HPLC/QTOF-MS metabolomics analysis applied to identify skin biomarkers of UVC-induced skin injury in mice and preventive effects of abietic acid

Abietic acid (AA) is a main constituent from pine resin, which has definite therapeutical effects for treating skin ulcers and tumor. Here, we explored the metabolome changes in skin tissues of mice with UVC-induced skin injury treated with AA by a HPLC-QTOF-MS/MS method. Model mice were induced with UVC irradiation. Skin histopathological changes were examined by routine HE staining. Metabolomic analysis technology and pattern recognition statistical method were applied to analyze the metabolites in the skin tissues of mice to study the therapeutic effect of AA on UVC-induced skin injury in mice. Ceramides, sphingosines, glycyl-L-glutamine, dihydroorotic acid, adenosine, dCMP and lyso-phosphatidylcholines can be used as biomarkers of UVC-induced skin injury. AA can improve the pathological tissue from the pathway of skin lipid and purine pyrimidine metabolism to achieve the therapeutic effect. AA can effectively treat UVC-induced skin injury in mice

Protective effect of Pinus massoniana needle extract against oxidative stress in human dermal papilla cells via the nuclear factor-erythroid 2-related factor 2-antioxidant responsive element signaling pathway / 中华皮肤科杂志

Objective:To investigate protective effect of Pinus massoniana needle extract (PMNE) against oxidative stress in human dermal papilla cells (HDPC) , and to explore its mechanisms. Methods:As research objects, some cultured HDPC were treated with H 2O 2 at different concentrations of 0 (control group) , 0.1, 0.2, 0.4, 0.8 and 1.0 mmol/L, in order to establish the optimal condition for in vitro oxidative stress in HDPC; some other HDPC were transfected with nuclear factor-erythroid 2-related factor 2 (Nrf2) specific small interfering RNAs (siRNA1, siRNA2, siRNA3) or a Nrf2-overexpressing plasmid (pCMV6-XL5-Nrf2) , the HDPC transfected with a scrambled-siRNA and an empty plasmid pCMV6-XL5 served as the control siRNA group and control plasmid group respectively, and HDPC subjected to conventional culture served as the blank group; after the above treatment, real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine Nrf2 mRNA and protein expression, respectively; cell viability and apoptosis were detected in the above transfected cells after the treatment with H 2O 2 at an optimal concentration. In the subsequent experiment, some HDPC were divided into several groups: control group subjected to conventional culture, dihydrotestosterone group treated with 0.03 μg/ml dihydrotestosterone, proanthocyanidin group treated with 0.03 μg/ml dihydrotestosterone and 6.00 μg/ml proanthocyanidin B2, PMNE groups treated with 0.03 μg/ml dihydrotestosterone and PMNE at different concentrations of 1, 5, 25 and 100 μg/ml; after the above treatment, cell viability and apoptosis were detected, relative fluorescence intensity of intracellular reactive oxygen species (ROS) , malondialdehyde (MDA) content, mRNA and protein expression of Nrf2, quinone oxidoreductase 1 (NQO1) , heme oxygenase 1 (HO-1) , Kelch-like ECH-related protein 1 (Keap1) , transforming growth factor (TGF) -β1, Sma- and Mad-related protein 2/3 (Smad2/3) , phosphorylated Smad2/3 (p-Smad2/3) were determined in HDPC. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference- t test for multiple comparisons. Results:The viability of HDPC ranged from 75% to 85% after the treatment with 0.4 mmol/L H 2O 2, which was selected as the optimal condition for in vitro oxidative stress in HDPC. Compared with the blank group and control siRNA group, the Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups showed significantly decreased Nrf2 mRNA and protein expression (all P < 0.05) , but significantly increased apoptosis rate (Nrf2-siRNA1, Nrf2-siRNA2, Nrf2-siRNA3 groups, blank group and control group: 12.50% ± 0.05%, 26.07% ± 0.05%, 58.44% ± 1.03%, 10.38% ± 0.64%, 13.05% ± 0.12%, respectively; all P < 0.05) . Nrf2 protein expression was the lowest in the Nrf2-siRNA2 group, so Nrf2-siRNA2 was selected as the optimal interfering fragment for subsequent experiments. Compared with the blank group and control plasmid group, the Nrf2 overexpression group showed significantly increased Nrf2 mRNA and protein expression (both P < 0.05) , but a significantly decreased apoptosis rate (all P < 0.05) . After the treatment with 0.4 mmol/L H 2O 2, the Nrf2 overexpression group showed a significantly decreased apoptosis rate, but significantly increased cell viability compared with the empty vector group ( t = 3.66, 40.40, respectively, both P < 0.001) ; the Nrf2-siRNA2 group showed a significantly increased apoptosis rate, but significantly decreased cell viability compared with the control group ( t = 13.13, 67.37, respectively, both P < 0.001) . In the PMNE treatment experiment, the proanthocyanidin group and PMNE groups showed significantly increased cell viability, but significantly decreased apoptosis rates compared with the dihydrotestosterone group (all P < 0.01) ; proanthocyanidin and PMNE at different concentrations could significantly inhibit dihydrotestosterone-induced overexpression of ROS and MDA in HDPC (all P < 0.01) ; the protein expression of Nrf2, NQO1 and HO-1 was significantly higher in the proanthocyanidin group, 5-, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) , while the protein expression of Keap1 and TGF-β1, and the Smad2/3 phosphorylation level were significantly lower in the proanthocyanidin group, 25- and 100-μg/ml PMNE groups than in the dihydrotestosterone group (all P < 0.05) . Conclusion:Nrf2 plays an important role in protecting against oxidative damage in HDPC, and PMNE may exert marked protective effect on HDPC by activating the Nrf2-antioxidant responsive element signaling pathway.

Sealing effects of pinus massoniana bark extract on dentin tubule and its wear resistance in vitro / 西安交通大学学报(医学版)

Objective To observe the sealing effect of transient pinus massoniana bark extract (PMBE)pre-coating on dentin tubules and its wear resistance so as to provide an experimental basis for PMBE to be used to prevent and treat dentin hypersensitivity.Methods The model of dentin hypersensitivity was established by acid etching method.The samples were transient pre-coating with 80 g/L of PMBE ethanol solution,and then some of them experienced brushing wear treatment with ethanol group and fluorine vanish group as negative and positive controls.The surface and profile morphologies of the samples were observed using field emission scanning electron microscope (FESEM)in each group.The area of dentin tubular openings (ADTO)and percentage area of dentin tubular openings (PADTO)were also measured.Results The dentin tubules were opened completely and the lumen of dentin tubules was empty in ethanol group.The dentin tubules were completely or basically closed and some films or plugging could be seen in the lumen of dentin tubules in fluorine vanish/immediate group and PMBE/immediate group.The dentin tubules were partly opened and some wear marks could be seen on the dentin surface in fluorine vanish/wear groups,PMBE/wear groups.ADTO and PADTO in ethanol group were significantly higher than those in fluorine vanish groups and PMBE groups (P＜0.05).ADTO and PADTO did not significantly differ between fluorine vanish/wear groups and PMBE/wear groups (P＞0.05),which were all significantly higher than those in fluorine vanish/immediate group and PMBE/immediate group (P＜0.05).Conclusion Local transient pre-coating of PMBE ethanol solution can achieve fine immediate sealing effect of dentin tubules and some degree of wear resistance.

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.

Comparative study on the anti-demineralization effect of pinus massoniana bark extract and grape seed extract on human root dentin in vitro / 实用口腔医学杂志

Objective:To evaluate the effect of pinus massoniana bark extract (PMBE) and grape seed extract (GSE) on dentin demineralization caused by acid.Methods:40 root dentin blocks with half of the surface covered were randondy divided into 4 groups (n =10).All samples were subjected to pH cycling for 8 days,and deionized distilled water(DDW),0.1％NaF,12％ PMBE solution and 12％ GSE solution were used as the experimental solutions in the 4 groups.The dentin mineral density(DMD) of the both sides was determined using micro-computed tomography.The D-value of DMD between nn-demineralized and demineralized side (△DMD)was calculated.The samples were observed with field emission scanning electron microscops (FE-SEM).Results:The △DMD of DDW,NaF,PMBE and GSE groups was 198.64 ±59.97,45.94 ±24.21,90.23 ±28.77 and 105.07 ±29.53 respectively.The △DMD between PMBE and GSE groups had no significant difference (P ＞ 0.05),which were both higher than that of NaF group (P ＜ 0.05) and lower than that of DDW group(P ＜ 0.05).The FE-SEM revealed that the dentin tubules in DDW group were completely open,but in NaF group were essentially closed in PMBE group and GSE group were spindle shaped or narrow crack opening.Conclusion:PMBE and GSE had almost the same effect on improving the acid resistance of dentin.

Effect of Pinus massoniana needle extract on root dentin demineralization in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aims to evaluate the effects of Pinus massoniana needle extract (PMNE) on inhibiting demineralization of root dentin.</p><p><b>METHODS</b>Root dentin blocks were randomly divided into distilled deionized water (DDW) group, fluoride sodium (NaF) group, and 4%, 8% and 12% PMNE groups according to the experimental solution used in the process of pH cycling in each group. All specimens in each group experienced pH cycling for 8 d. The dentin mineral density (DMD) of the normal dentin and demineralized dentin and their D-value (ΔDMD) were determined using micro computed tomography. The morphology of dentin surface after pH cycling was also observed using a scanning electron microscope.</p><p><b>RESULTS</b>The ΔDMD values in all PMNE groups and the NaF group were considerably lower than the ΔDMD in the DDW group (P<0.05). The ΔDMD values of the 8% and 12% PMNE groups had no difference (P>0.05), both of which were lower than the ΔDMD in the 4% PMNE group and higher than that in the NaF group (P<0.05). The dentin tubules were partly opened in the PMNE groups. The opening degrees of the dentin tubule in PMNE groups were significantly less and smaller than the opening degree in the DDW group and were larger than that in the NaF group.</p><p><b>CONCLUSIONS</b>PMNE can inhibit the deminera-lization of root dentin and can slow down the reduction in DMD. PMNE has the potential to prevent caries, and 8% PMNE can effectively inhibit dentin demineralization.</p>

Production suitability regionalization study of Pinus massoniana / 中国中药杂志

The distribution, yield and sample information data of Pinus massoniana was obtained by document literature and sample investigation. Based on sample data from 12 provinces including 414 sample plots and environment factors in China,the distribution regionalization of P. massoniana was predicted by using Maxent and spatial analysis function of ArcGIS. The results showed that the northernmost distribution of P. massoniana was 33.5 degrees north latitude, and it mainly distributed in the southeast in China. Based on plant age, plant height, yield per plant and other growth index from 414 sample plots, combined vegetation form and other data, the growth regionalization of P. massoniana was carried out by using SPSS and related functions of ArcGIS. The results showed that Fujian, Guizhou and Guangxi had a lager distribution area of P. massoniana, meanwhile, it had a relatively higher yield of fresh pine needles. The relational model between environmental factors and shikimic acid,and procyanidin, and the total lignans was constructed by using SPSS regression analysis method. Then the spatial calculation function of ArcGIS was used tocarry out the quality regionalization of P. massoniana based on the relational model. The results showed that east of Sichuan, Guizhou, Chongqing had a good pine needles quality. Based on the distribution, growth and quality regionalization, the production suitability regionalization of P. massoniana was carried out. The results showed that the optimal planting base region mainly distributed in east of Sichuan, middle and east of Guizhou, and east of Guangxi.

Studies on chemical constituents from fresh pineneedles of Pinus massoniana / 中草药

Objective: To study the chemical constituents from the fresh pineneedles of Pinus massoniana. Methods: Certain chromatography means were used in the isolation and purification, and the structures of all the compounds were identified by means of spectroscopic analysis and physicochemical properties. Results: Fourteen compounds were elucidated respectively as (+)-catechin (1), (+)-gallcatechin (2), phlorin (3), tachioside (4), 3,4-dimethoxyphenyl-1-O-β-D-glucopyranoside (5), 3,4-dimethoxyphenyl-1- O-(3-O-methyl-α-L-rhamnopyranosyl)-1→2-β-D-glucopyranoside (6), citrusin D (7), (6S,7E,9R)-roseoside (8), raspberry ketone- O-β-D-glucopyranoside (9), (-)-oplopan-4-one-10-α-O-β-D-glucose (10), massonianoside D (11), massonianoside B (12), isolariciresinol-9'-O-α-L-arabinofuranoside (13), and (2R,3R)-taxifolin-3'-O-β-D-glucopyranoside (14). Conclusion: Compounds 2-6, and 10 are isolated from the plants of Pinus L. for the first time, and compounds 7-9 are obtained from P. massoniana for the first time.

Effect of Pinus massoniana Lamb. bark extract on lytic cycle of Epstein-Barr virus.

Pinus massoniana bark extract (PMBE) at a concentration of 60 μg/mL or more inhibits the expression of Epstein–Barr virus (EBV) lytic proteins, such as Rta, Zta, and EA-D. EBV lytic cycle was blocked by inhibiting the transcription of immediate-early genes. The results suggest that the PMBE has anti-EBV activity. Thus, the extract is potentially useful in preventing the lytic development of EBV in vitro.

Comparative study on bio-activity of polysaccharide from Pinus massoniana pollen and its sulfated derivative / 中国生化药物杂志

Purpose To study the polysaccharide from Pinus massoniana pollen(PPM)and to compare its anti-tumor,immune modulation activities and scavenging qualities of free radical with its sulfated derivative(S-PPM).Methods PPM Wag chemically modified by chlorosulfonic acid-pyfidine method and their bioactivities were compared.Results The substituting degree of S-PPM was 1.47.Results showed that S-PPM was more powerful in inhibiting the growth of tumor cell in vivo and in vitro and in promoting T,B lymphocytes than PPM.But there Wag no remarkable difference in promoting phagocytosis of macmphages.S-PPM was stronger in scavenging superoxide anion radical than PPM but it wag vice versa in scavenging hydroxyl radical.Conclusion S-PPM inhibited the cancer cell growth mainly through specific immunity.Sulfate of PPM influenced its quality of scavenging free radicals greatly.