Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Effect of isopentenyl pyrophosphate translocation on the biosynthesis of triptolide / 生物工程学报

Triptolide has wide clinical applications due to its anti-inflammatory, anti-tumor and immunosuppressive activities. In this study, we investigated the effect of blocking isopentenyl pyrophosphate (IPP) translocation on the biosynthesis of triptolide by exogenously adding D,L-glyceraldehyde (DLG) to the suspension cells of Ttripterygium wilfordii at different stages (7 d, 14 d). Subsequently, the cell viability, biomass accumulation, triptolide contents, as well as the profiles of the key enzyme genes involved in the upstream pathway of triptolide biosynthesis, were analyzed. The results showed that IPP translocation is involved in the biosynthesis of triptolide. IPP is mainly translocated from the plastid (containing the MEP pathway) to the cytoplasm (containing the MVA pathway) in the early stage of the culture, but reversed in the late stage. Blocking the translocation of IPP affected the expression of key enzyme genes involved in the upstream pathway of triptolide, which in turn affected the accumulation of triptolide. Understanding the characteristics and mechanism of IPP translocation provides a theoretical basis for further promoting triptolide biosynthesis through synthetic biology.

Simultaneous detemination of 8 terpenoids compounds in tripterygium wilfordii suspension cells by UPLC-MS/MS / 国际中医中药杂志

Objective To establish a UPLC-QQQ-MS/MS (ultra-high performance liquid chromatography triple quadrupole tandem mass Spectrometry) method for simultaneous detemination of 8 terpenoids compounds in Tripterygium wilfordii suspension cells. Methods The separation and analysis were performed on Water HSS T3 C18, with 0. 1％ acetic acid solution-acetonitrile as the mobile phase for gradient elution. The column temperature was set at 30 ℃ with flow rate was 0.3 ml/min. The electrospray ionization (ESI) source was applied and operated in alternating positive and negative mode. Multi-reaction monitor (MRM) mode was used to quantify the 8 compounds. Results The limits of detection and limits of quantitation in 8 compounds (triptolide, triptonide, dehydroabietlc acid, triptophenolide, demethylzeylasteral, tripteridine, pristimerin, celastrol) were 0.0096-0.2480 ng and 0.0192-0.4960 ng, respectively. The correlation coefficents (r) of calibration curves were 0.9966-0.9993. The average recovery rates were 95.04％-105.20％, and the relative standard deviation was 2.14％-6.31％. Conclusions The established quantitative method is simple, rapid, sensitive and accuracy. It can be used for simultaneous analysis of 8 terpenoids compounds in Tripterygium wilfordii suspension cells by using UPLC-QQQ-MS/MS, which affords methodology evidence for research and quality control of Triptervgium wilfordii Hook. f. and its plant tissue cultures.

Molecular cloning and functional identification of sterol C24-methyltransferase gene from

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from().(GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by thecDNA was expressed and purified as a recombinant protein from() and showed SMT activity. The expression ofwas highly up-regulated incell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis ofand will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of

Correlation Study on Storage Time and the Content of 5-Hydroxymethylfurfural in Corni Fructus / 中国中医药信息杂志

Objective To study the correlation between storage time and the content of 5-hydroxymethylfurfural (5-HMF) in Corni Fructus because of the color change caused by storage time. Methods Corni Fructus samples of different storage time with 0, 1, 2, 3, and 4 years were collected. The contents of 5-HMF were determined by HPLC. Results The HPLC determination method of 5-HMF in Corni Fructus was established. The contents of 5-HMF varied from undetected value to 0.292 8%, with the increase of storage time of 0, 1, 2, 3, and 4 years and the color gradually deepened from red, dark red, reddish brown to brown. The contents of 5-HMF in black wine-prepared Corni Fructus were 0.954 4%-1.837%. Conclusion The browning of Corni Fructus is related to the production of 5-HMF. With the extension of storage time of Corni Fructus, the color gradually deepens and the content of 5-HMF increase significantly. The storage time of Corni Fructus can be suggested to be one year.

Evaluation of Quality Coherence of Angelicae Dahuricae Radix (Baizhi) Decoction Pieces Based on HPLC Fingerprint / 中国中医药信息杂志

Objective To evaluate the quality coherence of commercial Angelicae Dahuricae Radix (Baizhi) decoction pieces. Methods The samples of 12 batches of commercial decoction pieces, 1 batch of sulphur fumigated Baizhi, and 1 batch of naturally dried Baizhi were collected. HPLC method was used to determine the fingerprints and the contents of imperatorin and isoimperatorin by C18 column (4.6 mm×250 mm, 5 μm) with a gradient mobile phase of acetonitrile-water solution system at the flow rate of 1.0 mL/min. The column temperature was set at 35 ℃, and the max plot of detection wavelength was in 210-800 nm. Similarity calculation was used to analyze the data. Results The HPLC fingerprint analysis method was established with 12 common peaks. The similarities of 12 batches of commercial decoction pieces of Baizhi were 0.840-0.973. Their similarities compared with the sulphur fumigated Baizhi were 0.672-0.908. Compared with naturally dried Baizhi, the similarities of fingerprint were 0.536-0.684. The contents of imperatorin and isoimperatorin in 12 batches of commercial decoction pieces were 0.035%-0.140% and 0.028%-0.069%, respectively. Conclusion The quality of Baizhi decoction pieces was consistent. It can be speculated that Baizhi decoction pieces were processed with sulphur fumigation.

Effects of Sulphur Fumigation on Content of Imperatorin in Angelicae Dahuricae Radix (Baizhi) / 中国中医药信息杂志

Objective To study whether sulphur fumigation can cause changes to the content of imperatorin in Angelicae Dahuricae Radix (Baizhi).Methods The fresh samples of Angelicae Dahuricae Radix were collected from some cultivation bases. Half of each batch root was fumigated with sulfur according to the conventional method, and another half was cut into slices and dried naturally (i.e. without sulfur fumigation). The content of imperatorin was determined by HPLC on C18 column (4.6 mm×250 mm, 5μm) with a gradient mobile phase of acetonitrile-water solution system at the follow rate of 1.0 mL/min, 35℃ of the column temperature, and the max plot in 210-800 nm of the detective wavelength.ResultsTotally 29 batches of fresh roots ofAngelica dahurica were collected. The average contents of imperatorin were 0.202% in the Angelicae Dahuricae Radix with sulfur fumigation and 0.120% in the Angelicae Dahuricae Radix without sulfur fumigation. Compared with the corresponding samples without sulfur fumigation, the content of imperatorin in every Angelicae Dahuricae Radix with sulfur fumigation decreased by 6.77%-77.56% with an average decrease of 39.86%.Conclusion The content of imperatorin decreased significantly in every batch of Angelicae Dahuricae Radix with sulphur fumigation. It shows that the sulphur fumigation method was not suitable for producing and processing Angelicae Dahuricae Radix from fresh roots ofA. dahurica.

Extracting Technology of Total Glycosides from Barks of Ilex rotunda / 中国中医药信息杂志

Objective To investigate an optimum extracting technology for total glycosides from dried barks of Ilex rotunda Thunb. Methods The yields of pedunculoside and syringin by high performance liquid chromatography (HPLC) determination were taken as the indexes. Some parameters of the extraction technology were evaluated with an L9(3)4 orthogonal design. The optimum extraction parameters were used to extract total glycosides in laboratory. The feasibility was checked by determining the chemical constituents by means of HPLC method. Results The optimum extracting conditions were 12 times volumes of 50% ethanol as the solvent and refluxing 90 min each time for 3 times. An extract yield of total glycosides was 19.5% from barks of Ilex rotunda. Pedunculoside (292 mg/g), syringin (59.5 mg/g), sinapaldehyde glucoside, syringaresinol 4'-O-β-D-glucopyranoside, and syringaresinol 4',4'-di-O-β-D-glucopyranoside were detected in the extract. Conclusion Some glycosides were found in the extract with the optimum extraction technology in laboratory. The extraction technology is practicable and valid.

Chemical constituents contained in Populus tomentosa / 中国中药杂志

<p><b>OBJECTIVE</b>To separate and identify chemical constituents from stem barks of male plants of Populus tomentosa.</p><p><b>METHOD</b>Fresh stem barks of P. tomentosa were extracted with methanol to obtain extracts which were suspended in water and blended successively with petroleum ether, ethyl acetate and n-butanol. Various chromatographic techniques were used to separate and purify the constituents extracted with ethyl acetate and n-butanol fractions. Their structures were identified on the basis of their physicochemical properties and spectral data.</p><p><b>RESULT</b>Twelve compounds were separated with ethyl acetate and n-butanol fractions and identified as benzoic acid (1), daucosterol (2), tremuloidin (3), rhamnocitrin (4), sakuranetin (5), 7-O-methylaromadendrin (6), isograndidentatin A (7), siebolside B (8), sakuranin (9), micranthoside (10), alpha-D-glucopyranose (11), and sucrose (12).</p><p><b>CONCLUSION</b>Compounds 4-12 were separated from this plant for the first time. Of them, compound 10 was separated from this plant genus for the first time.</p>

Difference of volatile constituents contained in female and male flowers of Trichosanthes kirilowii by HS-SPME-GC-MS / 中国中药杂志

<p><b>OBJECTIVE</b>To compare and analyze volatile constituents from flowers of Trichosanthes kirilowii, in order to point out characteristic differences between female and male flowers.</p><p><b>METHOD</b>Blooming female and male flowers were collected in the same place. Volatile constituents were extracted from the flower by solid phase micro-extraction (SPME), then separated and analyzed by gas chromatography-mass-spectrometry (GC-MS).</p><p><b>RESULT</b>Fifty-two and forty-five chromatographic peaks were separated from the female and male flowers, respectively. Forty seven constituents were identified and their relative percentage compositions were determined with the peak area normalization method. Linalool, alpha-farnesene, benzene methanol, and (Z)-2-methylbutanal oxime were the main volatile constituents. The contents of linalool and alpha-farnesene in female flower were remarkably higher than those in male. In contrast, the content of benzene methanol in male flower was remarkably higher than that in female.</p><p><b>CONCLUSION</b>In the first study on chemical constituents from flowers in genus Trichosanthes, 37 compounds are separated from T. kirilowii. Contents of linalool, alpha-farnesene and benzene methanol show the characteristic differences of volatile constituents contained in male and female flowers of T. kirilowii, which enriches the basic studies on dioecious plant.</p>

Effect of Tween 80 on yuxingcao injection and volatile oils from Houttuynia cordata / 中国中药杂志

<p><b>OBJECTIVE</b>To research the effect of polysorbate 80 (Tween 80) on Yuxingcao injection and volatile oils from Houttuynia cordata.</p><p><b>METHOD</b>1H-NMR spectra of aldehydic and new matter in Yuxingcao injection, volatile oils of H. cordata, and solutions of Tween 80 and volatile oil of H. cordata are determined and compared from various angles of growing origin, storage temperature, and storage time.</p><p><b>RESULT</b>Three aldehydic singlets in 1H-NMR spectra of every volatile oil from 4 aerial part of H. cordata were observed. These aldehydic peaks were basically disappeared and a new peak at delta 8.30 was found in 1H-NMR spectra of the volatile oil solutions in tween 80. Any obvious aldehydic peak in 1H-NMR spectra did not be observed in Yuxincao injection. A weak peak at 8 8.30 was found in 1H-NMR spectra in Yuxincao injection, and the peak high of delta 8.30 was remarked gone up when the injection was stored in 40 degrees C for 1 to 3 months.</p><p><b>CONCLUSION</b>Tween 80 might cause the obvious reduce of aldehydic compounds contents and the production of a novel singal at delta 8.30 in 1H-NMR spectra when it was mixed with the volatile oil from the aerial part of H. cordata. The novel signal at delta 8.30 in 1H-NMR spectra existed in Yuxincao injection and was very small, but was increased remarkably when the Yuxincao injection was stored at 40 degrees C for 1 month at least.</p>

Acupuncture and Moxibustion Treatment of Urinary Incontinence after Stroke Clinical Observation / 浙江中医药大学学报

[Objective] To explore the treatment of urinary incontinence after stroke.[Methods]To use the Acupucture and Moxibustion method to treat the 35 patients who met the inclusion criteria,compared before and after treatment of urinary incontinence in patients with the degree of improvement.[Results] After two courses of treatment,the total effective rate 94.286%.[Conclusion]Acupuncture and Moxibustion treatment of urinary incontinence after stroke have a positive effect,and this method is simple and few side effects.