Synthesis and Biological Evaluation of Gentiopicroside Derivatives as Novel Cyclooxygenase-2 Inhibitors with Anti-Inflammatory Activity.

Purpose: Nonsteroidal anti-inflammatory drugs cause a series of adverse reactions. Thus, the search for new cyclooxygenase-2 selective inhibitors have become the main direction of research on anti-inflammatory drugs. Gentiopicroside is a novel selective inhibitor of cyclooxygenase-2 from Chinese herbal medicine. However, it is highly hydrophilic owing to the presence of the sugar fragment in its structure that reduces its oral bioavailability and limits efficacy. This study aimed to design and synthesize novel cyclooxygenase-2 inhibitors by modifying gentiopicroside structure and reducing its polarity. Materials and Methods: We introduced hydrophobic acyl chloride into the gentiopicroside structure to reduce its hydrophilicity and obtained some new derivatives. Their in vitro anti-inflammatory activities were evaluated against NO, TNF-α, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Molecular docking predicted that whether new compounds could effectively bind to target protein cyclooxygenase-2. The inhibitory activity of new compounds to cyclooxygenase-2 enzyme were verified by the in vitro experiment. Results: A total of 21 novel derivatives were synthesized, and exhibit lower polarities than the gentiopicroside. Most compounds have good in vitro anti-inflammatory activity. The in vivo activity results demonstrated that 8 compounds were more active than gentiopicroside. The inhibition rate of some compounds was higher than celecoxib. Molecular docking predicted that 6 compounds could bind to cyclooxygenase-2 and had high docking scores in accordance with their potency of the anti-inflammatory activity. The confirmatory experiment proved that these 6 compounds had significant inhibitory effect against cyclooxygenase-2 enzyme. Structure-activity relationship analysis presumed that the para-substitution with the electron-withdrawing groups may benefit the anti-inflammatory activity. Conclusion: These gentiopicroside derivatives especially PL-2, PL-7 and PL-8 may represent a novel class of cyclooxygenase-2 inhibitors and could thus be developed as new anti-inflammatory agents.

Synthesis, and anti-inflammatory activities of gentiopicroside derivatives.

A series of 26 novel derivatives have been synthesized through structural modification of gentiopicroside, a lead COX-2 inhibitor. And their in vivo and in vitro anti-inflammatory activities have been investigated. The in vitro anti-inflammatory activities were evaluated against NO, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by LPS. Results showed that most compounds had good inhibitory activity. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Results demonstrated that several compounds were more active than the parent compound gentiopicroside. The inhibition rate of the most active compound P23 (57.26%) was higher than positive control drug celecoxib (46.05%) at dose 0.28 mmol·kg-1. Molecular docking suggested that these compounds might bind to COX-2 and iNOS. Some of them, e.g P7, P14, P16, P21, P23, and P24, had high docking scores in accordance with their potency of the anti-inflammatory activitiy, that downregulation of the inflammatory factors, NO, PGE2, and IL-6, was possibly associated with the suppression of iNOS and COX-2. Therefore, these gentiopicroside derivatives may represent a novel class of COX-2 and iNOS inhibitors.

[Review of chemical constituents,pharmacological effects and clinical applications of Danggui Buxue Decoction and prediction and analysis of its Q-markers].

Danggui Buxue Decoction is a classic prescription of Qi and blood tonification, which is mainly applied in treating fatigue, internal damage Qi weakness, blood deficiency, and outward going of floating Yang. Modern pharmacology shows that it can promote hematopoiesis, regulate immunity, and protect heart and cerebral vessels. The prescription, often used for the treatment of anemia and other diseases in clinic, is composed of Astragali Radix and Angelicae Sinensis Radix at a dosage ratio of 5∶1. It is a modern compound prescription for invigorating Qi and generating blood. Based on the review of the chemical constituents, pharmacological effects, and clinical applications of Danggui Buxue Decoction, its Q-marker was predicted and analyzed according to the "five principles" of Chinese medicine Q-marker--quality transmissibility and traceability, ingredient specificity, component validity, component measurabi-lity, and formula compatibility environment. The results suggested that calycosin, calycosin-7-O-ß-D-glucoside, formononetin, ononin, astragaloside A, ferulic acid, and ligustilide could be used as Q-markers of Danggui Buxue Decoction, which provides reference for establishing the quality system of Danggui Buxue Decoction.

[Review of chemical constituents, pharmacological effects and clinical applications of Suanzaoren Decoction and prediction and analysis of its Q-markers].

Suanzaoren Decoction is a classic prescription for nourishing the heart and liver, nourishing blood and tranquilizing the mind. It has the functions of sedation and hypnosis, anti-anxiety, anti-depression, anti-convulsion and so on. Modern clinic is mostly used to treat different types of insomnia, depression, neurasthenia, tension headache and vertigo. In this paper, the chemical consti-tuents, pharmacological effects and clinical application of Suanzaoren Decoction are reviewed. Based on this, the quality marker(Q-marker) of Suanzaoren Decoction was predicted and analyzed according to the "five principles" of Q-marker of traditional Chinese medicine--transmission and traceability, specificity, effectiveness, measurability and compatibility environment of compound prescriptions. The results indicated that jujuboside, spinosin, ferulic acid, senkyunolide Ⅰ, sarsasapogenin, mangiferin, liquiritoside and glycyrrhizic acid were predicted and analyzed, and those can be used as Q-markers of Suanzaoren Decoction. Subsequently, the above components can be selected as indicators to control and evaluate the quality of Suanzaoren Decoction and its preparations, and establish a quality traceability system.

Spectrum-effect relationship between HPLC fingerprints and antioxidant activity of Angelica sinensis.

Angelica sinensis is one of the most important traditional Chinese medicines and has antioxidant activities that greatly contribute to its pharmacological action. However, the compounds responsible for its antioxidant activity remain unknown. In this study, the fingerprints of 10 batches of A. sinensis collected from different locations in China were established with HPLC to identify the common peaks. The antioxidant activities of these 10 batches were evaluated with 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and ferric-reducing antioxidant power assays. The spectrum-effect relationship between HPLC fingerprints and antioxidant effect of A. sinensis was examined by the partial-least-square regression analysis and the variable importance in projection method. Results showed that the antioxidant effect of A. sinensis results from the synergistic effect of various compounds, and peaks X3 and X7-X18 were the main substances responsible for antioxidant efficacy. This study successfully identified the spectrum-effect relationship between HPLC fingerprints and the antioxidant effect of A. sinensis. This relationship can provide methods for establishing the quality standards for A. sinensis and developing new and effective products of A. sinensis based on its antioxidant ingredients.

[The therapeutic effects of Yougui pill on knee osteoarthritis and the expression of Wnt signal pathway related factors in rats].

OBJECTIVES: To observe the effects of Yougui pill (Traditional Chinese Medicine) on the related factors of Wnt signal pathway of rats with knee osteoarthritis (KOA), and explore its protective mechanism. METHODS: Sixty SPF SD rats were randomly divided into the sham-operative group, model group, glucosamine sulfate group, high-dose, middle-dose, low-dose of Yougui pill treated group (n=10). KOA model was established by modified Hulth method for six weeks. The rats in the high, middle and low-dose of Yougui pill group were treated with Yougui pills at the doses of 20,10 and 5 g/kg respectively by gastrogavage once a day for 8 weeks, while equal volume of normal saline was given to those in the sham and model control group and an equal volume of glucosamine sulfate (1.7 g/kg·d) was given to those in glucosamine sulfate group for 8 weeks. The knee joint was removed after the last dose of drug. The pathological changes of cartilaginous tissues were observed under a microscope. The mRNA levels of Dickkopf homolog 1(DKK1), Wnt induced secreted protein 1(WISP1), Wnt1, low density lipoprotein receptor related protein 5(LRP5) and beta -catenin in rats cartilaginous tissues were analyzed by using RT-PCR method, and the protein contents of DKK1, WISP1, Wnt1, LRP5 and beta-catenin in cartilaginous tissues were detected by Western blot. RESULTS: Compared with the sham group, the articular cartilage was severely damaged, the Mankin score was increased significantly (P<0. 05), the mRNA and protein expression levels of DKK1 in cartilaginous tissue were markedly decreased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were increased significantly in model group(P<0.05). Compared with model group, the articular cartilage lesions was light (P<0.05), the Mankin Score was decreased significantly(P<0.05), and the mRNA and protein levels of DKK1 in cartilaginous tissue were increased(P<0.05), while those of WISP, Wnt1, LRP5 and beta-catenin were decreased in Yougui pill high-dose group and glucosamine sulfate group (P<0.05). CONCLUSIONS: Yougui pill has protective effects on the KOA by inhibiting the expressions of WISP, Wnt1, LRP5, beta-catenin and increasing the expression of DKK1 cytokine in the Wnt signaling pathway.

Gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, Gentiana macrophylla Pall have been prescribed for the treatment of pain and inflammatory conditions. In addition, it is a common Tibetan medicinal herb used for the treatment of tonsillitis, urticaria, and rheumatoid arthritis (RA), while the flowers of G. macrophylla Pall have been traditionally treated as an anti-inflammatory agent to clear heat in Mongolian medicine. The secoiridoid glycosides and their derivatives are the primary active components of G. macrophylla and have been demonstrated to be effective as anti-inflammatory agents. MATERIALS AND METHODS: Solvent extraction and D101 macroporous resin columns were employed to concentratethe gentiopicroside. Gentiopicroside cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the toxicity of gentiopicroside in chondrocytes was reconfirmed using Hoechst staining. Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were utilized to explore the protective effects and mechanisms of gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte. RESULTS: The MTT assay demonstrated that 50, 500, and 1,500 µg/mL of gentiopicroside exhibited no significant toxicity to chondrocytes (P>0.05) after 24h. Using immunohistochemistry, ELISA, RT-PCR, Western blot method to explore the protective effect and mechanism of gentiopicroside on chondrocytes induced by IL-1ß. The results showed some pathways of IL-1ß signal transduction were inhibited by gentiopicroside in rat chondrocytes: p38, ERK and JNK. Meanwhile, gentiopicroside showed inhibition in the IL-1ß-induced release of MMPs while increasing Collagen type II expression. CONCLUSIONS: The current study demonstrated that gentiopicroside exhibited a potent protective effect on IL-1ß induced inflammation response in rat articular chondrocyte. Thus, gentiopicroside could be a potential therapeutic strategy for treatment of OA.

[Optimation the extracting technology of Angelica sinensis by central composite design and response surface methodology].

OBJECTIVE: To optimize the extracting technology from Angelica sinensis by central composite design-response surface methodology. METHODS: On the basis of the single factor,independent variables were ethanol concentrations, solvent ratio and ultrasonic time, while dependent variable was the OD value of extraction rates of ferulic acid and liqustilide. A two-order polynomial equation was fitted to estimate the relationship between independent and dependent variables. Response surface method was used to optimize the extracting process. RESULTS: The optimum extraction conditions for Angelica sinesis were obtained as follows: the extracting solvent was methanol concentrations of 70%, 30 fold solvent, extracting for once and for 40 minutes. The deviation between observed and predicted values was 1.23%. CONCLUSION: The result indicates that the central composite design and response surface methodology is simple, convenient and reliable for optimizing the extraction process of Angelica sinesis with higher precision.

[Study on quality evaluation of Angelica sinensis by grey incidence degree method].

OBJECTIVE: To evaluate the quality of Angelica sinensis based on Grey incidence degree method. METHODS: Grey model was set up by determining four main compositions contained in the samples. RESULTS: The result of quality evaluation on 21 samples by this model was as same as that of genuine medicinal materials. CONCLUSION: Grey incidence degree method and the model can be used to evaluate the quality of Angelica sinensis.

[Chemical constituents from leaves of Sterculia foetida].

OBJECTIVE: To study the chemical constituents from the leaves of Sterculia foetida. METHOD: Compounds were isolated by chromatographic techniques. Their structures were elucidated by spectroscopic methods. RESULT: Eight compounds were identified as 5,7,8-tetrahydroxy-4'-methoxyflavone-8-O-beta-D-glucoside (1), 5,7,8-tetrahydroxy-4'-methoxyflavone-7-O-beta-D-glucoside (2), quercetin-3-O-beta-D-glucoside (3), apigenin-6, 8-di-C-beta-D-glucoside (4), puerarin (5), 5,7,8,3'-tetrahydroxy-4'-methoxyflavone (6), 5,7,8-tetrahydroxy-3',4'-dimethoxyflavone (7), 5,7,8-tetrahydroxy-4'-methoxyflavone (8). CONCLUSION: Compounds 1, 2 and 4-8 were isolated from this plant for the first time.

Two flavonoid glycosides and a phenylpropanoid glucose ester from the leaves of Sterculia foetida.

Two new flavonoid glycosides, hypolaetin 4'-methyl ether 8-O-beta-d-glucuronide 2''-sulfate (1) and hypolaetin 4'-methyl ether 3'-O-beta-d-glucoside (2), and a new phenylpropanoid glucose ester, 1,6-diferuloyl glucose (3), were isolated from the leaves of Sterculia foetida L. Their structures were elucidated by spectroscopic analysis and chemical evidence.

[Chemical constituents from flowers of Carthamus tinctorius].

OBJECTIVE: To study the chemical constituents from the flowers of Carthamus tinctorius. METHOD: Compounds were isolated by chromatographic techniques. Their structures were elucidated by spectral methods. RESULT: Ten compounds were identified as 7,8-dimethylpyrazino [2,3-g] quinazolin-2, 4-(1H, 3H) -dione (1), adenosine (2), adenine (3), uridine (4), thymine (5), uracil (6), roseoside (7), 4'-O-dihydrophaseic acid-beta-D-glucopyranoside methylester (8), 4-O-beta-D-glucopyranosyloxy-benzoic acid (9) and p-hydroxybenzoic acid (10). CONCLUSION: Compounds 1 and 8 were isolated from natural plants for the first time, and compounds 7, 9 and 10 were isolated from this plant for the first time.

[Preparation and evaluation of sustained-release microsphere of Sanguis Draconis in vitro].

OBJECTIVE: To prepare sustained-release microsphere containing extract of Sanguis Draconis and to measure its dissolution in vitro. METHOD: Sustained-release microsphere was prepared with polylactic acid (PLA) as carriers using the oil-in-water (O/W) emulsion solvent evaporation method. The powder particle's characteristics of sustainded-release microsphere were evaluated comprehensively, and its dissolution characteristics in vitro were studied. RESULT: The microsphere was round and its surface was smooth, drug-loading rate was 21.97% and the entrapment rate was 55.76%, the accumulative release percentage was 76. 71% in 16 hours. CONCLUSION: The sustained release effect of Sanguis Draconis microspheres was formed with potentially wide applications.