| Purposes:Natural traditional Chinese medicine has always been the treasure house for the development of new anti-cancer drugs.Qinglongyi,also known as walnut(Juglands ragia L.),is the dry skin of immature fruits(Juglands mandshurica Maxim.)and Juglans mandshurica[1].Qinglongyi was first published in "Kaibao Materia Medica",which is sexually spicy,bitter,astringent,and flat.It has the effects of detoxification,detoxification,analgesia,and pain relief[2].Qinglongyi has a long history of use in our country,but it has limited its use because of the lack of accepted methods of use.Juglone(Juglone)is the most potent anti-tumor herbal monomer in Qinglongyi extract,but it is not used clinically because of its poor water solubility and toxic side effects.The development of new dosage forms based on nanotechnology can improve drug targeting,reduce toxic side effects,and optimize bioavailability.This project is intended to conduct research through the following two parts:1.Screening and acute toxicity test of Qinglongyi's best anti-tumor extraction method.Second,the construction of juglone nanoparticles and its preliminary evaluation of the anti-tumor effect of gastric cancer.Methods:1.Selection and acute toxicity test of Qinglongyi's best anti-tumor extraction method.1.1 Qinglongyi heat reflux water extraction,Qinglongyi heat reflux alcohol extraction,Qinglongyi cold extraction alcohol extraction,Qinglongyi ultrasonic alcohol extraction four different extraction methods to prepare crude extract liquid,neutral filter paper filter liquid,filtered drug The solution was concentrated at 40? by rotary evaporation.The extract was placed in a negative 80? freezer for 24 hours and then taken out.The low temperature vacuum drier was dried to obtain 4 kinds of crude dry extract powder.Four kinds of bold dry extracts were weighed separately,and the content of solid substances per unit volume was calculated(formula:extraction rate = weight/volume of dry solids),and the extraction rate was calculated.The standard curve of Juglans mandshurica was established by HPLC,and the content of juglone in different extraction methods was further calculated and compared according to the regression curve formula(the above experiment was repeated 3 times).1.2 Four extraction methods The clean cream was dissolved and diluted to different concentrations.MTT colorimetry was used to compare four extraction methods in vitro to inhibit cell proliferation.The IC50 values of the four extracts were calculated.1.3 Compared with MTT colorimetric method,the cold ethanol extract of Qinglongyi had the most significant inhibitory effect on the proliferation of human gastric cancer cell MKN-45 cells in vitro.The IC50 results of RTCA dynamic observation(24 hours,48 hours,72 hours)were:24 hours:0.000202046g/mL(R=0.987215),48 hours:7.74913E-005g/mL(R=0.968029),72 hours:5.95942E-005g/mL(R=0.968114),the above experiment was repeated 3 times,each concentration 3 complex holes.Qinglongyi cold-extracted ethanol extract increased the number of MKN-45 cells arrested in S phase,and the proportion of S in high-dose group increased more than that in blank control group and low-dose group.Qinglongyi Cold Extract Alcohol Extract Induces Apoptosis(Significantly Increased Q2+Q4 Ratio)1.4 Based on the extraction method obtained in the above experiment,the mouse acute toxicity test was further performed to evaluate its safety.ICR mice were intragastrically administrated from 0-high-dose 6 group and fasted for 12 hours before gavage.The maximum intragastric administration volume was 0.4 mL/10 g body weight,15 min,30 min,1 h,2 h,4 h,8 h,and 12 h after intragastric administration.The mice were observed at 24h,48h,72h-14d,and weighed at 12h,24h,48h and 72h-14d.Blood was collected from the abdominal aorta for 14 days to determine the effect of the drug on the blood system.The mice were sacrificed and formalin,liver,spleen,lung and kidney were fixed in formaldehyde and embedded in paraffin.HE staining was used to observe whether there were toxic side effects on the main organs.The construction of juglone PLGA nanoparticles and its preliminary evaluation of the anti-tumor effect of gastric cancer.2.1 MTT colorimetric method to verify whether there is a synergistic effect of juglone monomer and iRGD(tumor penetrating peptide)in vitro.2.2 PLGA nanoparticles loaded with Juglans mandshurica were constructed by single emulsion method,dynamic light scattering,transmission electron microscopy and other techniques were used to characterize the study of juglans nanoparticles.Human gastric cancer MKN-45 cells were used to establish a subcutaneous tumor model in nude mice,and a comparison was made between the Qinglongyi primary extract gavage group,the tail vein injection of Juglans monosperma,PLGA-juglone nanoparticles,PLGA-juglone nanoparticles and iRGD co-administration,and the control group.In vivo anti-tumor efficacy of the tumor-bearing nude mice model of MKN-45 cells was analyzed and the experimental results were analyzed to reflect the improvement suggestions.Result:1.Qinglongyi hot reflux water extraction,Qinglongyi thermal reflux alcohol extraction,Qinglongyi cold extraction alcohol extraction,Qinglongyi ultrasonic alcohol extraction are used to weigh four kinds of crude dry extracts respectively,and the content of solid substances per unit volume is calculated(formula:Extraction rate = weight/volume of dry solid matter),calculate the extraction rate as follows:Qinglongyi hot reflux water supply 27.335%(n=3),Qinglongyi hot reflux alcohol supply 27%(n=3),Qinglongyi Cold soaked alcohol was extracted by 11.6%(n=3)and Qinglongyi ultrasonic alcohol by 12.8%(n=3).After being compared by HPLC,the contents of walnuts in the four extraction methods were 0.412 ?g/mL(n=3)in Qinglongyi hot reflux water,0.534 ?g/mL(n=3)in Qinglongyi hot reflux alcohol,and cold leaching in Qinglongyi.Alcohol was extracted at 23.030 ?g/mL(n=3)and Qinglongyi ultrasound was extracted at 5.967 ?g/mL(n=3).2,Qinglongyi cold-leaf ethanol extract on the human gastric cancer cell MKN-45 cell proliferation in vitro inhibition is the most obvious,Qinglongyi cold-leaf alcohol extracts make the number of MKN-45 cells arrested in the S phase increased.Qinglongyi cold-extracted alcohol extracts induce apoptosis(significant increase in the proportion of Q2+Q4).3.There was no death in the mice with intragastric administration up to 117mg/20g/day,and there was obvious loss of appetite,weight loss and activity in mice fed 78mg/20g/day group and 117mg/20g/day groups.cut back.From the fourth day onwards,he resumed eating and his body weight began to increase.No significant body weight changes were observed on the 14th day.After further examination of their blood routine,platelet count groups were different(n=3,some P values<0.05),white blood cell counts and hemoglobin were within normal values,and there was no significant difference between groups(n=3,P<0.05).His heart,liver,spleen,lung and kidney had no obvious HE staining.Blood routines need to be monitored during the administration of Qinglongyi drugs,and the dose changes dynamically according to patient conditions.However,the platelet count is subject to experimental operation and other causes of error,and further experimental verification is needed.4.There was a synergistic effect of juglone monomer and iRGD in the inhibition of MKN-45 cell proliferation in vitro,which was dose-dependent with Juglans mandshurica(P<0.0001),and was not related to the dose of iRGD.5.The formula for calculating the entrapment efficiency of nanoparticles is(encapsulation rate =amount of drug/total drug dose),and the formula for drug loading is(drug loading = drug content/total amount).When 10 mg PLGA contained 1 mg juglone monomer,the drug loading and entrapment efficiency was the highest,the entrapment efficiency was 60.8%,and the drug loading was:9.09%(n=3,P<0.05).When 10 mg PLGA was loaded with 1 mg of juglone monomer,the nanoparticle size was about 149.6 nm(n=5).Dynamic light scattering and transmission electron microscopy showed that the particle size was stable,and the nanoparticles rapidly released 50%at the constant temperature of 37? and then slowly released for 7 days.Human gastric cancer MKN-45 cells tumor-bearing mice model Qinglongyi cold-leaf ethanol extract intragastric administration group,Juglomern monotonic tail vein injection group,PLGA-juglone nanoparticle tail vein injection group,PLGA-juglone nanoparticles and iRGD common tail In the intravenous injection group and the control group,except for the control group,the other four groups had the effect of inhibiting tumor growth,and the difference was statistically significant(P<0.05,n=6).Two nude mice in the Juglone monomer group died on the second and third days of administration,respectively,and there were obvious toxicities in the internal organs.Both PLGA-juglone nanoparticles,PLGA-juglone nanoparticles,and iRGD co-administered both effectively inhibited tumour growth in tumor-bearing nude mice,but there was no statistical difference between the two groups.The molecular weight of iRGD may be small compared to PLGA-juglone.Co-administration was preceded by the metabolism of nanoparticles,which did not play a role in good targeted delivery.Therefore,further consideration was given to the preparation of PLGA-juglone-PEG-iRGD nanoparticles encapsulated by red blood cell membranes.The iRGD intercalated with PEG on the surface of the red blood cell membrane enters the long cycle with the PLGA-juglone nanoparticles and can theoretically achieve better drug delivery effect.Conclusion:The crude extract of Qinglongyi obtained by cold-leaf ethanol extraction has the highest content of juglone,and its role in the human gastric cancer MKN-45 cell line is to arrest cell cycle at S phase and induce apoptosis.The PLGA-loaded Pinl inhibitor Juglans mandshurica nanoparticles exert a good anti-tumor effect on human gastric cancer MKN-45 tumor-bearing mice,and they are safe and effective.It can not only increase the solubility of Juglans mandshurica but also exert passive targeting.The co-administration of PLGA-juglone nanoparticles,PLGA-juglone nanoparticles and iRGD both effectively inhibited tumor growth in tumor-bearing nude mice,but there was no statistical difference between the two groups.RBCs-PLGA-juglone-PEG-iRGD is expected to provide potential for the treatment of high-expression of pinl in gastric cancer and even more solid tumors,and has certain clinical application prospects. |
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