Comparison of catalytic functions and expression patterns of two pinene synthases from Wurfbainia villosa / 中国中药杂志

Wurfbainia villosa fruit is rich in volatile terpenoids, among which pinene is one of the main components and has anti-inflammatory, antibacterial, anti-tumor, and other pharmacological activities. This research group found that W. villosa fruits were rich in α-pinene by GC-MS, and terpene synthase(WvTPS63, formerly known as AvTPS1) with β-pinene as the main product was cloned and identified, but α-pinene synthase had not been identified. In this study, based on the genome data of W. villosa, we screened and found WvTPS66 with highly similar sequences to WvTPS63, identified enzyme functions of WvTPS66 in vitro, and performed a comparative analysis of sequence, catalytic function, expression pattern, and promoter with WvTPS63. Multiple sequence alignment showed that the amino acid sequences of WvTPS63 and WvTPS66 were highly similar and the conservative motif of terpene synthase was almost identical. In vitro enzymatic experiments on catalytic functions showed that both could produce pinene, and the main product of WvTPS63 was β-pinene, while that of WvTPS66 was α-pinene. Expression pattern analysis showed that WvTS63 was highly expressed in flowers, WvTPS66 was expressed in the whole plant, and the highest expression level was found in the pericarp, which indicated that it might be mainly responsible for the synthesis of α-pinene in fruits. In addition, promoter analysis revealed the presence of multiple regulatory elements related to stress response in the promoter regions of both genes. The findings of this study can provide a reference for the functional study of terpene synthase genes and new genetic elements for pinene biosynthesis.

Feasibility of vapor permeation technology for separating of Asari Radix et Rhizoma essential oil-bearing water / 中草药

Objective: To verify the feasibility of vapor-permeable membrane technology for the separation of water bodies containing essential oil of Asari Radix et Rhizoma (ARR) essential oil, and then to apply vapor permeate technology to the separation of more essential oils of traditional Chinese medicine. Methods: The polydimethylsiloxane/polyvinylidene fluoride (PDMS/PVDF) composite flat membrane and polyvinylidene fluoride (PVDF) flat membrane were collected as the membrane material. The oil-bearing water body of ARR volatile oil was separated by vapor permeate technology, and the oil penetration rate of two kinds of membranes was calculated. At the same time, the changes of the composition and content of the essential oil before and after the membrane were analyzed by gas chromatography-mass spectrometry (GC-MS). Results: The results showed that the essential oil penetration rate was significantly higher than that of PDMS/PVDF membrane when PVDF membrane was used as membrane material. GC-MS qualitative analysis results showed that the composition of the essential oil in the penetrants of the two membranes was basically the same as that of the essential oil obtained by the traditional steam distillation method. The content of α-pinene, β-pinene, 3,5-dimethoxytoluene, and methyl eugenol were determined by double internal standard method. The results showed that the content of each component in the PVDF membrane permeation was significantly higher than that of the PDMS/PVDF membrane permeation solution. Conclusion: It is feasible to separate the oil containing water from the essential oil of ARR by vapor permeation membrane technology. Compared with the PDMS/PVDF membrane, the PVDF membrane is more suitable for separating the oil containing water of the essential oil of ARR.

Phytochemical and Antimicrobial Evaluation of the Essential Oil of Croatian Salvia brachyodon Vandas

Backgraund: This study was designed to evaluate the phytochemical profile and the antimicrobial potential of the essential oil of Salvia brachyodon Vandas growing wild in Croatia. Short tooth sage (S. brachyodon Vandas),an endemic species that grows in the south east areas of the Adriatic coast in Croatia, Bosnia and Herzegovina, and Montenegro. It is a perennial plant that grows up to 70-80 cm in height and flowers from July to September. Methodology: The phytochemical components of the essential oil were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The antimicrobial activity was assessed against a panel of representative Gram-positive and Gram-negative bacteria as well as fungi. The antimicrobial activities of the oil against pathogenic microorganisms were determined by using agar disc diffusion and broth microdilution methods. Results: From the thirty-eight identified constituents representing 95.7% of the oil, 1,8-cineole (16.7%), β-pinene (19.7%) and α-pinene (7.6%), were the major components. The levels of oxygenated monoterpenes such as camphor (5.6%), borneol (4.2%), myrtenol (2.4 %) and terpinen-4-ol were significant. Other important compounds were sesquiterpenes hydrocarbons β-caryophyllene (6.6%), α-humulene (4.9%), viridiflorol (3.0%), spathulenol (2.9%) and aromadendrene. Preliminary antimicrobial screening revealed that the oil exhibited a very interesting antimicrobial profile. The oil exhibited moderate in vitro antibacterial activity after it was tested against twenty pathogenic bacteria and fungal strains, but high antimicrobial activity observed against medically important pathogens such as E. coli O157:H7, Listeria monocytogenes and Candida albicans. Conclusion: Results presented here may suggest that the essential oil of S. brachyodon possess antimicrobial properties, and is, therefore, a potential source of antimicrobial ingredient in food and pharmaceutical industry. The obtained results are preliminary and a further research is needed in order to obtain information regarding the practical effectiveness of essential oil to prevent the growth of foodborne and spoilage microbes under specific application conditions.

Synthesis, biological activity, computer aided drug design of alpha-pinene derivatives / 中国中药杂志

Based on the anticancer mechanism of biological alkylating agent, we designed and synthesized two alpha pinene derivatives:(1R,5S)-(6,6-dimethylbicyclo[3,1,1]hept-2-en-2-yl)methyl benzenesulfonate and (1R,5S)-(6,6-dimethylbicyclo[3,1,1]hept-2-en-2-yl)methyl 4-methylbenzenesulfonate, of which structures were confirmed by ¹H-NMR, HPLC and MS date. These two compounds showed a good inhibition of tumor cells' proliferation. Further, the computer siuulation of molecular docking and metabolic kinetics indicated that these two copounds may have stable molecular complexation with protein CDK2, which closely related to the cell cycle.

Simultaneous Determination of α-pinene,β-pinene and Linalool in Volatile Oil from Hedychium flavum by GC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of α-pinene,β-pinene and linalool in volatile oil of Hedychium flavum. METHODS:The volatile oil was extracted from H. flavum according to steam distillation stated in Chinese Pharmacopoeia(2015 edition volume Ⅳ). GC method was adopted to determine the content. The determination was performed on HP-5 capillary chromatographic column(30 m×0.032 mm×0.25 μm)with hydrogen flame ion detector(FID),nitrogen gas as carrier gas,with injector temperature of 200 ℃,and detector temperature of 250 ℃,column flow rate of 0.8 mL/min, samples size of 1 μL and split ratio of 40:1 by programmed temperature. RESULTS:The linear range of α-pinene,β-pinene and linalool were 0.090 5-2.413 3 mg/mL(r=0.999 9),0.098 3-2.620 0 mg/mL(r=0.999 9),0.169 1-4.510 0 mg/mL(r=0.999 8), respectively. RSDs of precision,stability(12 h),reproducibility tests were no more than 2.0%(n=6). The average recoveries were 99.84%(RSD=0.49%,n=6),100.24%(RSD=1.38%,n=6),99.41%(RSD=1.67%,n=6),respectively.The contents of α-pinene,β-pinene and linalool ranged 0.214 4-1.325 0,0.766 2-3.172 1,0.357 4-1.518 7 mg/g in volatile oil from 23 batches of H. flavum produced in Guizhou province. CONCLUSIONS:The method established in this experiment is rapid,simple and accurate.It can be used for the content determination of α-pinene,β-pinene and linalool in volatile oil from H.flavum.

Anti-cariogenic Properties of α-Pinene, a Monoterpene in Plant Essential Oil

Dental caries is the most common chronic disease in the dental field. Streptococcus mutans (S. mutans) is the most important bacteria in the formation of dental plaque and dental caries. In a previous study, we confirmed that the essential oil of Chrysanthemum boreale has antibacterial activity against S. mutans. Alpha-pinene is one of the major chemical components of Chrysanthemum boreale essential oil. In the present study, we investigated the inhibitory effects of α-pinene on cariogenic properties such as growth, acid production, biofilm formation, and bactericidal activity on S. mutans. Alpha-pinene at a concentration range of 0.25-0.5 mg/mL significantly inhibited the growth of S. mutans and acid production of S. mutans. Biofilm formation was significantly inhibited at < 0.0625 mg/mL α-pinene, similar to the data from scanning electronic microscopy. Under confocal laser scanning microscopy, the bacterial viability was decreased by α-pinene in a dose-dependent manner. These results suggested that α-pinene may be a useful agent for inhibiting the cariogenic properties of S. mutans.

Effects of α-pinene extracted from pine needle on expression of miR-221 and its potential target genes in human hepatocellular carcinoma cells / 中国中药杂志

To investigate the anti-hepatoma mechanism of α-pinene, HepG2 cell was treated with α-pinene and the change of cell cycle was examined by flow cytometry. The expression of miR-221, which was related the regulation of G₂/M phase, was detected by quantitative Real-time PCR. Meanwhile, TargetScan and other online bioinformatics methods were used to analyze and predict the target genes of miR-221, then the expression level of related target genes were detected by quantitative Real-time PCR. The results showed that α-pinene inhibited the proliferation of HepG2 cells in dose-dependent manner. It was also proved that HepG2 cells were arrested at G₂/M phase by α-pinene (P<0.05). The expression of miR-221 was down-regulated in α-pinene treated HepG2 cell. The bioinformatics analysis showed that CDKN1B/P27 and CDKN1C/P57 may be the protential targets of miR-221 and both of them were significantly up-regulated（P<0.001,P<0.05）by α-pinene treatment. According to these results, it was believed that α-pinene may inhibit the proliferation of hepatocellular carcinoma cells through arrest the cell at G₂/M phase, which may be associated with the down-regulate of the miR-221 expression and up-regulate of the CDKN1B/P27 and CDKN1C/P57 expression.

Comparative Analysis on Simultaneous Determination of α-pinene, β-pinene, Eucalyptol and Linalool in Baeckea Frutescens Preparations by Reference Substances Method and Reference Extract Method / 中国中医药信息杂志

Objective To determinate the contents of α-pinene, β-pinene, eucalyptol and linalool in Baeckea frutescens by reference substances method and reference extract method respectively; To explore the feasibility of replacing single component reference by control extracts in assay. Methods The GC system consisted of a quartz column DB-5 (60 m×0.25 mm×0.25 μm); The temperature programming rose from 80 ℃ (15 min) to 90 ℃ by 1 ℃/min, lasting 2 min, then 10 ℃/min to 110 ℃, then 25 ℃/min to 240 ℃, lasting 8 min in the end; The temperature of the entrance of capillary vessel column was 250 ℃, and the temperature of the detector was 250 ℃. Results α-pinene, β-pinene, eucalyptol and linalool were in a good linear relationship within each concentration scope (r≥0.999). The average recovery rates were in the range of 96.5%–102.2%. The results of t-test demonstrated that there is no significant difference between the two methods. Conclusion The reference extract method can be used as a quality evaluation pattern for Baeckea frutescens.

Chemical Composition and Locomotors Activity of Essential Oils from the Rhizome, Stem, and Leaf of Alpinia malaccencis (Burm F.) of Indonesian Spices.

The essential oils of three parts of the plant Alpinia malaccencis (Burm f.) (Zingiberaceae) collected from Sumedang, West Java Province has been studied. Determination of essential oil components from the rhizome, stem, and leaf of Alpinia malaccencis (Burm f.) was performed by confirmation of the linear retention index (LRI) and comparing the NIST library peak with data reported in the literature, the mass spectrum peaks with literature data. The essential oils obtained from rhizome, stem, and leaf of Alpinia malaccencis (Burm f.) by steam distillation was 1.22%, 0.25%, and 0.7%, (w/w), respectively. Rhizome oil composition was almost similar with stem oil with methyl cinnamate as the major component. Composition of leaf oil is different from rhizome and stem oil which dominated α-pinene (30.57%). The rhizome in doses 0.1 ml (47.09 %) had stronger locomotor inhibition activity compare than stem (26.97 %) and leaf oils (32.23 %) in doses 0.1 mL.

Antibacterial activity of the essential oils of myrtle leaves against Erysipelothrix rhusiopathiae

Objective: To evaluate the antibacterial activity of the essential oil of Myrtus communis (M.communis Methods: Wild populations of M. communis collected from Khuzestan and Lorestan provinces, Southwest Iran, were examined for antibacterial activity and chemical variability in leaves. Thein vitro antibacterial activity against E. rhusiopathiae was performed by agar disc diffusion and micro-dilution assays.Results:) L. against Erysipelothrix rhusiopathiae (E. rhusiopathiae) in vitro. in both assays. The results showed that the major components of the oil were α-pinene (22.3%-55.2%), 1,8-cineole (8.7%-43.8%) and linalool (6.4%-14.5%). The inhibition zones and MIC values for bacteria which were sensitive to the essential oils of M. communis were in the range of 14.7-27.0 mm and 0.031-0.25 mg/mL, respectively.Conclusions:This study demonstrates that products with valuable antibacterial activity can be The essential oils of M. communis have strong antibacterial against E. rhusiopathiae produced from leaves of M. communis against E. rhusiopathiae.

Antibacterial activity of the essential oils of myrtle leaves against Erysipelothrix rhusiopathiae

<p><b>OBJECTIVE</b>To evaluate the antibacterial activity of the essential oil of Myrtus communis (M. communis) L. against Erysipelothrix rhusiopathiae (E. rhusiopathiae) in vitro.</p><p><b>METHODS</b>Wild populations of M. communis collected from Khuzestan and Lorestan provinces, Southwest Iran, were examined for antibacterial activity and chemical variability in leaves. The in vitro antibacterial activity against E. rhusiopathiae was performed by agar disc diffusion and micro-dilution assays.</p><p><b>RESULTS</b>The essential oils of M. communis have strong antibacterial against E. rhusiopathiae in both assays. The results showed that the major components of the oil were α-pinene (22.3%-55.2%), 1,8-cineole (8.7%-43.8%) and linalool (6.4%-14.5%). The inhibition zones and MIC values for bacteria which were sensitive to the essential oils of M. communis were in the range of 14.7-27.0 mm and 0.031-0.25 mg/mL, respectively.</p><p><b>CONCLUSIONS</b>This study demonstrates that products with valuable antibacterial activity can be produced from leaves of M. communis against E. rhusiopathiae.</p>

Chemical composition and antifungal activity of essential oils isolated from Juniperus Oxycedrus L.

The chemical composition of essential oils isolated from the leaves of Juniperus oxycedrus by hydrodistillation was analyzed by GC-MS. 42 compounds, representing 96.73% of total oil, were identified. J. oxycedrus oil was found to be rich in α-pinene (39.63%), manoyl oxide (12.34) and z-caryophyllene (4.1%) and characterized by relatively high amounts of monoterpenes hydrocarbons and sesquiterpenes. Results of the antifungal testing by in vitro contact assay showed that the oil significantly inhibit the growth of nine plant pathogenic fungi.

Phytochemical composition of the essential oil of different populations of Stachys lavandulifolia Vahl

<p><b>OBJECTIVE</b>To examine the chemical variability in inflorescences of wild populations of Stachys lavandulifolia Vahl (S. lavandulifolia) collected throughout two provinces (Isfahan and Chaharmahal va Bakhtiary), Southwest Iran.</p><p><b>METHODS</b>The essential oils of S. lavandulifolia Vahl from seven locations were obtained by hydro-distillation and analysed by gas chromatography and gas chromatography-mass spectrometry.</p><p><b>RESULTS</b>The results revealed that distinct differences in the content of compounds depending on region of sample collection. The main constituents of the essential oils were α-thujone (0.3%-32.3%), α-pinene (trace to 37.3%), myrcene (0.5%-15.9%), β-phellandrene (1.1%-37.9%), germacrene D (0.4%-11.3%), Δ-cadinene (trace to 11.6%) and 1, 4-methano-1 H-indene (trace to 10.1%).</p><p><b>CONCLUSIONS</b>The results of the present study indicated that essential oil components of S. lavandulifolia Vahl can be varied with genetic (ecotype), environmental conditions and geographic origin. In general, the essential oils of various populations of S. lavandulifolia Vahl were rich in monoterpenoids and sesquiterpenoids.</p>

Phytochemical composition of the essential oil of different populations of Stachys lavandulifolia Vahl

Objective: To examine the chemical variability in inflorescences of wild populations of Stachys lavandulifolia Vahl (S. lavandulifolia) collected throughout two provinces (Isfahan and Chaharmahal va Bakhtiary), Southwest Iran. Methods: The essential oils of S. lavandulifolia Vahl from seven locations were obtained by hydro-distillation and analysed by gas chromatography and gas chromatography-mass spectrometry. Results: The results revealed that distinct differences in the content of compounds depending on region of sample collection. The main constituents of the essential oils were α-thujone (0.3%-32.3%), α-pinene (trace to 37.3%), myrcene (0.5%-15.9%),β-phellandrene (1.1%-37.9%), germacrene D (0.4%-11.3%),?-cadinene (trace to 11.6%) and 1, 4-methano-1 H-indene (trace to 10.1%). Conclusions:The results of the present study indicated that essential oil components of S. lavandulifolia Vahl can be varied with genetic (ecotype), environmental conditions and geographic origin. In general, the essential oils of various populations of S. lavandulifolia Vahl were rich in monoterpenoids and sesquiterpenoids.