Extraction, Isolation, Assaying Of Pinellin And Its Effect On Hela Cell

Rhizoma Pinelliae (Pinellia ternata (Thunb.)Breit.) is a plant of the Pinellia Tenore ofAraceae. As a traditional Chinese herbal medicine, Pinellia ternata has many founctions such asdrying dampness to eliminate phlegm, calming the adverse-rising energy to stop vomiting anddissipating palpable and removing stasis. And mainly active components of which are sterols,alkaloids, active polysaccharides, amino acids, organic acids and proteins, but Pinellia ternataagglutinin (PTA) is one of the main components of total protein of Pinellia ternata. Recentstudies show that pinellin with multiple biological activites is the main active ingredient ofPinellia ternata in insect resistance, anti-tumour and anti-fertility, which make it a research focus.In this paper, the center of attention is protein and PTA including the dynamic changes of totalprotein of Pinellia ternata and the effect of PTA on Hela cells in vitro.In this study, four kinds of commonly used total protein extraction methods (acetoneprecipition method,95%(NH4)2SO4salting-out method, TCA-acetone precipitation method andTris-saturated phenol method) were used to extract total protein of Pinellia ternata in equalconditions.Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis was used todetect the total protein extracted by the four methods. The electrophoresis results showed that:the electrophoretic behavior of the total protein extracted by the four methods had no significantdifference. Also, Bradford protein assay kit was used to determine the total protein content andthe results showed that:TCA-acetone precipitation method (1.082mg/mL)ú95%(NH4)2SO4salting-out method (0.678mg/mL) úTris-saturated phenol method (0.493mg/mL) úacetoneprecipition method (0.369mg/mL). So TCA-acetone precipitation method is the best one fortotal protein extraction of Pinellia ternata.TCA-acetone precipitation method was used to extract the total protein of Pinellia ternatasamples of different growth stages (bud stage, seeding stage, bulbil stage, spathe stage andmaturation stage). And the samples collected in different periods were divided into two groups offresh and dried. SDS-PAGE electrophoresis and Bradford protein assay kit were used to detect the total protein in different growth stages. The results of SDS-PAGE electrophoresis of the twogroups showed that: the total protein composition was different in different growth periods infresh group. And the protein bands of samples of fresh group were at least10bands more thanthat of the dried group. But samples of dried group had some protein bands which were not existin fresh group, such as the proteins with the apparent molecular weight of26kDa and35kDa.However, the proteins with the apparent molecular weight of12kDa and24kDa were in largeproportion in all the samples of the two groups.The determination results showed that: total protein content of samples of fresh groupchanges in a manner like this: maturation stageúspathe stageúbud stageúbulbil stageúseeding stage, while that of dried group as maturation stageúbulbil stageúbud stageúspathestageúseeding stage. It can be concluded that total protein content is highest in maturation stagebut lowest in seeding stage. The results showed that total protein components were different indifferent growth periods of Pinellia ternata, so does the same protein content. In addition, totalprotein content and types also had some differences between the fresh and dried groups.One kind of mannose-Sepharose4B affinity gel was designed to isolate PTA from totalprotein of Pinellia ternata. The results of SDS-PAGE electrophoresis showed that its apparentmolecular weight was12kDa. Further studies showed that the lectin is a glycoprotein, and canagglutinate erythrocytes in mice.In this paper, anti-tumor activity of the lectin was further studied. Hela cells were treatedby five different concentrations (0.004mg/mL,0.02mg/mL,0.1mg/mL,0.5mg/mL and1mg/mL) of PTA, and MTT method was used to detect cell viability. The results showed that lowconcentrations lectin (0.004mg/mL,0.02mg/mL and0.1mg/mL) can promote Hela cellproliferation, but the promoting proliferation effect weakended with concentration and timeincreased, the maximum efficiency of promoting proliferation23.36%was at the time when cellswere cultured for48hours. But high concentrations lectin (0.5mg/mL and1mg/mL) inhibitedHela cell proliferation, and the effect increased in a dose and time dependent manner, themaximum inhibition rate71.89%was at the time when cells were cultured for72hours.At the same time, Hela cells of logarithmic phase growth were treated with the two concentrations lectin (0.004mg/mL and1mg/mL) with promoting and inhibition proliferationeffect, PBS as the control, and cultured for24hours,48hours and72hours, respectively. Thenthe cells growth state was observed under inverted microscope photography, the results showedthat: cell treated by0.004mg/mL lectin was all growth better than the control group at the threeperiods. While cells treated by1mg/mL lectin were in a poor state. When cultured24hours,some cells were still in division and proliferation state, and some cells dead. But cell morphologywas significantly different from the control group. When cultured48hours and72hours, all thecells were inhibited, and began to shrink to death, suspending in the culture medium.