Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli / 中国中药杂志

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.

Quality research of Gastrodiae Rhizoma with UPLC fingerprint and content determination of multicomponent / 中草药

Objective To establish the analysis method of UPLC fingerprint and simultaneous determination of five active components (gastrodin, 4-hydroxybenzyl alcohol, parishin B, parishin C, and parishin A). Methods The analysis was performed on a Waters Acquity with a Waters ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column. The gradient mobile phase was acetonitrile and 0.1% formic acid-water solution, flow rate was set at 0.3 mL/min, the injection volume was 0.2 μL and the temperature of column was 35 ℃. The detection wavelengths were set at 270 nm (fingerprint), 220 nm (content determination). The 28 batches of samples fingerprint were analyzed by similarity evaluation, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). The identification and content determination of the main common peaks were carried out by comparison with reference. Results We set up the common mode of the fingerprint with 20 common peaks, and six of them were identified, which were gastrodin (1), 4-hydroxybenzyl alcohol (3), p-hydroxybenzaldehyde (7), parishin B (14), parishin C (15), and parishin A (18). The similarities among 28 samples were all above 0.90. PCA was used to divide the samples into five groups, and six different substances were found. The content of the same component and the fluctuation range among different components were all different. The content of the five components of each origin was Guizhou 2.52% > Yunnan 2.49% > Shaanxi 2.33% > Hubei 2.10% > Zhejiang 1.90% > Anhui 1.65%.Conclusion The establishment of UPLC fingerprint and simultaneous determination of five active components provide a reference for quality control of Gastrodia Rhizoma.

Chemical Constituents Analysis of Morchella and Determination of 4-Hydroxybenzyl Alcohol / 世界科学技术-中医药现代化

Objective: To analyze the chemical constituents of Morchella esculenta, and to determine the main activeingredient of it, so as to better control the quality of Morchella esculenta and promote the development and utilization of Morchella. Methods: The compounds were extracted and percolated by ethanol. The samples were separated using silicagel and identified by13C-NMR data. HPLC was used to assay the contents of 4-Hydroxybenzyl alcohol. Results: A totalof 5 compounds were isolated and their structures were identified as 11, 15, 19-trimethyl-5, 9-eicosadienoic acid, cis-13-Docosenoic acid&Erucic acid, 4-Hydroxybenzyl alcohol, Ergosta-5, 7, 22-triene-3β-ol and Mannitol. Conclusion: 4-Hydroxybenzyl alcohol was first isolated from the Morchella esculenta fruiting body, and HPLC method for thedetermination of 4-Hydroxybenzyl alcohol was established. The content of 4-Hydroxybenzyl alcohol were no less than0.40％. The determination method can be used for quality control of Morchella esculenta.

Simultaneous determination of five constituents in Gastrodiae Rhizoma from seven growing areas by RP-HPLC / 中成药

AIM To develop a RP-HPLC method for the simultaneous content determination of five constituents in wild Gastrodia elata Blume,G.elata Bl.form.glauca S.Chow,G.elata Bl.form.elata and G.elata Bl.form.flavida S.Chow from seven growing areas (Zhaotong and Lijiang in Yunan,Lu'an in Anhui,Hanzhong in Shannxi,Dejiang in Guizhou,Guangyuan in Sichuan,and Yichang in Hubei).METHODS The analysis of Gastrodiae Rhizoma 70％ methanol extract was performed on a 35 ℃ thermostatic Waters Sunfire C18 column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of methanol-0.1％ formic acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 270 nm.RESULTS Adenosine,gastrodin,4-hydroxybenzyl alcohol,4-hydroxybenzaldehyde and parishin A showed good linear relationships within their own ranges (r≥0.999 5),whose average recoveries were 99.34％-101.33％ with the RSDs of 1.85％-3.28％.Their contents were the highest in G.elata Bl.form.glauca S.Chow cultivated in Zhaotong (collected in 2013),Lu'an (collected in 2013),Yichang (collected in 2013),Zhaotong (collected in 2014) and Yichang (collected in 2013),respectively,and the total content was the highest in G.elata Bl.form.glauca S.Chow cultivated in Yichang (collected in 2013).CONCLUSION This accurate,sensitive and reliable method can be used for the quality control of Gastrodiae Rhizoma.

Simultaneous determination of eight active components in Gastrodiae Rhizoma by HPLC-MS / 中草药

Objective: To establish an HPLC method for the simultaneous determination of eight active components (gastrodin, 4-hydroxybenzyl alcohol, vanillyl alcohol, vanillin, p-hydroxybenzaldehyde, parishin B, parishin C, and parishin A). Methods: The analysis was performed on an Agilent 1220 system with a Hypersil C18 column (250 mm × 4.6 mm, 5 μm). The eight components were separated in 75 min with gradient mobile phase consisting of 0.5% CH3COOH-H2O (A) and 0.5% CH3COOH-CH3OH (B): 0-10 min, 98% A; 10-60 min, 98%-60% A; 60-75 min, 60% A. The temperature of column was 30 ℃, and the injection volume was 20 μL. The multiple-reaction monitoring (MRM) scanning was employed for the quantification with switching electrospray ion source polarity in negative mode. The ion spray voltage was set at -4500 V and the turbo spray temperature was maintained at 550 ℃. Results: The eight components had the good linearity (r ≥ 0.999 2) within the linear ranges. The average recovery rate was 94.51%-102.70% with RSD < 3.50%. The contents of the eight components of Gastrodiae Rhizoma varied according to the different habits. The contents of parishins A, B, and C were high while the contents of vanillyl alcohol and vanillin were low. Conclusion: The developed HPLC-MS method is simple, sensitive, and accurate, and has the good repeatability in separation, which is available for the quality control of Gastrodiae Rhizoma.