Screening Of Antitumor Active Components From Seeds Of Taxus Wallichiana Var.mairei And Preparation Of Nano Micelles

Background:Cancer remains a significant challenge for human health today.Current treatment approaches mainly include radiation therapy,immunotherapy,surgical removal,and chemotherapy,among others.Chemotherapy drugs have a certain therapeutic effect on cancer at early,middle,and late stages,and they are often used in combination with other treatment approaches,making them a common strategy for treating cancer.However,the toxicity of chemotherapy drugs is well known.Therefore,finding chemotherapy drugs with better anti-tumor efficacy and lower toxicity becomes crucial.Natural plants are considered as reservoirs containing rich bioactive compounds.Studies have shown that Taxus wallichiana var.mairei possesses various pharmacological activities,with its most notable effect being its anti-tumor properties,and the seeds of Taxus wallichiana var.mairei are rich in resources and exhibit excellent anti-cancer activity.Therefore,focusing on the screening of ideal anti-cancer active ingredients from Taxus wallichiana var.mairei seeds is an effective approach for cancer treatment.Polymer micelles have shown numerous advantageous properties in the advancement of anticancer drugs,including improved solubility,enhanced targeting specificity,reduced toxicity,increased bioavailability,and heightened anti-tumor effectiveness.Utilizing polymer micelles to deliver natural products into the body can significantly augment the therapeutic outcomes of anticancer medications.Thus,in this study,Taxus wallichiana var.mairei seeds were selected as the source material for screening potential anticancer drugs.Subsequently,these drugs were delivered using polymer micelles,aiming to enhance anti-tumor efficacy and mitigate toxicity.Methods and results:1.Antitumor active fractions and monomeric compounds were isolated from the ethanolic extract of Taxus wallichiana var.mairei seeds using organic solvent extraction,silica gel column chromatography,thin layer chromatography and other means of separation and purification,combined with the method of MTT.The compound was then identified as ecdysteroid（EC）through various analytical techniques.The antitumor activity of ecdysteroid（EC）and the high-content fraction（BE）of EC were evaluated using the MTT assay.The results showed that EC exhibited good antitumor effects against Hep G2,with half maximal inhibitory concentration(IC₅₀)of 68.88μg/m L.However,it demonstrated weaker antitumor effects against A549 and U251.On the other hand,BE showed significant antitumor effects against Hep G2,A549,and U251,with IC₅₀ values of 34.34μg/m L,35.25μg/m L,and 61.56μg/m L,respectively.2.The High-Performance Liquid Chromatography（HPLC）analysis method for the determination of ecdysteroid（EC）from Taxus wallichiana var.mairei seed extract was developed.Methodological investigations were carried out to assess its suitability and performance.The results confirmed the successful development of the HPLC analysis method for EC,and meet the required methodological standards.The content of EC in the BE was determined to be（62.00±0.21）%using the developed analysis method.The extraction process of BE was optimized using a combination of single-factor and central composite response surface methods,with the extraction yield of EC as the indicator.Based on the results obtained from the single-factor experiments,the optimal extraction conditions for BE were determined as follows:a temperature of 60°C,ethanol concentration of 50%,extraction time of 9 hours,solid-liquid ratio of 1:10,and a single extraction cycle.The fitted model from the central composite response surface analysis showed a significant P-value of 0.0003 and a high R² value of 0.9147,indicating that the regression model is statistically significant and effectively represents the experimental results.Finally,the optimized extraction conditions were determined to be a temperature of 70°C,ethanol concentration of 75%,and an extraction time of 6 h.The resulting extraction yield of EC was（607.74±15.18）μg/g.3.The HPLC method for the analysis of BE was successfully established.The results showed that the analytical method met the methodological requirements and was suitable for quantitative analysis of BE in-vitro.To construct BE micelles（BE-M）,the solvent injection method was employed,and a single-factor approach was utilized to optimize the preparation process.The optimized BE micelles were assessed for particle size,polydispersity index（PDI）,encapsulation rate,drug loading,transmission electron microscopy,critical micelle concentration（CMC）,X-ray diffraction analysis（XRD）,Fourier-transform infrared analysis（FT-IR）,and stability under ambient temperature conditions.Additionally,the cumulative release of BE-M was investigated using the dialysis method in hydrochloric acid solution（p H 1.2）,phosphate buffer solution（p H6.8）,and phosphate buffer solution（p H 7.4）.The results from the single-factor optimization revealed that the optimal temperature was 50°C,the speed was 1600 rpm/min,and the ratio of BE to m PEG-PCL to TPGS was 10 mg:20 mg:10 mg.In vitro characterization demonstrated that BE-M comprised uniformly dispersed spherical particles with a particle size of 34.37±0.11 nm and a PDI of 0.152±0.003.The encapsulation efficiency was determined to be 86.53±1.54%,with a drug loading of 21.63±0.87%.The low CMC value of BE-M（0.881μg/m L）indicated its excellent resistance to dilution.XRD and FT-IR analysis confirmed the successful encapsulation of BE within BE-M,thus validating the successful preparation of BE-M.Stability studies revealed that BE-M remained stable for a minimum of 7 days under ambient temperature conditions.In vitro release studies indicated that BE-M exhibited favorable release behavior in all three release medias and had a sustained release effect on BE.4.The HPLC method for the detection of BE in different organs of mice was successfully established and its selectivity was evaluated.The distribution of BE and BE-M in different organs of mice was investigated.The anti-tumor activity of BE-M was evaluated using Hep G2,A549,and U251 tumor cells.The safety of BE-M was assessed using zebrafish as a model organism.The results show that HPLC has good selectivity for the detection of BE in mouse tissues and can effectively analyze and detect BE in different tissues.Tissue distribution studies showed that BE-M increased BE accumulation in various tissues,with the highest accumulation in liver and brain,suggesting that BE-M has a targeted effect on these organs.Additionally,BE-M extended the retention time of BE in different tissues.The MTT assay results showed that the IC₅₀ values of BE-M against Hep G2,A549,and U251 were 25.87μg/m L,28.04μg/m L,and 48.54μg/m L,respectively,while the IC₅₀ values of BE against Hep G2,A549,and U251 were 34.2μg/m L,34.98μg/m L,and 62.75μg/m L,respectively.This indicates that BE-M significantly improved the in vitro anti-tumor effect of BE,highlighting the superiority of BE-M.The safety evaluation in zebrafish revealed that lethal concentration(LC₅₀)of BE-M on the 3rd day(LC₅₀=219.45μg/m L)was higher than that of BE(LC₅₀=138.06μg/m L),indicating that BE-M reduced the toxicity of BE and exhibited good safety.