted mainly in the urine.

Effect of forsythin in inhibiting renal cell carcinoma via PI3K/Akt signaling pathway / 中草药

Objective: To investigate the mechanism of forsythin in inhibiting the growth, migration and invasion of human renal cancer cells (786-0). Methods Human renal cancer cells (786-0) were cultured in vitro, and different concentrations of forsythin were added. Cell viability was detected by MTT assay, cell apoptosis was detected by AO/EB assay, cell migration and invasion abilities were respectively investigated by wound healing and transwell migration assays. The expression levels of PI3K, p-PI3K, Akt, p-Akt, FOXO3a, p-FOXO3a, p21, p27, Fasl, Bim, MMP-2, and MMP-9 were detected by Western bloting. Results:In 786-0 cells, forsythin effectively inhibited the growth of renal cancer cells, promoted apoptosis, interfered with cell cycle, and upregulated expression levels of p21, p27, Fasl, and Bim, when compared with control group (P < 0.05, 0.01); Compared with the control group, different concentrations of forsythin can significantly inhibit the migration and invasion of renal cancer cells, and reduce the synthesis of MMP-2 and MMP-9 (P < 0.05, 0.01). Meanwhile, forsythin can significantly inhibit the phosphorylation of PI3K, Akt and FOXO3a in a concentration-dependent manner (P < 0.05, 0.01). Conclusion: Forsythin can regulate apoptosis and cell cycle, and inhibit the growth of renal cancer cells by down-regulating the PI3K/Akt signaling pathway. At the same time, forsythin can effectively inhibit the ability of renal cell migration and invasion through the PI3K/Akt pathway.

Study on Changes of Extraction Rates of Forsythiaside A and Forsythin in Forsythia suspense Compatible with Other Medicinal Materials of Menshi Huwei Formula before and after Decoction / 中国药房

OBJECTIVE： To investigate the changes of extraction rates of forsythiaside A and forsythin in Forsythia suspensa compatible with other medicinal material of Menshi huwei formula before and after decoction. METHODS： HPLC method was used to determine the extraction amounts and to calculate the extraction rates of forsythiaside A and forsythin in F. suspensa （5 g×7 doses）， F. suspensa （5 g×7 doses） compatible with Pinelliae rhizoma praeparatum cum zingibere et alumine （PRZA）， Menshi huwei formula [including 6 ingredients as F. suspense （5 g×7 doses）， PRZA] after decocted with water. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-0.2％ formic acid solution （gradient elution）. The detection wavelength was set at 278 nm， and the column temperature was 30 ℃. The flow rate was 1.0 mL/min. RESULTS： The linear range of forsythiaside A and forsythin were 0.61-6.1， 0.246-2.46 μg （r＝0.999 7， 0.999 9）， respectively； RSDs of precision， stability （within 20 h） and reproducibility tests were all lower than 2％ （n＝6）. Average recovery rates were 96.10％-99.37％ （RSD≤2.36％，n＝6） respectively. In F. suspensa， extraction rates of forsythiaside A and forsythin were 96.90％ and 66.67％. In F. suspensa compatible with PRZA， extraction rates of them were 101.61％ and 54.55％. In Menshi huwei formula， extraction rates of them were 98.39％ and 84.85％. CONCLUSIONS： After F. suspensa is compatible with PRZA， the extraction rates of forsythiaside A is increased while forsythin is decreased. After compatible with other medicinal material in Menshi huwei formula， extraction rates of both are increased slightly.

[Optimization of method for determination of Forsythia Fructus and discussion on the establishment of quality control standard for green (old) Forsythia Fructus].

In order to obtain the optimum method for content determination of Forsythia Fructus (FF), a variety methods for the sample preparation of FF were evaluated by the content determination methods of Chinese Pharmacopoeia. And an optimum method was screened and as follows: 30 times with 70% ethanol solution in ultrasonic extractor for half an hour. The method can achieve the best effect of simultaneously extracting forsythoside A and forsythin. Then, a HPLC method for simultaneous determination of forsythoside A and forsythin was established by methodology. The HPLC chromatographic conditions: the mobile phase consisted of acetonitrile (A)-0.4% acetic acid solution (B) with gradient elution [0-33 min，15%A，33-43 min，15%-25%A，43-60 min，25% A] was at the flow rate of 1 mL·min⁻¹, the column temperature was 25 °C, and the detection wavelength was 330 and 277 nm. Moreover, the contents of forsythoside A and forsythin for 10 Green Forsythia Fructus (GF) and 5 Old Forsythia Fructus (OF) were determined by this method and Chinese Pharmacopoeia. The result not only displayed that the established method is effective, rapid, and simple, but also showed that the contents of forsythoside A and forsythin for GF and OF were significantly different. Which implied that the forsythoside A and forsythin limit standard for GF and OF should be controled by different values. This studies provide an important basis for the establishment of the content determination of FF and the quality control standard for GF and OF.

[Simultaneous determination of 9 components in Xiao'er Chiqiao Qingre granules by UPLC].

To establish a method for the simultaneous determination of 9 components, namely gardenoside, paeoniflorin, forsythoside A, baicalin, forsythin, glycyrrhizic acid, rhein, honokiol, and magnolol in Xiao'er Chiqiao Qingre granules（XECQ Gra）. Ultra performance liquid chromatography (UPLC) was used on an Acquity UPLC HSS T3 C18 column (2.1 mm×100 mm, 1.8 µm) with 0.1% phosphoric acid acetonitrile (A)-0.1% phosphoric acid solution (B) as mobile phase for gradient elution. The flow rate was 0.3 mL·min⁻¹ ; the column temperature was set at 30 °C, and the determination wavelength was set at 220 nm. All the 9 compounds were well separated, and showed good linear relationship within their concentrations (r>=0.999). The average recoveries were between 95.84%-101.4% and the RSD values were all less than 3.0%. The method is simple, reliable, and accurate, and could be used for the quality control of XECQ Gra.

Simultaneous determination of 9 components in Xiao'er Chiqiao Qingre granules by UPLC / 中国中药杂志

To establish a method for the simultaneous determination of 9 components, namely gardenoside, paeoniflorin, forsythoside A, baicalin, forsythin, glycyrrhizic acid, rhein, honokiol, and magnolol in Xiao'er Chiqiao Qingre granules（XECQ Gra）. Ultra performance liquid chromatography (UPLC) was used on an Acquity UPLC HSS T3 C₁₈ column (2.1 mm×100 mm, 1.8 μm) with 0.1% phosphoric acid acetonitrile (A)-0.1% phosphoric acid solution (B) as mobile phase for gradient elution. The flow rate was 0.3 mL·min⁻¹ ; the column temperature was set at 30 °C, and the determination wavelength was set at 220 nm. All the 9 compounds were well separated, and showed good linear relationship within their concentrations (r>=0.999). The average recoveries were between 95.84%-101.4% and the RSD values were all less than 3.0%. The method is simple, reliable, and accurate, and could be used for the quality control of XECQ Gra.

Optimization of method for determination of Forsythia Fructus and discussion on the establishment of quality control standard for green (old) Forsythia Fructus / 中国中药杂志

In order to obtain the optimum method for content determination of Forsythia Fructus (FF), a variety methods for the sample preparation of FF were evaluated by the content determination methods of Chinese Pharmacopoeia. And an optimum method was screened and as follows: 30 times with 70% ethanol solution in ultrasonic extractor for half an hour. The method can achieve the best effect of simultaneously extracting forsythoside A and forsythin. Then, a HPLC method for simultaneous determination of forsythoside A and forsythin was established by methodology. The HPLC chromatographic conditions: the mobile phase consisted of acetonitrile (A)-0.4% acetic acid solution (B) with gradient elution [0-33 min，15%A，33-43 min，15%-25%A，43-60 min，25% A] was at the flow rate of 1 mL·min⁻¹, the column temperature was 25 °C, and the detection wavelength was 330 and 277 nm. Moreover, the contents of forsythoside A and forsythin for 10 Green Forsythia Fructus (GF) and 5 Old Forsythia Fructus (OF) were determined by this method and Chinese Pharmacopoeia. The result not only displayed that the established method is effective, rapid, and simple, but also showed that the contents of forsythoside A and forsythin for GF and OF were significantly different. Which implied that the forsythoside A and forsythin limit standard for GF and OF should be controled by different values. This studies provide an important basis for the establishment of the content determination of FF and the quality control standard for GF and OF.

Evaluating the safety of forsythin from Forsythia suspensa leaves by acute and sub-chronic oral administration in rodent models.

OBJECTIVE: To access the toxicity of forsythin from Forsythia suspensa leaves and evaluate its safety. METHODS: Acute toxicity was determined by oral administration of a single dose of 18100 mg/kg forsythin in NIH mice. Sub-chronic toxicity was evaluated by oral administration of several doses of forsythin for 30 days at does of 0, 540, 1620, and 6480 mg/kg in SD rats. RESULTS: In the acute toxicity study, mortality was not observed after 14 days. In addition, clinically relevant adverse effects, or variations in body weight or food consumption were not observed. Similarly, after 30 days in the sub-chronic toxicity study, no mortality or significant toxicological effects such as decreased food consumption, body weight, biochemical parameters and vital organs etc. were noticed. CONCLUSION: The results revealed that the forsythin from Forsythia suspensa leaves has low or no toxicity via oral administration, and therefore is suitable for further development and applications.

Evaluating the safety of forsythin from Forsythia suspensa leaves by acute and sub-chronic oral administration in rodent models

Objective To access the toxicity of forsythin from Forsythia suspensa leaves and evaluate its safety. Methods Acute toxicity was determined by oral administration of a single dose of 18 100 mg/kg forsythin in NIH mice. Sub-chronic toxicity was evaluated by oral administration of several doses of forsythin for 30 days at does of 0, 540, 1 620, and 6 480 mg/kg in SD rats. Results In the acute toxicity study, mortality was not observed after 14 days. In addition, clinically relevant adverse effects, or variations in body weight or food consumption were not observed. Similarly, after 30 days in the sub-chronic toxicity study, no mortality or significant toxicological effects such as decreased food consumption, body weight, biochemical parameters and vital organs etc. were noticed. Conclusion The results revealed that the forsythin from Forsythia suspensa leaves has low or no toxicity via oral administration, and therefore is suitable for further development and applications.

Evaluating the safety of forsythin from Forsythia suspensa leaves by acute and sub-chronic oral administration in rodent models

OBJECTIVE@#To access the toxicity of forsythin from Forsythia suspensa leaves and evaluate its safety.@*METHODS@#Acute toxicity was determined by oral administration of a single dose of 18100 mg/kg forsythin in NIH mice. Sub-chronic toxicity was evaluated by oral administration of several doses of forsythin for 30 days at does of 0, 540, 1620, and 6480 mg/kg in SD rats.@*RESULTS@#In the acute toxicity study, mortality was not observed after 14 days. In addition, clinically relevant adverse effects, or variations in body weight or food consumption were not observed. Similarly, after 30 days in the sub-chronic toxicity study, no mortality or significant toxicological effects such as decreased food consumption, body weight, biochemical parameters and vital organs etc. were noticed.@*CONCLUSION@#The results revealed that the forsythin from Forsythia suspensa leaves has low or no toxicity via oral administration, and therefore is suitable for further development and applications.

Content Determination of 3 Components in Sangju Ganmao Pills by HPLC-MS/MS / 中国药房

OBJECTIVE:To establish the method for simultaneous determinations of rutin,forsythin and platycodin D in Sangju ganmao pills.METHODS:HPLC-MS method was adopted.The determination was performed on Waters Atlantis C18 column with mobile phase consisted of acetonitrile-0.1％ formic acid (gradient elution) at the flow rate of 0.2 mL/min.The column temperature was set 35 ℃,and sample size was 10 μL.The ionization mode was electrospray ion,and the reaction mode was multi-reaction monitoring.By positive ion detection mode,the drying gas and nebuliser gas were all high purity nitrogen.The drying gas temperature was 270 ℃.The drying gas flow rate was 25 L/min.The sheath gas flow rate was 10 L/min.The capillary voltage was 4 500 V.The nozzle voltage was 2 000 V and the scanning time was 0.1 s.RESULTS:The linear range of rutin,forsythin and platycodin D were 0.010 82-2.164 μg/mL (r=0.999 7),0.010 18-2.036 μg/mL (r=0.999 4),0.010 27-2.054 μg/mL (r=0.999 7),respectively The limits of quantification were 1.250,0.260,2.720 ng/mL,and the limits of detection were 0.380,0.078,0.820 ng/mL.RSDs of precision,stability and reproducibility tests were all no more than 3.0％.The recoveries were 97.88％-99.88％ (RSD=0.72％,n=6),98.48％-103.13％ (RSD=1.91％,n=6),98.79％-101.41％ (RSD=1.05％,n=6).CONCLUSIONS:This method is simple,precise,stable and reproducible,and can be used for simultaneous determination of rutin,forsythin and platycodin D in Sangju ganmao pills.

Kegan Liyan oral liquid ameliorates lipopolysaccharide-induced acute lung injury through inhibition of TLR4-mediated NF-κB signaling pathway and MMP-9 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Kegan Liyan oral liquid (KGLY), a Chinese prescription modified from classic formulas Yin-Qiao-San (from TCM classic Wenbing Tiaobian) and Shen-Jie-San (first mentioned in Shanghan Wenyi Tiaobian), has been reported to exert heat-clearing and detoxifying effects and used extensively for the treatment of severe pulmonary diseases in clinics including influenza, cough and pneumonia. AIM OF THIS STUDY: The purpose of this study was to investigate the protective effect of KGLY on lipopolysaccharide (LPS) induced acute lung injury (ALI) in mice and to illuminate the underlying mechanisms. MATERIALS AND METHODS: Mice were orally administrated with KGLY (50, 100 and 150mg/kg) before intratracheal instillation of LPS. 24h post LPS challenge, lung tissues and the bronchoalveolar lavage fluid (BALF) were collected for lung wet/dry (W/D) weight ratio, histopathological examinations and biochemical analyses. The cell counts, protein concentration, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2) in BALF, superoxide dismutase (SOD), glutathione (GSH), myeloperoxidase (MPO) and malondialdehyde (MDA) levels were detected. Meanwhile, the activation of toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), as well as matrix metalloproteinases 9 (MMP-9) were determined by western blot assay. RESULTS: KGLY significantly prolonged mice survival time and ameliorated LPS-induced edema, thickening of alveolar septa and inflammatory cell infiltration in a dose-dependent manner. Additionally, KGLY markedly attenuated LPS-induced acute pulmonary inflammation via decreasing the expressions of cytokines and chemokines (IL-1ß, IL-6, TNF-α, and MIP-2), enhanced the activities of anti-oxidative indicators (SOD and GSH), suppressed the levels of MPO and MDA, and down-regulated the expressions of TLR4, NF-κB and MMP9. CONCLUSIONS: The results suggested that the relieving effect of KGLY against LPS-induced ALI might be partially due to suppression of oxidative stress and inflammatory response, inhibition of TLR4-mediated NF-κB activation, and down-regulation of MMP9 expression, indicating it may be a potential therapeutic agent for ALI.

Gan-Lu-Siao-Du-yin, a prescription of traditional Chinese medicine, inhibited enterovirus 71 replication, translation, and virus-induced cell apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gan-Lu-Siao-Du-yin (GLSDY) is a prescription of traditional Chinese medicine. GLSDY contains 11 ingredients and is commonly used for endemic diseases. Enterovirus 71 (EV71) is an endemic disease that can cause meningoencephalitis with mortality and neurologic sequelae without any effective management. It is unknown whether GLSDY is effective against EV71 infection. AIM OF THE STUDY: To test the hypothesis that GLSDY can protect cell from EV71-induced injury. MATERIALS AND METHODS: Effects of a hot water extract of GLSDY on EV71 were tested in human foreskin fibroblast cells (CCFS-1/KMC) and human rhabdomyosarcoma cells (RD cells) by plaque reduction assay and flow cytometry respectively. Inhibition of viral replication was further examined by reverse quantitative RT-PCR (qRT-PCR). Its effect on viral protein translation and virus-induced apoptosis were examined by western blot. RESULTS: GLSDY was dose-dependently effective against EV71 infection (p<0.0001) in both CCFS-1/KMC cells and RD cells. GLSDY was highly effective when supplemented after viral inoculation (P<0.0001) with an IC50 of 8.7µg/mL. GLSDY inhibited viral RNA replication (P<0.0001), formation of viral structural proteins (VP0, VP1, VP2 and VP3) and non-structural proteins (protease 2B and 3AB). Furthermore, 300µg/mL GLSDY is effective to inhibit virus-induced apoptosis possibly through direct inhibition of caspase-8 and indirectly by inhibition of Bax. CONCLUSIONS: GLSDY is cheap and readily available to manage EV71 infection by inhibiting viral replication, viral protein formations, and EV71-induced apoptosis.

Simultaneous Determination of Five Active Components in Sijiganmao Tablets by HPLC / 中国药师

Objective: To develop an HPLC method for the simultaneous determination of five active components ( 5-O-methyl-visammioside, prim-O-glucosycimifugin, hesperidin, rosmarinic acid and forsythin) in Sijiganmao tablets. Methods:The samples were separated on an Agilent C18 column (250 mm × 4. 6 mm,5 μm) by acetonitrile-0. 1% formic acid solution with gradient elution. The detection wavelength was 283nm,the flow rate was 1. 0μl·min-1 ,the column temperature was 30℃,the detection wavelength was 283 nm,and the sample size was 10μl. Results:The complete separation was obtained for the five active compounds. The five regression e-quations showed good linear relationships. The average recoveries of the compounds were between 95. 0% and 105. 0%. Conclusion:The established method is accurate, reliable, simple and effective, which can be used in the quality control of Sijiganmao tablets.