Data on Pine Needle Extraction and Bioactivity Testing

Description

The current study employed solid-liquid extraction using hexane to separate biomolecules from pine needles and GC/ MS QP2010 PLUS, Shimadzu for their identification. The solid and liquid cultures for microbial growth and UV-Vis to measure growth. Protein extraction was done using physical-chemical methods, separation using gel electrophoresis and analysis by MALDI-QUAD-TOF-MS-MS followed by Mascot software search for library comparison.

Steps to reproduce

The pine needles were harvested within the college campus near to university library. The needles were removed from the main veins, cut into small pieces, washed and exposed to atmospheric nitrogen overnight. Then, the needles were partitioned into reagent tubes and subjected to liquid nitrogen and frozen. The frozen needles were further grounded to powder and weighed. 100gm of powdered needles were mixed with 4ml of plant lytics (LY007) to break the cell wall so that during the extraction, the solvent should penetrate intracellular parts. The mixture was divided into two portions. Each portion was transferred into a reagent bottle and extracted using two fractions of 120ml of hexane was added in each bottle, Shaked at130 rpm for 12 hours, and the supernatant was collected and subjected to a rotary evaporator to get the solid extract. Identifying biomolecules in pine needle hexane extracts was done using GC/ MS QP2010PLUS, Shimadzu. An aliquot of 1µL of pine needle hexane extract was injected and separated by capillary gas chromatography. The ionization energy of 70ev was used to break down the eluents from capillary GC. The GC-MS analysis of pine needle hexane extract was achieved by searching against the NIST database and comparing the retention time of standard compounds with those of experimental results. Solid cultures and protein extraction The current study used solid culture, and yeast was stored at -20 ºC. Stored yeast was then subjected to liquid culture. Then 0.017g/ml, 0.025g/ml, 0.030g/ml and 0.050g/ml of pine needle extract were added to different flasks, followed by 4µl of yeast, 4µl of yeast without needle extract was used as control and shifted to cultivating shaker for 24hours under 30 ºC and 120rpm. The rate of yeast growth in samples and control was measured after 24hrs in terms of absorbance in UV-Vis light (580nm) at two up to three hours. When the absorbance was reached around 0.6, half of the liquid media in each flask of control was shifted into new four conical flasks and kept in the refrigerator together with one flask of each needle extract concentration, while other flasks were left on the shaker until the absorbance reached 1.5. Extraction and measurement of protein concentration The yeast cell pellets, obtained from 0.5 to 0.6 absorbance, were taken and placed into caped 1.5ml microtubes. Then, about 5ml of washing buffer (Pbs) was subjected to each microtube for washing. All the microtubes were then placed in the centrifuge machine for 5minutes at 3000rpm. The supernatant was removed from the microtubes and dumped. Following these steps, 100µl mammalian lytics (LY006) were added to each microtube and shook well. Then, a small amount of glass beads was added and vortexed for 3 minutes 4 times. The mixture in all microtubes was transferred into a centrifugation machine at the rotation of 12000rpm for 20 minutes at 4ºC.

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