Using dynamic molecular switches for shikimic acid production in Escherichia coli / 生物工程学报

Shikimic acid is an intermediate metabolite in the synthesis of aromatic amino acids in Escherichia coli and a synthetic precursor of Tamiflu. The biosynthesis of shikimic acid requires blocking the downstream shikimic acid consuming pathway that leads to inefficient production and cell growth inhibition. In this study, a dynamic molecular switch was constructed by using growth phase-dependent promoters and degrons. This dynamic molecular switch was used to uncouple cell growth from shikimic acid synthesis, resulting in the production of 14.33 g/L shikimic acid after 72 h fermentation. These results show that the dynamic molecular switch could redirect the carbon flux by regulating the abundance of target enzymes, for better production.

Study on the Synthesis of Shikimic Acid Derivatives and Their Reversal Effects on Paclitaxel-resistant Human Breast Cancer Cells / 中国药房

OBJECTIVE：To struc turally modify shikimic acid ，and to investigate the reversal effects of its derivatives on paclitaxel-resistant human breast cancer cells MCF- 7/PTX. METHODS ：Using shikimic acid as the lead structure ，1-position carboxyl group was structurally modified to synthesize a series of shikimic acid derivatives through esterification ，amidation， hydrogenation and reduction ，etc. Using non-drug resistant cells MCF- 7 as reference ，MTT assay was used to screen derivatives with inhibitory activity as well as half-inhibitory concentration （IC50）and reversal index （RI）of derivatives to MCF- 7/PTX. With the drug resistance-related transgelin 2 as the target ，the molecular docking of the active derivatives with the drug resistance-related protein was carried out by using Glide 1.0 computer-aided design software. RESULTS ：Totally 15 derivatives were obtained （T1-T15）， of which T 4-T15 were obtained for the first time. MTT assay showed that （3R， 4S， 5R） -N-benzyl-3， 4， 5-trihydroxy-1-cyclohexene-1-formamide（T7），（3R，4S，5R）-N-（3，4，5-trihydroxy-1-cyclohexenylmethyl）-benzylamine（T14）， （3R，4S，5R）-3，4-O-isopropyl-5-O-acetyl-1-cyclohexene-1-methyl formate （T15）inhibited MCF- 7 and MCF- 7/PTX cells to a certain extent ；IC50 values of T 7，T14 and T 15 combined with pacliaxel to MCF- 7/PTX cells were significantly lower than that in negative control （Paclitaxel alone ）group（P＜0.05）. RIs of T 14 and T 15 were higher ，and RIs of the highest dose were 8.8 and 9.3， which were equivalent to positive control verapamil （10.8）. Th e results of molecular docking showed that the hydroxyl groups at positions 3，4 of T 7 could form multiple hydrogen bonds with ； Arg625 and Asp 627 in the catalytic region of transgelin 2. In addition to the hydrogen bond mentioned above at T 7，the mail：batistuta28@126.com secondary amine side chain at position 1 of T 14 could also form hydrogen bond with Glu 657 of transgelin 2. When the hydroxyl group on the T 15 mother nucleus was derived from the donor group ，the binding of the hydroxyl group to transgelin 2 was closer and the inhibition was enhanced. CONCLUSIONS ： The derivatives T 7，T14 and T 15 have certain reverse activity to paclitaxel-resistant human breast cancer cells. The polyhydroxy structure of the mother nucleus is the main structural region of the hydrogen bond between shikimic acid and its derivatives and transgelin 2. The derivation of its power supply group or the introduction of secondary amines and hydrophobic groups into the 1-carboxyl group of shikimic acid is benifit for enhancing the drug resistance reversal effect of derivative .

Studies on chemical constituents of Chloranthus fortunei / 中草药

Objective: To study the antitumor constituents from Chloranthus fortunei. Methods: Various chromatographic techniques and spectroscopic methods were applied to investigate the chemical constituents from C. fortunei, and some of the compounds were screened for their antitumor activities by MTT method. Results: Sixteen compounds were obtained from the whole plants of C. fortunei and identified as rosmarinic acid (1), 2’-hydroxy-4,3’,4’,6’-tetramethoxychalcone (2), flavokawain A (3), cycloshizukaol A (4), atractylenolide III (5), 4β-hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione (6), (8α)-6,8-dihydroxycadina-7 (11),10 (15)-dien-12-oic acid γ-lactone (7), curcolonol (8), 11-hydroxyldrim-8,12-en-14-oic acid (9), friedelin (10), isovanillic acid (11), 6β-hydroxystigmast-4-en-3-one (12), 3,4-dihydroxybenzoic acid (13), shikimic acid (14), scopolin (15) and N-acetyltyramine 1-O-β-D-glucoside (16). Compounds 4 and 5 showed weak cytotoxicity with IC50 ranged from 46 to 85 μmol/L. Conclusion: Compounds 2, 10, 11, and 13-15 are obtained from the genus Chloranthus for the first time and compounds 1-3 and 6-16 are isolated from C. fortunei for the first time. Some sesquiterpenoids from C. fortunei exhibited weak antitumor activities.

Research progress on synthesis of anthraquinones based on shikimic acid/o-succinylbenzoic acid pathway and polyketone pathway / 中草药

Anthraquinone and its derivatives are very important secondary metabolites in plants. They have many functions such as photoprotection and improvement of plant disease resistance. They also have very important applications in the fields of medicine and chemical engineering. Efficiently and quickly obtaining anthraquinones and improving the synthesis efficiency of anthraquinones in plants have become one of the research focuses of modern synthetic biology. However, the synthetic pathway of anthraquinones is more complicated. At present, it is generally believed that anthraquinones are formed in plants by the shikimic acid/o- succinylbenzoic acid pathway and polyketone pathway. This article focuses on the recent research advances in the skeleton synthesis of anthraquinone via shikimic acid/o-succinylbenzoic acid pathway and polyketone pathway in plants, and provides a certain theoretical basis for studying the synthesis and regulation of anthraquinone metabolites in plants.

Shikimic Acid Promotes Oligodendrocyte Precursor Cell Differentiation and Accelerates Remyelination in Mice / 神经科学通报·英文版

The obstacle to successful remyelination in demyelinating diseases, such as multiple sclerosis, mainly lies in the inability of oligodendrocyte precursor cells (OPCs) to differentiate, since OPCs and oligodendrocyte-lineage cells that are unable to fully differentiate are found in the areas of demyelination. Thus, promoting the differentiation of OPCs is vital for the treatment of demyelinating diseases. Shikimic acid (SA) is mainly derived from star anise, and is reported to have anti-influenza, anti-oxidation, and anti-tumor effects. In the present study, we found that SA significantly promoted the differentiation of cultured rat OPCs without affecting their proliferation and apoptosis. In mice, SA exerted therapeutic effects on experimental autoimmune encephalomyelitis (EAE), such as alleviating clinical EAE scores, inhibiting inflammation, and reducing demyelination in the CNS. SA also promoted the differentiation of OPCs as well as their remyelination after lysolecithin-induced demyelination. Furthermore, we showed that the promotion effect of SA on OPC differentiation was associated with the up-regulation of phosphorylated mTOR. Taken together, our results demonstrated that SA could act as a potential drug candidate for the treatment of demyelinating diseases.

Chemical constituents from Clematis apiifolia / 中草药

Objective: To investigate the chemical constituents from the stem of the Clematis apiifolia. Methods: A variety of chromatographic Methods: were applied in isolation and purification including silica gel, ODS, Sephadex LH-20 gel, and HW-40C and so on. Their structures were identified by the nuclear magnetic resonance (NMR), mass spectrometry and so on. Results: These compounds were isolated and determined as hederagenin (1), α-hederin (2), sapindoside B (3), stigmast-22-en-3β, 6β, 9β-triol (4), pleuchiol (5), stigmasterol-3-O-β-D-glucopyranoside (6), stimasterol (7), siderin (8), shikimic acid (9), ethyl-α-D-glucopyranoside (10), ethyl-β-D-glucopyranoside (11), D-gulonic acid (12), 3,5-dihydroxyl-4-valerolactone (13), 2-formyl-5-hydroxymethylfuran (14), uracil (15), uridine (16), tricosanol (17), triacontanol (18), hexacosanoic acid (19), and monostearin (20). Conclusion: All compounds are obtained from C. apiifolia for the first time including firstly reported compound 3 from Clematis plants and compounds 4, 5, 9-12 from Ranunculaceae family.

Content Determination of Total Flavonoids and Shikimic Acid in Pine Needles of Pinus massoniana Lamb. in Wudang Area / 中国中医药信息杂志

Objective To establish a method to determine the contents of total flavonoids and shikimic acid in pine needles of Pinus massoniana Lamb.in Wudang Area.Methods Rutin was used as reference standard,and the content of total flavonoids in pine needles of Pinus massoniana Lamb. was determined by UV spectrometry at wavelength of 500 nm. The content of shikimic acid was determined by HPLC-DAD. The Fortis Xi C8 column (5 μm, 250 mm × 4.6 mm) was adopted with acetonitrile - 0.4% phosphoric acid solution (8:92, V/V) as mobile phase at the flow rate of 0.9 mL/min. The detection wavelength was 213 nm; the column temperature was 30 ℃; the injection volume was 20 μL. Results The linear range was 8.26-49.54 μg for rutin (r=0.999 4) with an average recovery of 99.2%, RSD=1.94%. The linear range was 10.26-61.56 μg for shikimic acid (r=0.999 4) with an average recovery of 99.5%,RSD=1.93%.The contents of total flavonoids in pine needles of Pinus massoniana Lamb.was 28.33 mg/g, and shikimic acid was 15.25 mg/g, respectively. Conclusion The method is simple, rapid, accurate and reproducible. It can be used for the content determination of total flavonoids and shikimic acid in pine needles of Pinus massoniana Lamb. in Wudang Area.

Degranulation and anti-inflammatory effects of shikimic acid in Chaenomeles speciosa on peritoneal mast cells in rats / 中国免疫学杂志

Objective:To investigate the effect of Chaenomeles speciosa total extract shikimic acid(SA) on the degranulation of compound 48/80(C48/80) stimulated rat peritoneal mast cells and the anti-inflammatory.Methods: Qualitative analysis of shikimic acid in the total extract of Chaenomeles speciosa by high performance liquid phase;RPMC were identified by toluidine blue,immunohistochemistry and electron microscopy;CCK-8 method was used to detect the proliferation of RPMC in each concentration group.The release rate of β-hexosidase was determined by substrate method.The content of histamine in supernatant was detected by ELISA.Results: SA had no significant effect on the growth of RPMC at the concentration of 0-90 μg/ml.Compared with the positive control group,SA could effectively inhibit the release of β-hexosidase and inhibit the secretion of histamine.Conclusion: SA inhibits the release of histamine in inflammatory mediators by inhibiting the degranulation of RPMC stimulated by C48/80,and then exerts anti-inflammatory effects.

Simultaneous purification of shikimic acid and total flavonoids from pine needles of Cedrus deodara by macroporous adsorption resin / 中草药

Objective: To research the simultaneous purification process of shikimic acid and total flavonoids from pine needles of Cedrus deodara (Roxb.) G. Don. Methods: Taking the purity of shikimic acid and total flavonoids as the evaluation indicator, the purification effect of six macroporous resins were evaluated. Orthogonal design L9(34) and single factor experiments were employed to optimize the purification conditions by comprehensive scoring. The purification capacity of the best resin was investigated by the sample mass concentration, the volume flow of the sample, the ratio of the resin to the drug, the amount of water for washing, the concentration of ethanol, and the elution of ethanol. Results: XAD 7HP macrophous resin offered better purification effect of shikimic acid and total flavonoids from pine needles of C. deodara than other macrophous resins. The optimum purification condition was confirmed as follows: The concentration of shikimic acid in the sample was 11.59 mg/mL, and total flavonoids concentration was 6.9 mg/mL; The flow rate was 8 BV/h, and the sample volume was 2.0 mL/g; The shikimic acid was eluted with loading capacity and 4 BV of water; The total flavonoids was eluted with 4 BV of 70% ethanol successively. The purity of shikimic acid can be increased from 19.25% to 28.98%, and the purity of total flavonoids can be increased from 11.92% to 54.45%. Conclusion: The optimized purification process is stable, feasible and suitable for pilot enrichment of shikimic acid and total flavonoids from pine needles of C. deodara.

Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli / 中国天然药物

Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

Polymorphism of shikimic acid / 中国中药杂志

This study was performed to systematically investigate the polymorphism of shikimic acid. Through optimizing the recrystallization solvent, solvent volume, recrystallization temperature, time and pressure, three crystal forms were discovered and prepared. The differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffraction (PXRD) and infrared spectrometry (IR) were used to characterize these solid states. Furthermore, the influencing factor experiments were used to explore the stability of these polymorphisms and the transformation among them. Three new polymorphisms were prepared and identified. The results indicated that only PXRD could identify different polymorphisms and there was no solvent in all three crystal forms. The composition, thermodynamic property and transformation of these crystal forms were described in this work. Furthermore, an effective method for qualitative analysis of these crystal forms was established.

Chemical constituents in n-butanol extract from pine needles of Cedrus deodara / 中草药

Objective: To investigate the chemical constituents in the n-butanol extract of pine needles of Cedrus deodara. Methods: Chemical constituents were separated and purified by silica gel and Sephadex LH-20 chromatography column. The structures were elucidated on the basis of physicochemical properties and spectral data (1H-NMR, 13C-NMR, and DEPT). Results: The compounds were identified as 1-(4'-hydroxy-3'-methoxyphenyl)-2-[4″-(3-hydroxypropyl)-2″-methoxyphenoxy]-1, 3-propanediol (1), (7S, 8R)-9, 9'-dihydroxy-3, 3'-dimethoxy-7, 8-dihydro-benzofuran-1'-propanol base neolignan-4-O-β-D-glucoside (2), (7R, 8R)-3', 9, 9'- trihydroxy-3-methoxy-7, 8-dihydro-benzofuran-1'-propanol base neolignans-9-O-α-L-rhamnoside (3), (6R, 9R)-6-hydroxy-3-oxo-α- ionol-9-O-β-D-glucopyranoside (4), (6R, 9R)-3-oxo-α-ionol-9-O-β-D-glucopyranoside (5), shikimic acid butyl ester (6), quinic acid butyl ester (7), (6S, 9R)-6-hydroxy-3-oxo-α-ionol-9-O-β-D-glucopyranoside (8), 5-p-trans-coumaroylguinic acid (9), and (E)-1-O-p- coumaroyl-α-D-glucopyranoside (10). Conclusion: Compounds 1-7 are isolated from C. Trew for the first time.

Experimental Study on the Anti-inflammation Effect of 3,4-oxo-isopropylidene-shikimic Acid / 中国中医药信息杂志

Objective To study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on transcription factor STAT1, STAT3 and NF-?B in human cervix cancer Hela cell. Methods The cell proliferation was assessed by MTT assay. The luciferase activity of transcription factor STAT1, STAT3 and NF-?B was determined by the Dual-Luciferase Reporter Assay System. Results ISA can not inhibited the growth of Hela cell. The luciferase activity of transcription factor NF-?B in Hela cells treated with ISA was inhibited, while the luciferase activities of transcription factor STAT1 and STAT3 were not inhibited. Conclusion ISA can inhibit inflammation, which may be related with suppression of NF-?B transcriptional activity.