Researches On The Active Components In Fermented Croton Seeds For Reducing Toxicity And Keeping Efficacy

Fructus crotonis is the dried ripe fruit of Croton tiglium L.,a plant of Euphorbiaceae family recorded in the current Chinese Pharmacopoeia.Fructus crotonis has two types of toxic ingredients.The first type,with the fatty oil(croton oil)up to 60%,is both toxic and active,and can separate free croton oil out from the intestines by oral administration,which would enhance the intestinal secretion and motility and cause drastic diarrhea.The other one is a protein(crotin),which lyses the erythrocytes and leads to local necrosis.These two toxic ingredients would decrease,degenerate or lose their activity when being heated,therefore the oil cream(croton cream)is usually prepared by boiling its kernels,which produces positive effects on relieving its toxicity.There are many ways to process Fructus crotonis,but there is only one mothed in the current pharmacopoeia,namely defatted croton seed powders.Its effective dose and toxic dose is very close,and further more,traditional method is too complicated,the content of oil observed empirically,so the final results differ greatly and can not guarantee drug safety.There may be poisoning and even life-threatening if used improperly or overdose.Considering this,it is necessary to find a new better ways to solve such problems.Bidirectional fermentation,a method created by the author's laboratory personnel in 1980s for the solid fermentation of combination of medicinal fungi and medicinal herbs,paves a new path in decreasing the toxicity in those toxic herbal medicines.In preliminary research,it has achieved some success.in researches on the active components in Strychos nux-vomica fermented by Trametes Cinnabarina for reducing toxicity and keeping efficacy.The technology of bidirectional fermentation was adopted at the primary stage to carry out a toxicity release experiment,which found a significant decrease in the toxicity of Ganoderma lucidum(Curtis:Fr.)P.Karst,and the fermented products of croton(LBJZ).The topic "Researches on the Active Components in Fermented Croton seed for Reducing toxicity and Keeping efficacy"on the basis of the pre-project,was funded by Jiangsu Provincial Bureau of pharmaceutical science and technology projects,project number was LZ09018.Starting from the Content difference of Toxic substances among the Fructus crotonis,traditional processed products and fermentation products,focusing on the material basis,this thesis has explored the possibility of fermentation processing method and mainly includs the following sections:1.Toxicological comparison between defatted croton seed powder and two fermentation products of Fructus crotonisIn order to compare the toxic potency between processed and fermentation products of Croton tiglium,the acute oral toxicity,induced inflammatory and hematocytolysis were observed among raw Semen crotonis(seed kernel),sterilized Semen crotonis,raw Fructus crotonis(whole fruit),sterilized Fructus crotonis,Testa et pericarpium crotonis(fruit shell and seed coat),Semen crotonis pulveratum(defatted croton seed powder)and the two fermentation products of Fructus crotonis solid-fermented by Ganoderma lucidum and Beauveria bassiana(called Lingba Junzhi and Baiba Junzhi in Chinese,respectively).After being administrated by i.g.once or many times one day,the median lethal dose(LD50)of mice for Lingba Junzhi,Baiba Junzhi,sterilized whole fruit,defatted croton seed powder,sterilized seed kernel,raw whole fruit and raw seed kernel was 1.541 g/kg,1.495 g/kg,0.894 g/kg,0.787 g/kg,0.728 g/kg,0.38 g/kg,0.308 g/kg by large to small order;while the fruit shell and seed coat was low-toxic and its maximum dosage to infuse to mice was about 12.5 g/kg.When applying their ether extracts on the mouse ears,the raw seed kernel,sterilized seed kernel,raw whole fruit,sterilized whole fruit and defatted croton seed powder induced swelling(inflammation)of the mouse ears;but the fruit shell and seed coat,Lingba Junzhi and Baiba Junzhi didn't.Adding their saline extracts to 2%rabbit erythrocyte suspension to investigate hemolysis in vitro,only the raw seed kernel and raw whole fruit caused obvious hemolysis;the sterilized seed kernel,sterilized whole fruit,defatted croton seed powder fruit shell and seed coat,Lingba Junzhi and Baiba Junzhi didn't.The experimental results have shown that the toxicity of the both processed and fermented Croton tiglium are lower than that of the original crude drug,and the fermentation products of Fructus crotonis have a much lower toxicity.2.Effects of fermented Croton in mouse gastrointestinal propellant rate and the tumor inhibitory effect of croton different extract fractions of LBJZ in vitroCompare effects of mouse gastrointestinal propellant rate among Fructus Crotonis,Semen Crotonis Pulveratum and fermented Croton:LBJZ and BBJZ.Explore whether or not they keep toxicity and keep efficacy after fermentation.As a result,after fed mice with solution LBJZ and BBJZ.The mouse gastrointestinal propellant rates of LBJZ and BBJZ was far less than Fructus Crotonis and had Significant differences(P<0.01),but had no Significant differences with Semen Crotonis Pulveratum(P>0.05).Research Inhibition of croton extracts for proliferation of A549 cells and MKN-45 cells through culture cell in vitro.The inhibition is different in different extract fractions for proliferation of A549 cells while have shown some inhibition for proliferation of MKN-45 cell.Comparing with virus group,1 ?mol/mL Croton triazole glycoside can significantly inhibit the HSV-1 TK mRNA expression in 6h through Real-time quantitative RT-PCR(P<0.01)and lost the antiviral effect in 48h.vitro experiments showed croton triazole glycoside had early anti-HSV-I replication effect.3.Studies on solid fermentation process for Fructus CrotonisTo investigate effects of fermentation time on the contents of two main toxic ingredients including fatty oil and total protein in Lingba Junzhi(LBJZ,a croton product fermented by Ganoderma lucidum(Curtis:Fr.)P.Karst.).By using gravimetric analysis and coomassie brilliant blue G-250 staining method,the contents of fatty oil and total protein in LBJZ fermented at day 0,12,16,20,23,26,29 and 32 were determined and compared,respectively.After methyl esterification,GC-MS was adopted to further analyze the principal components of fatty oils quantitively and qualitatively whose relative contents in percentage were determined with area normalization method.The contents of fatty oil and total protein in LBJZ were all decreased on the whole with prolonging the fermentation time.The total protein and the fatty oil content were lowered to the lowest level from day 12 to day 32 and from day 23 to day 32 of fermentation respectively,and retained at a rather stable level.42 chemical compounds in total were isolated and identified from fatty oils by GC-MS during the whole fermentation,among which 11 compounds' comparatively contents were more than 1.0%.Compared with the first 0-day fermentation,contents of 6 compounds increased while 5 compounds decreased on the 32nd of fermentation,and at least 8 new substances were formed.The optimal fermentation time of LBJZ is around 23 to 26 days.4.Comparison of the toxic ingredient contents of fermented croton fruits,crude croton fruits and defatted croton seed powdersTo lay the material basis for synergism and attenuation of applying fermentation to deal with Croton tiglium L.By using gravimetric analysis and Coomassie brilliant blue G-250 dye binding,the fatty oil and total protein contents of two fermentation products of croton fruits(The two products fermented by Ganodenna lucidum(Curtis:Fr.)P.Karst,and Beauveria bassiana(Bals.)Vuill were named Lingba Junzhi(LBJZ)and Baiba Junzhi(BBJZ)in Chinese,respectively.),crude croton fruits and defatted croton seed powders were determined,respectively,and GC-MS method was then used to analyze the chemical constituents of the fatty oils from the four samples mentioned above.The toxic substances of croton fruits,fatty oils and total proteins were greatly lowered by means of fermentation by G.lucidum and B.bassiana,was varied,and even lower than defatted croton seed powders.There were 39,24,36 and 42 chemical components identified in fatty oils from crude croton fruits,defatted croton seed powders,LBJZ and BBJZ by GC-MS analysis.Compared with crude croton fruits,the fatty oil composition and content of the two fermented croton fruits changed significantly,and there were five new substances emerged in LBJZ and in BBJZ,respectively.The content of toxic substances in croton fruits reduced significantly after fermentation.5.Studies on chemical constituents of fermented croton fruitsusing methods of solvent extraction,column chromatography,preparative chromatography and recrystallization etc for Its methanol extract fractions in fermented croton fruits,six kinds of compounds were isolated and identified by 1HNMR and 13CNMR:eicosanoic acid(1),heptadecanoic acid(2),phorbol esters(3),crotonic ester(4),crotonoside(5),and Croton triazole glycoside(6),compounds 4 and 6 are new compounds.6.Studies on Croton triazole glycoside resist HSV-? in vitroTo establish a method for determining the contents of Croton triazole glycosides in fermented croton fruits and defatted croton seed powders by RP-HPLC.The analysis was performed on a column(5 ?m,4.6 mm×250 mm)applying octadecylsilyl silica gel as loading materials.The mobile phase was a mixture of acetonitrile(A)and aqueous solution(B),gradient elution:3%A?10%A(0?15 min),10%A?15%A(15?25 min),15%A?20%A(25?30 min),20%A?30%A(30?35 min),30%A?95%A(35?40 min),,stop time:50 min.The volumetric flow rate was 1.0 mL·min-1,the column temperature at 35 ? and the detection wavelength at 260 nm.The linear range of Lot was 1.02?16.32 ?g·mL-1 and R2 was more than 0.999.The average recovery of Lot was 98.46%.Just fermented croton fruits and defatted croton seed powders contain Croton triazole glycosides and the order of content is:fermented croton fruits>defatted croton seed powders,LBJZ>BBJZ;With the extension of the fermentation time,the Content of Croton triazole glycoside in fermented croton fruits(LBJZ)showed an upward trend,and Content level of 20 days to 23 days reached a maximum and remained relatively stable state(the average level is about 0.075%).The method has good accuracy,specificity and reproducibility in application of quantitative analysis of Croton triazole glycoside in fermented croton fruits and defatted croton seed powders.Study the effect and mechanism of the anti-HSV-I of Croton triazole glycoside In vitro through MTT and CPE.Using real-time quantitative RT-PCR experiments investigate the drugs resist effect of the HSV-1 TK mRNA in 6,12,24,48 h.The 1mol/mL Croton triazole glycoside can inhibit the CPE becauseof HSV-I infection markedly in vero in 24h,the effect is almost with Positive drug acyclovir and lesions appear as +,while,the lesions of Virus group is + + +.And the effect decreased with decreasing drug concentration.Antiviral activity Croton triazole glycoside decreased in 48 to 72h,Compared with the virus group,there was no significant difference in CPE.The drugs did not prevent the HSV-i infection and there was no direct killing effect.