Chromosome-scale genome assembly of Rhododendron molle provides insights into its evolution and terpenoid biosynthesis.

BACKGROUND: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. RESULTS: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-ß) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. CONCLUSIONS: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle.

A Novel Network Pharmacology Strategy to Decode Mechanism of Lang Chuang Wan in Treating Systemic Lupus Erythematosus.

Complex disease is a cascade process which is associated with functional abnormalities in multiple proteins and protein-protein interaction (PPI) networks. One drug one target has not been able to perfectly intervene complex diseases. Increasing evidences show that Chinese herb formula usually treats complex diseases in the form of multi-components and multi-targets. The key step to elucidate the underlying mechanism of formula in traditional Chinese medicine (TCM) is to optimize and capture the important components in the formula. At present, there are several formula optimization models based on network pharmacology has been proposed. Most of these models focus on the 2D/3D similarity of chemical structure of drug components and ignore the functional optimization space based on relationship between pathogenetic genes and drug targets. How to select the key group of effective components (KGEC) from the formula of TCM based on the optimal space which link pathogenic genes and drug targets is a bottleneck problem in network pharmacology. To address this issue, we designed a novel network pharmacological model, which takes Lang Chuang Wan (LCW) treatment of systemic lupus erythematosus (SLE) as the case. We used the weighted gene regulatory network and active components targets network to construct disease-targets-components network, after filtering through the network attribute degree, the optimization space and effective proteins were obtained. And then the KGEC was selected by using contribution index (CI) model based on knapsack algorithm. The results show that the enriched pathways of effective proteins we selected can cover 96% of the pathogenetic genes enriched pathways. After reverse analysis of effective proteins and optimization with CI index model, KGEC with 82 components were obtained, and 105 enriched pathways of KGEC targets were consistent with enriched pathways of pathogenic genes (80.15%). Finally, the key components in KGEC of LCW were evaluated by in vitro experiments. These results indicate that the proposed model with good accuracy in screening the KGEC in the formula of TCM, which provides reference for the optimization and mechanism analysis of the formula in TCM.

RhoA/Rock2/Limk1/cofilin1 pathway is involved in attenuation of neuronal dendritic spine loss by paeonol in the frontal cortex of D-galactose and aluminum­induced Alzheimer's disease­like rat model.

Alzheimer's disease (AD) has become the most prevalent neurodegenerative disorder. Given the pathogenesis of AD is unclear, there is currently no drug approved to halt or delay the progression of AD. Therefore, it is pressing to explore new targets and drugs for AD. In China, polyphenolic Chinese herbal medicine has been used for thousands of years in clinical application, and no toxic effects have been reported. In the present study, using D­galactose and aluminum­induced rat model, the effects of paeonol on AD were validated via the Morris water maze test, open field test, and elevated plus maze test. Neuronal morphology in frontal cortex was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. RhoA/Rock2/Limk1/cofilin1 signaling pathway­related molecules were determined by Western blotting. Cofilin1 and p­cofilin1 were analyzed by immunofluorescence. Results showed that pre­treatment with paeonol attenuated D­galactose and aluminum­induced behavioral dysfunction and AD­like pathological alterations in the frontal cortex. Accompanied by these changes were the alterations in the dendrite and dendritic spine densities, especially the mushroom­type and filopodia­type spines in the apical dendrites, as well as actin filaments. In addition, the activity and intracellular distribution of cofilin1 and the molecules RhoA/Rock2/Limk1 that regulate the signaling pathway for cofilin1 phosphorylation have also changed. Our data suggests that paeonol may be through reducing Aß levels to alleviate the loss of fibrillar actin and dendrites and dendritic spines via the Rho/Rock2/Limk1/cofilin1 signaling pathway in the frontal cortex, and ultimately improving AD­like behavior.

[New thoughts on material basis and mechanism of Yin-tonifying traditional Chinese medicine in treatment of diabetes mellitus and its complications].

Diabetes mellitus is a serious chronic metabolic disease, and the patient's hyperglycemia is often accompanied by complications. In the circles of medical science, traditional Chinese medicine(TCM) has the earliest knowledge and research about diabetes. According to TCM, the clinical characteristics of diabetes mellitus were basically the same as "Xiaoke". TCM also believes that "Yin deficiency and dryness heat" was the main pathogenesis of diabetes. Therefore, Yin-tonifying TCMs is widely used in clinical treatment of diabetes mellitus, including Dendrobii Caulis, Lilii Bulbus, Ophiopogonis Radix, Polygonati Rhizome. The effective component for treating diabetes in the above Chinese materia medica is polysaccharides, which is used to treat complications of diabetes mellitus, like vascular disease, nephropathy, retinopathy, peripheral neuropathy. According to literature reports, except for specific some Yin-tonifying TCMs with effective ingredients for preventing and treating diabetes, other Yin-tonifying TCMs only contain water, alcohol extracts or polysaccharides in the treatment of diabetes. However, due to unclear material basis, dose-effect relationship and mechanism target of hypoglycemic drugs, Yin-tonifying TCMs are restricted in clinical application, with certain difficulties in in-depth studies. In this paper, the literatures related to the treatment of diabetes mellitus and its complications with Yin-tonifying TCM are analyzed and summarized, the existing problems are analyzed, and the research ideas and methods based on chromatographic technology and metabonomics are put forward, in order to provide reference for the application and development of Yin-tonifying TCM.

Efficacy of Aeschynomene indica L. leaves for wound healing and isolation of active constituent.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, the aerial parts of Aeschynomene indica L. (AIL) have been used for wound healing, and to treat urinary tract infection, hepatitis, enteritis, dysentery, nyctalopia, conjunctivitis, urticaria, and furuncle. However, no scientific investigation has been conducted on its wound healing potential. AIM OF THE STUDY: To investigate the effects of AIL extract on wound healing, isolate the active constituent and reveal the possible mechanism of enhancing wound healing. MATERIALS AND METHODS: The circular excision wound healing model was used to evaluate in vivo wound-healing activity. Hematoxylin and eosin staining was applied to assess inflammatory cells infiltration, angiogenesis, fibroblast proliferation, collagen synthesis, collagen remodeling, and skin appendages generation. Sirius red-picric acid staining was employed for quantitative analysis of the ratio of collagen I/III. Immunohistochemical staining for CD68, CCR7 (CD197), CD163, TGF-ß1 and α-SMA was performed to determine macrophages phenotypes transition (M1-to-M2) and prove the scar-improving effect of AIL on wound healing. RESULTS: We successfully isolated the active constituent (Sub-Fr0.2) for wound healing from AIL extract, circular excision wound healing experiment and hematoxylin & eosin staining showed Sub-Fr0.2 has a significant promoting effect on wound healing. Results of sirius red-picric acid staining demonstrated a reduced ratio of collagen I/III in the Sub-Fr0.2 group as compared with the vehicle group. Immunohistochemical staining for CD68, CCR7 (CD197), and CD163 in the Sub-Fr0.2 group exhibited an elevated speed of macrophages transiting from M1 phenotype to M2 phenotype, when compared with the vehicle group. Besides, the expression of TGF-ß1 and α-SMA were inhibited on wounds treated with the ointment containing Sub-Fr0.2. CONCLUSION: Leaves of AIL and its active constituent (Sub-Fr0.2) effectively promoted wound healing and reduced scar formation, this efficacy might be exerted by accelerating macrophages phenotypes transition and inhibiting TGF-ß1 and α-SMA expression.

Novel derivative of Paeonol, Paeononlsilatie sodium, alleviates behavioral damage and hippocampal dendritic injury in Alzheimer's disease concurrent with cofilin1/phosphorylated-cofilin1 and RAC1/CDC42 alterations in rats.

Alzheimer's disease (AD) is a typical hippocampal amnesia and the most common senile dementia. Many studies suggest that cognitive impairments are more closely correlated with synaptic loss than the burden of amyloid deposits in AD progression. To date, there is no effective treatment for this disease. Paeonol has been widely employed in traditional Chinese medicine. This compound improves learning behavior in an animal model; however, the mechanism remains unclear. In this study, Paeononlsilatie sodium (Pa), a derivative of Paeonol, attenuated D-galactose (D-gal) and AlCl3-induced behavioral damages in rats based on evaluations of the open field test (OFT), elevated plus maze test (EPMT), and Morris water maze test (MWMT). Pa increased the dendritic complexity and the density of dendritic spines. Correlation analysis indicated that morphological changes in neuronal dendrites are closely correlated with behavioral changes. Pa treatment reduced the production of Aß, affected the phosphorylation and redistribution of cofilin1 and inhibited rod-like formation in hippocampal neurons. The induction of D-gal and AlCl3 promoted the expression of RAC1/CDC42 expression; however, the tendency of gene expression was inhibited by pretreatment with Pa. Taken together, our results suggest that Pa may represent a novel therapeutic agent for the improvement of cognitive and emotional behaviors and dendritic morphology in an AD animal model.

Improving 10-deacetylbaccatin III-10-ß-O-acetyltransferase catalytic fitness for Taxol production.

The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-ß-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-ß-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a ß-xylosidase to obtain an in vitro one-pot conversion of 7-ß-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml-1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.

[Research progress on chemical constituents, pharmacological mechanism and clinical application of Guizhi decoction].

Guizhi Decoction was one of the most commonly used traditional Chinese Medicine which possesses the effects of "jie-ji-fa-biao, regulating Ying and Wei". It was mainly used to treat mind-cold due to exogenous evils such as fever, headache, sweating, hate the wind, et al. Modern studies indicated that the chemical constituents of Guizhi decoction mainly include phenylpropanoid, monoterpenes, organic acids, flavonoids, triterpenoid saponins and so on. Pharmacological experimental studies had shown that Guizhi decoction could play a big role in dual-directional regulation on sweat gland, body temperature, immune function, gastrointestinal peristalsis, and blood pressure, and could also play the role of anti-inflammatory, antibacterial, antiviral, anti-allergic, analgesic, hypoglycemic, and cardiovascular protection. Many diseases such as internal, external, gynecological and pediatric diseases were treated in the clinical by using Guizhi decoction and its analogous formulae involving circulatory, immune, urinary, reproductive, endocrine, digestive, nervous and other systems. This article reviews the latest research progress of Guizhi decoction from three aspects: chemical constituents, pharmacological mechanism and clinical application. It will provide reference for further research and development of Guizhi decoction.

[Expression, subcellular localization and characterization of dammarenediol-â ¡ synthase of Panax ginseng in Saccharomyces cerevisiae].

To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2,3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g(-1) to 12.24 mg·g(-1). This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S.cerevisiae.

In vitro and in vivo antitumor activity of a novel porphyrin-based photosensitizer for photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is a promising treatment in cancer therapy, based on the use of a photosensitizer activated by visible light in the presence of oxygen. Nowadays significant research efforts have been focused on finding a new photosensitizer. In the present paper, the antitumor effects of a novel porphyrin-based photosensitizer, {Carboxymethyl-[2-(carboxymethyl-{[4-(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]-methyl}-amino)-ethyl]-amino}-acetic acid (ATPP-EDTA) on two types of human malignant tumor cells in vitro and a gastric cancer model in nude mice, were evaluated. METHODS: The PDT efficacy with ATPP-EDTA in vitro was assessed by MTT assay. The intracellular accumulation was detected with fluorescence spectrometer, and the intracellular distribution was determined by laser scanning confocal microscopy. The mode of cell death was investigated by Hoechst 33342 staining and flow cytometer. BGC823-derived xenograft tumor model was established to explore the in vivo antitumor effects of ATPP-EDTA. RESULTS: ATPP-EDTA exhibited intense phototoxicity on both cell lines in vitro in concentration- and light dose-dependent manners meanwhile imposing minimal dark cytotoxicity. The accumulation of ATPP-EDTA in two malignant cell lines was time-dependent and prior compared to normal cells. It was mainly localized at lysosomes, but induced cell death by apoptotic pathway. ATPP-EDTA significantly inhibited the growth of BGC823 tumors in nude mice (160 mW/cm(2), 100 J/cm(2)). CONCLUSIONS: Present studies suggest that ATPP-EDTA is an effective photosensitizer for PDT to tumors. It distributed in lysosomes and caused cell apoptosis. ATPP-EDTA, as a novel photosensitizer, has a great potential for human gastric cancer treatment in PDT and deserves further investigations.

Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea.

HPLC analysis of samples from four major fermentation procedures of Jing-Wei Fu brick tea showed that the level of major tea catechins epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) dropped increasingly to about 1/3 in the final product. Phytochemical study of the final product led to the discovery of four new B-ring fission metabolites of catechins (BRFCs) Fuzhuanin C-F (1-4) together with three known BRFCs (5-7), six known catechins (8-13), five simple phenols (14-18), seven flavones and flavone glycosides (19-25), two alkaloids (26, 27), three triterpenoids (28-30) and one steroid (31). The structures were elucidated by spectroscopic methods including 1D and 2D NMR, LC-HR-ESI-MS, IR, and CD spectra. Five compounds (16-18, 28, 29) were reported for the first time in tea. Possible pathways for the degradation of major tea catechins and the generation of BRFCs were also provided.

[The role of CYP2E1 in the protection of garlic oil's from n-hexane-induced neurotoxicity].

OBJECTIVE: To study the role of CYP2E1 in the protective effects and mechanism of garlic oil (GO) on the peripheral nerve injuries induced by n-hexane. METHODS: Fifty male Wistar rats were randomly divided into five groups (n = 10): the control, the GO (80 mg/kg) control, the n-hexane (2000 mg/kg) model, the low dose GO (40 mg/kg) plus n-hexane, and the high dose GO (80 mg/kg) plus n-hexane groups. All rats were treated by intragastric administration 6 times a week for 10 weeks. The gait scores were determined every two weeks for monitoring the peripheral neurotrosis. All rats were sacrificed in 10 weeks, the activities and expression levels of hepatic CYP2E1 and 2, 5-HD in serum were examined. RESULTS: As compared with control group, the content and activity of hepatic CYP2E1 in GO control group reduced by 83.1% and 48.3% respectively (P < 0.01), the content and activity of hepatic CYP2E1 in model group increased by 112.5% and 72.2% respectively (P < 0.01). As compared with model group, the contents of hepatic CYP2E1 in low dose and high dose GO groups reduced by 32.9% and 39.1% respectively, the activities of hepatic CYP2E1 in low dose and high dose GO groups reduced by 27.4% and 44.5% respectively (P < 0.01); the contents of serum 2,5-HD in low dose and high dose GO groups reduced by 47.7% and 78.7% respectively (P < 0.01). The gait scores in model, low dose and high dose GO groups were significantly lower than that in control group, but the gait scores in low dose and high dose GO groups were significantly lower than that in model group (P < 0.05). CONCLUSION: Garlic oil can effectively reduce the peripheral neurotrosis induced by n-hexane due to the decreased content and activity of hepatic CYP2E1, resulting in the reduced formation of 2, 5-HD from n-hexane.

[Protective effects of garlic oil on n-hexane-induced neurotoxicity in rats via inhibition of hepatic alcohol dehydrogenase activity].

OBJECTIVE: To study the protective effects of garlic oil (GO) on the peripheral nerve injuries induced by n-hexane. METHODS: Male Wistar rats were randomly divided into four groups (10 rats in each group): the control, the n-hexane treatment (2000 mg/kg), the low dose GO, and the high dose GO groups. The rats in the low and high doses of GO groups were pretreated with GO (40 and 80 mg/kg) before exposure to n-hexane (2000 mg/ kg), while the animals of the n-hexane treatment group were given normal saline and then 2000 mg/ kg n-hexane. The rats were exposed to GO and n-hexane 6 times a week for 10 weeks. The gait scores and staying time on the rotating rod for all rats were detected every two weeks. The rats were sacrificed at the end of ten weeks, then the levels of alcohol dehydrogenase (ADH), maleic dialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase(GSH-Px), total antioxidation capacity(T-AOC) and the ability of inhibition of *OH in livers were examined. RESULTS: The gait scores increased significantly and the time staying on the rotating rod obviously decreased in rats of n-hexane treatment group, as compared with control group (P < 0.05 or P < 0.01). In the hepatic tissues of n-hexane group, the levels of MDA and ADH significantly increased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously decreased, as compared to control group (P < 0.05 or P < 0.01). In 2 GO groups, the gait scores and the staying time on the rotating rod were significantly improved, the levels of MDA and ADH significantly decreased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously increased, as compared with n-hexane group (P < 0.05 or P < 0.01 ). CONCLUSION: ADH could play an important role in the protective effects induced by garlic oil on the peripheral nerve injuries produced by n-hexane.

[Analysis on resistance testing and technological research of acupoints].

In order to summarize and analyze the progresses and problems of resistance testing and technology research on acupoints, investigate the related solutions, articles on resistance testing from 1990 to 2011 were collected, and advantages and shortcomings of different types of testing instruments were discussed in this article as well. Detection technology of acupoint impedance has under updating changes, which are often applied on locating acupoints and clinical diagnosis. The progresses of those technologies are mainly embodied in improvement of circuit model, which enable a longer testing duration, less media interference and so on. The previous studies often focus on resistance testing of the skin. Since dermal resistance does not equal to acupoint resistance, the designation of equivalent circuit does not embody the condition of cells, tissues and nerves at acupoints, and testing technology is often affected by factors such as polarization of electrodes, contact medium and anatomical characteristics of acupoints, the resistance of acupoints can not be taken as the effective framework of the diagnostic standard and judgement of therapeutic effect in clinic. Based on the key problems in resistance testing of acupoints, it is proposed by the article that the techniques and designation of circuit model should be reformed, and mathematical model should be established in the study to meet the requirement of the life system. Inductance of human body should be considered in resistance research of acupoints, and interference factors should be eliminate as far as possible in designation of equipments. Thus, the change law of acupoint resistance in the inner part of the body can be fully embodied by the designation of experiments.

[Research prospect for sensing needle in Chinese medicine].

The domestic and abroad research literatures on sensing needle in Chinese medicine in recent years are retrieved. The design principle and application situation of calcium sensing needle, temperature sensing needle, pH sensing needle and oxygen partial pressure sensing needle are summarized. The findings suggest that the sensing needle can be used in the measurements of in vivo, location positioning, fixed-point, real time, and dynamic state with little or no damage to the human body. It can be used as a transducer or a device for treatment. This paper proposes that the sensing needle can be used in the measurements in clinical practices of western medicine; it will facilitate the development of Chinese and western integrative medicine.

[Purification of total flavones from Morus alba L.by macroporous adsorbents and kinetic model for the process].

OBJECTIVE: To establish the separation and purification technology for the total flavones from Morus alba L.(MTF), and present a kinetic model for this process. METHODS: Three types of macroporous adsorbents were tested to separate and purify MTF, and to evaluated by adsorption capacity, elution ratio and product purity. The curves of dynamic adsorption-elution process was plotted and the kinetic equations were presented. RESULT: Among three types of macroporous adsorbents, the NKA-9 type showed better property with the adsorption capacity of 43.4 mg.g(-1)and the elution ratio of 98.2%. The purity of MTF product reached to 58.2 % in the technological conditions following: extract sample concentration of 20 mg.ml(-1), 6 times washing water and elution reagent of 70% ethanol. The adsorbent could be used for 4 times repeatedly. The kinetic equation was y=u(1 - e(-kx)). CONCLUSION: The NKA-9 type of macroporous absorbent is suitable to purify the MTF.

[Microwave-assisted extraction of flavonoids in Mulberry leaf and root-bark].

OBJECTIVE: To extract flavonoids in Mulberry leaf and White Mulberry root-bark by microuave-assisted method. METHODS: An orthogonal table was used to find the best procedure for microwave-assisted extraction (MAE) of flavonoids. The advantages of MAE were explored by comparing with the commonly used extraction methods. The different contents of flavonoids in Mulberry Leaf and Mulberry Root-bark were also compared. RESULTS: The best condition for procedure was extracting 20 min at 60 degrees C with 12 times of 70% ethanol. Among all factors, the ethanol concentration was significant This experiment implied that flavonoids in Mulberry leaf were 9 times as that in Mulberry Root-bark probably. Moreover, the flavonoids extracted from Mulberry Leaf and White Mulberry root-bark by MAE were 55% and 32% more than that from commonly used extraction method individually. CONCLUSION: MAE is advantageous than commonly used extraction method. It can be used for batch production.