Analysis Of Hormone-like Ingredient In The Extract Of Pine Pollen Disruption Powder

Pine pollen is a dry pollen of Pinus massoniana,Pinus tabulaeformis or several species of plants.Pine pollen contains large amounts of nutrients and biological active substances such as proteins,amino acids,vitamins,minerals,nucleic acid as well as lecithin.In addition,it also has the function of regulating gastrointestinal,improving immunity,and relieving fatigue.Therefore,Pinus massoniana pollen was well known as the "green gold" in the area of health food.On the basis of that the clinical trials have detected that the hormone-like ingredient existed in the extract of pine pollen,we systematically isolated and purified the extract of pine pollen disruption in order to determine the type,structure and content of hormone-like ingredient in extract.The research results are as follows: 1? I22 separation.The Compound 1 was systematically analyzed by 1HNMR,13 CNMR,DEPT90 and HPLC-MS.The results indicated that the Compound 1 was two isomers including DBP and DIBP and it was not hormone-like compound.2? Determination of sex hormones.To analyze I22,three sex hormones including Estradiol(E2),Testosterone(T)and Progesterone(P)using UPLC-Q-TOF-MS.The results showed that the hormone-like ingredients of pine pollen disruption powder were E2,T and P.We used the Chemiluminescence Immunoassay method to determine the content of E2,T and P in the extract of pine pollen disruption powder.And the optimum conditions were determined by optimizing the volume of the substrate and the time of immune reaction.The best conditions were that testosterone of 200?L;progesterone of 200?L;and estradiol were and of 250?L,in addition,60 min was used as the reaction time.The proposed Chemiluminescence Immunoassay method was validated.The results showed that the relative deviation of precision and stability were less than 15%.Moreover,it also have a good linearity.The sensitivity of E2,P and T were 2.78 pg/m L,1.42 pg/m L and 0.15 pg/m L,respectively.The contents of E2,P and T in the extract of pine pollen disruption powder were 18.92 ng / g,16.45 ng / g and 32.03 ng / g,respectively.3? Determination of plant hormone content.We firstly find the gibberellin in the extract of pine pollen disruption powder.I22 samples and three plant hormone standards were analyzed by UPLC-Q-TOF-MS.The results showed that the extract of pine pollen disruption powder contained gibberellin-like hormone.A UPLC-Q-TOF-MS method was established for determining the content of gibberellin in the extract of pine pollen disruption powder.The method was carried out on a C18 column and acetonitrile and water were used as mobile phase.Then the effects of mobile phase solvent system and elution condition were further investigated.The chromatographic separation conditions were determined as: 0-0.5 min,20% acetonitrile;0.5-1 min,90% acetonitrile;1-5.5 min,90% acetonitrile;5.5-6 min,20% acetonitrile;6-6.5 min,20% acetonitrile.Furthermore,the proposed UPLC-Q-TOF-MS method was validated and the results showed that the average recoveries of gibberellin in the range of 50 ~ 250 ?g/m L were good(R2?0.9994),the average recoveries ranged from 88.4% to 95.5%,RSD was 2.9% ~ 5.6%.The results showed that the content of gibberellin the extract of pine pollen disruption powder was 0.599 mg/g.The method has the advantages of simple operation,high sensitivity as well as good stability.Thus it could provide the theoretical basis to determine the trace plant hormones of plants.In conclusion,we firstly found gibberellin in the extract of pine pollen disruption powder in this study.UPLC-Q-TOF-MS method was also well established for qualitative and quantitative analysis of plant hormones.This method possess the characteristics of high efficiency,high sensitivity and accuracy,it lays the foundation of the further investigation of plant hormones.