Comparison of Methods for Protein Extraction from Pine Needles

Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid （TCA）/acetone （TCA method） to determine their efficiency in separating pine needle proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and two-dimensional PAGE （2D-PAGE）. Proteins were then separated by SDS-PAGE. Among three methods the method using sucrose extraction buffer showed the highest efficiency and highest quality in separating proteins. In addition, clearer and more stable strips were detected by SDS-PAGE using sucrose extraction buffer. When the proteins extracted using sucrose extraction buffer were separated by 2D-PAGE, more than 300 protein spots, with isoelectric points （PI） ranging from 4.0 to 7,0 and molecular weights （MW） from 6.5 to 97.4 kD, were observed. This confirmed that the method with sucrose extraction buffer was an efficient and reliable method for extracting proteins from pine needles.

Study on Antibacterial Effects of Extracts from Pinus massoniana Lamb.Needles

[Objectives]To study the antibacterial effects of extracts from Pinus massoniana Lamb.needles.[Methods]In this experiment,the components from Pinus massoniana Lamb.needles were extracted by systematic solvent extraction method,and the antibacterial activity against four common bacteria,Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Aspergillus flavus and the antibacterial active component were examined for by punch method.[Results]Different solvent extraction rate was different,the rates of petroleum ether,chloroform,ethyl acetate,n-butanol,water extracts were 4.2%,16.7%,17.4%,21.1%,40.6%.All extracts showed inhibitory effect against test bacteria.It was observed that the inhibition of G+was stronger than G-,and the extracts had the best antibacterial activity to Staphylococcus aureus while the weakest to Aspergillus flavus.The antibacterial activity of the components decreased in the order:ethyl acetate extract>n-butanol extract>chloroform extract>petroleum ether extract>aqueous phase.The extracts were stable under ultraviolet radiation(UV)light and long term storage.The antibacterial activity of the extracts was weaker with the increase of pH value when the pH value≤8.[Conclusions]It is inferred that the antibacterial components in the extract of Pinus massoniana needles are widely distributed,and the components with medium polarity or above are the main antibacterial components.

A New Flavonoid in Pine Needles of Cedrus deodara

Objective To study the chemical constituents of flavonoids in pine needles of Cedrus deodara.Methods Flavonoids were isolated and purified from ethyl acetate extract of pine needles by chromatography on silica gel and Sephadex LH-20.Their structures were identified on the basis of spectroscopic analysis and chemical evidence.Results Five flavonoids were isolated and purified.Their structures were identified as cedrusone A(1),myricetin (2),2R,3R-dihydromyricetin(3),quercetin(4),and 2R,3R-dihydroquercetin(5).Conclusion Compound 1 is a new compound.Compounds 2-5 are isolated from pine needles of this genus for the first time.

Colorimetric Assay to Determine In Vitro Antibacterial Activity Against Clinical Isolates： Enhanced Activity in Damaged Chinese Masson Pine Needles

A colorimetric assay for antibacterial susceptibility testing of clinical isolates （Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae） is described based on the reduction of a novel tetrazolium salt, 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2Htetrazolium （MTS）, in the presence of phenazine methosulfate （PMS） as an electron-coupling agent. The combination of 200 μg/mL MTS with 25 μmol/L PMS resulted in production of large amounts of formazan within 1 h of exposure. In this setting, fractions extracted from Chinese Masson pine （Pinus massoniana Lamb.） needles damaged by the pine caterpillar Dendrolimus punctatus Walker were found to have enhanced levels of antibacterial activity. These fractions, which were designated ＂Master＂, ＂Technique＂, and ＂Strength＂, were isolated and identified by reverse-phase C18 cartridge concentration, gel filtration, and affinity chromatography. Two fractions purified from healthy and undamaged needles were designated H1 and H2, respectively. For all test bacteria species. Technique produced the lowest minimal inhibitory concentrations （MICs）, ranging from 2 to 32 μg/mL, and H2 produced the highest values, with four of the six M ICs being higher than 128 μg/mL. We found that the Rmax model fitted the data well in that the r^2 ranged between 0.87 and 0.96 （median, 0.92） and no statistically significant deviations from the model were found （P= 0.23）. The median coefficient of variation of the log RC50 values and the slope m of the fitted model for all six strains among the replicates were 38 and 41%, respectively. In the course of the investigation, the physiological and functional factors involved in pest damage to plants were also explored. In summary, the MTS-PMS colorimetric assay has advantages over existing methods for the examination of antibacterial activity, and could be developed further such that it would be suitable for screening new antibiotic molecules.

Preparation of Paraffin Section of Pinus sylvestris Needles

[Objective] Taking Pinus sylvestris needles as materials, a set of test con-ditions suitable for the preparation of paraffin sections of Pinus sylvestris needles was determined. [Method] Based on the traditional method for paraffin sections preparation, steps including fixation, dehydration, adhering to slides and staining were investigated taking the structural characteristics of pine needles into considera-tion. [Result] 70% ethanol was used in the FAA fixative; before affixed on slides, the cut sections were first expanded in a 40 ℃ water bath and taken out by using Su-perfrost Plus slides, fol owed by drying in a 35 ℃ oven for 24 h; pine needles were cut into 8 μm thick sections; staining was achieved by immersion in eosin for 5 min and in hematoxylin for 1 min. [Conclusion] The conditions obtained above can en-hance the effect of fixation and dehydration; adhering to slides is easy to operate and the sections are not easy to drop; the dyeing effect is relatively preferable.

Effect of Pine Needle on Immunity and Serum Cholesterol of Chickens

The research aimed to study the effects of pine needle on immunity and serum cholesterol of chickens. Totally 240 healthy Roman cocks （one day old） were randomly divided into four groups, three replicates each group and 20 chickens per replicate. The chickens in contrel group were fed with basal diet and nor- real drinking water; the chickens in groups I and II were fed with the basal diet added with 0.5 and 1.0 g/ehieken pine needle powder, respectively; the chickens in group HI were fed with pine needle decoction at the dose of 0.5 g/chicken. The pre-trial lasted 4 d, and the former trial lasted 70 d. The results showed that the percentages of CD3＋ CD4 ＋ T lymphocytes in groups I, II and III were higher than that in control group at the 15th day after the first immunization, but the percenta- ges in groups 1 and II were lower than that in control group at the 30th , 45th and 60th days, and the percentage in group III was lower than that in control group at the 45th day. The percentages of CD3＋ CD8 ＋ T lymphocytes in groups I, II and III were higher than that in control group at the 15th, 30th and 45th days after the first immunization, but the percentages in groups I and III were lower than that in control group at the 60th day. At the 14th and 21st days after the first immunization, the levels of NDV antibody in groups I and II were lower than that in control group, but the levels at the 28th and 35th days were higher than that in control group. The level of NDV antibody in group III was higher than that in control group at the 14th and 21st days while it was lower at the 7th and 35th days. The levels of serum total cholesterol and trigtyceride in groups I, II and III had lower trend compared with control group, and the levels of serum triglyceride in groups I and II were significantly lower than that in control group and group III （P 〈0.05 ）. The levels of serum low density lipoprotein in groups II and III were lower than that in control group, while the level of serum high density lipoprotein in group II was higher than that in control group, but there were no significant differences （P 〉 0.05 ）. In conclusion, pine needle has many functions as follows ： promoting the proliferation and differentiation of T lymphocytes, improving the content of CD3＋CD4 ＋ and CD3 ＋ CD8＋ lymphocyte, collaborating with vaecine to enhance the specific immune, and reducing the serum cholesterol level in different degrees.

Pool of Biological Resources for Potential Applications in Solid State Fermentation Obtained from a Forest Plantation of Pinus pseudostrobus Lindl, Mexico

A forest plantation, product of the reforestation of pine trees, represented a pool of biological resources for the implementation of a solid state fermentation process. The trees were identified as Pinus pseudostrobus Lindl from which lignocellulosic material in the form of pine needles was collected. Soil fungi, responsible for plant litter decomposition, were cultured at laboratory conditions and tested for their ability to grow on cellulose and hemicellulose as the sole carbon sources. A fungal strain, belonging to the genus Penicillium, was selected for growing it on pine needles as the substrate in a solid state culture. After following the culture for six days, the newly isolated strain exhibited a much higher capacity for spore production and holocellulose degradation, compared to a purchased strain of Penicillium chrysogenum and two control conditions. This work marks the beginning of future studies focused on commercial applications and represents the first report of a biotechnological process based on pine needles and their degradation by an ascomycetes species belonging to the genus Penicillium.