Study On Anti-lymphoma Pharmacodynamics Of Volatile Oil From Houttuyniae Herba And Preparation Of Its Solid Lipid Nanoparticles

Non Hodgkin's lymphoma(non-Hodgkin lymphoma,NHL)captured 90% of Lymphoma that was a hematologic malignancy.NHL could be divided into T cells NHL and B cells NHL with incidence rate of 80%.Diffuse large Bcelllymphoma(DLBCL)was the most common aggressive lymphoma with poor treatment and prognosis,so the breakthrough on pathogenesis and clinical treatment was urgently needed.Houttuynia cordata Thunb stemmed from saururaceae plants with spicy,slightly cold from the sight of Traditional Chinese Medicine,which was mainly used for the treatment of carbuncle spit lung abscess,heat dysentery,sore and swollen pain in treating symptoms.The Houttuynia cordata contained a large number of physiologically active substance,of which the volatile oil taking the main components.The volatile oil from Houttuynia cordata Thunb Herba(HHO)had a good curative effect on bronchitis,pneumonia,upper respiratory tract infection,chronic cervicitis and whooping cough,and had a certain curative effect on acute conjunctivitis and urinary tract infection.Therefore,study on the HHO had become a hot research topic.Previous experiments showed that HHO could significantly inhibit the growth and proliferation of diffuse large B cell lymphoma SUDHL-4 cells.In this paper,the effect of HHO on non Hodgkin's lymphoma in vitro and vivo and the preparation of solid lipid nanoparticles were inspected which could provide a scientific basis for the study of Houttuynia as a new anti-tumor medicine.1.Apoptosis of SUDHL-4 cells induced by HHO in vitroCell growth inhibition rate induced by HHO was detected by CCK-8 method,and the apoptosis rate and cell cycle changes were observed by FITC-Annerxin V/PI and PI staining.Optical microscope and fluorescent microscope were used to see the cell morphological changes and apoptosis death state induced by HHO.Apoptosis pathway was finally got through phosphorylation of antibody chip.The results of CCK-8 assay showed that HHO had significant inhibitory effect on SUDHL-4 cells in a time and concentration dependent manner.The inhibition rate of 24 h,48h.and 72 h were 77.55%,84.35% and 90.94% respectively;IC50 were 0.3198,0.2329 and 0.2001 ?L/mL respectively,the difference between groups was statistically significant(P < 0.05).After induced SUDHL-4 cells with the concentration of 0.10?L/mL,0.15?L/mL,0.20?L/mL,0.25?L/mL,0.30?L/mL HHO,48 h cell apoptosis rate was respectively(5.95 ± 1.35)%,(13.12 ± 0.28)%,(27.15 ± 0.55)%,(36.35 ± 0.93)%,(76.68 ± 1.01)% and(96.37 ± 0.87)%.The results showed that HHO could block SUDHL-4 cells in the G2/M phase,and speculated that the mechanism of inhibition of cell growth by HHO might be related to the inhibition in G2/M phase.After induced SUDHL-4 cells by different concentrations of HHO for 48 h,cell morphology and cell number decreased in a concentration dependent manner under optical microscope;red green cells and orange cells with cell membrane fragments damage increased with concentration increasing under fluorescence microscopy,indicating the HHO can promote the apoptosis of lymphoma cell line SUDHL-4.Application of protein chip technology in phosphoric acid,identify key protein antibodies in multiple signaling pathways in CSP100 chip to get different drug combination in blank group,by selecting the differences between samples of phosphorylation sites and its modulation ratio,mapped that HHO promoted the apoptosis of lymphoma cells by a key signaling pathway Raf-MEK1/2-ERK1/2-Bcl-2 and a key protein Bcl-2,which provided a effective scientific basis for the application of HHO on anti-lymphoma.2.Study on pharmacodynamics of HHO in vivoSUDHL-4 lymphoma tumor bearing mice model were established and randomly divided into 5 groups: vincristine group of 1mg/kg,HHO230mg/kg,150mg/kg,70mg/kg and negative control group.During the treatment,the living condition of mice was observed,the tumor weight was measured and the tumor inhibition rate was calculated.At the end of treatment,tumor and tissue were removed for HE staining to observe the changes of cell morphology.The results showed that HHO had a significant inhibitory effect on lymphoma in a concentration dependent manner.With the increase of the concentration of HHO,the inhibition rate of the drug to lymphoma was also increasing,which can reach up to 51.82%.Tumor sections showed that compared with the negative group,the cell gap was more and more biger,the contour structure had become increasingly blurred,the surrounding connective tissue grew,which indicating that HHO had inhibitory effect on lymphoma obviously.Mice visceral sections showed liver pathological changes when low concentrations of HHO administered to mice,while liver and spleen had different pathological changes at the high concentration of HHO.Therefore,HHO can cause liver and spleen injury in mice by intraperitoneal injection.3.Preparations and evaluation of Volatile oil from Houttuyniae Herba-Solid lipid nanopartcles(HHO-SLN)HHO-SLN was prepared by melt ultrasonic emulsification method.Single factor test and orthogonal test were used to select the best preparation process and the optimal prescription of preparation.This paper evaluated the HHO-SLN from the appearance,particle size,zeta potential,encapsulation drug loading,release and stability etc.The final optimized preparation process of prescription: glycerol monostearate was 0.25%,compound emulsifier was 3%,the ratio of poloxamer and egg yolk lecithin was 3:1,the HHO was 1%,the Tween 80 was 0.5%.Adding the aqueous phase into oil phase at the same temperature by constant temperature magnetic stirring slowly,stirring for 20 min for the formation of colostrum,and then making colostrum ultrasonic dispersion at the amplitude of 100% for 15 min.Filter the mixture with 0.22 nm microporous membrane after natural cooling at room temperature,and then the HHO-SLN was made successfully.HHO-SLN presented a regular spherical shape,small average particle size of(42.89±2.13)nm and uniform particle size distribution.The total number of nanoparticles of size between 10nm-100 nm nanoparticles accounted for the ratio of 91.8%,the average Zeta potential was(-20.4±0.78)mv,indiciting the system was relatively stable and uniform.The encapsulation efficiency and the drug loading were(91.49±2.40)% and(6.08±0.42)% respectively.The stability experiments show that the high temperature and strong light can affect the stability of the preparation with the particle size increasing and the encapsulation efficiency decreased.Long term experiments show that HHO-SLN should avoid light preservation under the condition of 4 °C,in order to maintain the appearance of preparation transparent and free of debris.The experimental results showed that HHO can induce the apoptosis of Diffuse large B-celllymphoma SUDHL-4 cells in vitro and in vivo with lower toxicity compared with the traditional Chinese medicine extract vincristine.The preparation of HHO-SLN could change HHO from liquid medicine into a solid state,and improve the stability and bioavailability of the drug,so the preparation of HHOSLN was stable and practical in production.