Studies Of Preparation Of Cryptoporous Volvatus Fermentation And Cryptoporus Polysaccharide And Their Anti-inflammatory Bioactivities

Cryptoporous volvatus is the sporophore of Cryptoporous volvatus[(Peck) Schear]which has been used as the folk remedy for tracheitis and asthma because of its activities of expelling wind and eliminating dampness,and dredging meridians to activate blood.It was early in the 15th century A.D.when the record of Cryptoporous volvatus appeared in Dian Nan Ben Cao(滇南本草).Its natural resource is limited. Currently,we have obtained a great quantity of Cryptoporous volvatus through fermentation culture technology.This substance is the same as natural resource by identifying its physical and chemical properties.Cryptoporous volvatus ferment substance(CFS) was proved to have special anti-inflammatory action and with low toxicity and without side effect by animal pharmacodynamics experiments. Development of this new Chinese medicine has good prospect.The dissertation was divided into four parts as below.Partâ… :Study of preparation of cryptoporous volvatus fermentation and anti-inflammatory bioactivity The results showed that the best carbon source of medium was glucose,the best nitrogen source was peptone and yeast extract paste,and pH was 4.8～5.0. The optimum fermentation condition:the optimum temperature was27～28℃,the agitation speed was 140 r/min,the fermentation period was 4～5 days.And the optimum culture medium dispensing of Cryptoporous volvatus submerged fermentation glucose 2.0%,peptone 0.5%, yeast extract paste 0.3%and KH₂PO₄ 0.1%were obtained by orthogonal experiment.Yield of Cryptoporous volvatus was affected by all kinds of factors in fermentation process.Total saccharides,reduced saccharides,pH value and aminenitrogen were each other in this process.At the end of fermentation,reducing sugar was less than or equal 0.6%,pH was less than or equal 3.8, and output attained more than or equal 27%(wet weight).The whole fermentation of Cryptoporous volvatus would last 3～5 days.These results would provide experimental evidence for production in large scale.The results of pharmacological experiments indicated that CFS by gavage could inhibite eosinophils in the BALF (bronchoalveolar lavage fluid) of the sensitized mice after antigen challenge in a dose-dependent manner,and significantly inhibite eosinophils accumulation in airway of the sensitized mice.It also indicated that CFS had active contents which could be absorbed by stomach.Partâ…¡:Pharmacological screen of active components in Cryptoporous volvatus This paper had extracted alkaloids,organic acids and polysaccharides separated from Cryptoporous volvatus fermentation,and had screened the bioactive ingredients which had special anti-inflammatory action using airway inflammatory cell of sensitized mice after antigen challenge as an activity monitoring parameter.The result of the experiments was that 0.13～0.20g alkaloid was obtained from 100g CFS by dilute hydrochloric acid extractation;1.2～4.0g organic acid was obtained from 100g CFS by ethyl acetate extractation;2.0～6.5g polysaccharide was obtained from 100g CFS with water extracting and ether de-estering.Screening these three constituents with eosinophils of sensitized mice after antigen challenge,the results showed that cryptoporus polysaccharide(CP) had stronger anti-allergic inflammation action;the order of anti-allergic inflammation action was as follow:polysaccharide＞CFS＞alkaloid＞organic acid.So CP was worth further studying.Partâ…¢:Primitive study of Cryptoporus polysaccharide purify and its bioavailability This paper was futherly purified with Cryptoporus polysaccharide,and was studied on the determination content,the molecular weight distribution and its effect on the inflammatory cell in the BALF(bronchoalveolar lavage fluid) of the sensitized mice after antigen challenge.And this paper was initially clarified with the bioavailability and effect intensity of CP by comparison of the inhibitory effect between ig and iv administration.CP was futherly purified by precipitation by PEG.The amount of total carbohydrates of CP,measured with phenol-sulfuric acid methed,was 87%.Accordinag to the molecular weight distribution by HPLC,CP had a characteristic peak different from the culture medium. The molecular weight of PC was in the range of 2000～200000,and it had a wide distribution of molecular weight.The chromatographic conditions were as follow:a gel filtration HPLC equipped with G2000SWxl column was applied for analysis;the mobile phase was 0.7%sodium sulfate;the flow velocity was 0.5ml/min and the temperature of colume was 29℃;refractive index detector was used.As the same time,the results showed that CP that was purified had significantly inhibitory effect on the inflammatory cell in the BALF(bronchoalveolar lavage fluid) of the sensitized mice after antigen challenge with different administrations and different usages.The bioavailability of CP in ig administration was about 20～40%.The effect intensity of CP in iv administration was about 2.5～3 times higher than that in ig administration. The results were that cryptoporus polysaccharide could be futherly purified and has stronger anti-allergic inflammation action than CFS.The main monosaccharide components of CP were fucose,xylose,mannose,galactose.Partâ…£:Primitive research on anti-imflammatory mechanism of Cryptoporus polysaccharide The time course of eotaxin and TNF-αmRNA expression in the lung tissue of sensitized mice after antigen challenge,and effects of Cryptoporus polysaccharide(CP) on these expressions were studied.Eotaxin and TNF-αmRNA expression were determined by semi-quantitative RT-PCR.The function implications of eotaxin mRNA and TNF-αmRNA expression were examed by detecting the number of total leucocyte and eosinophils in bronchoalveolar lavage fluid(BALF). The results showed that peak level of TNF-αmRNA expression appeared at 8h and eotaxin mRNA expression at 24h in the lung tissue of sensitized mice after antigen challenge.There was a significant difference comparison with control mice.Numbers of total leukocyte and eosinophil in BALF increased in sensitized mice comparison with normal mice.CP 3,10,30 mg·kg^-1 inhibited eotaxin and TNF-αmRNA expression,and inflammatory cells accumulation in airway of the sensitized mice in dose-manner.So inhibitory effects of CP on eotaxin and TNF-αmRNA expression might be one of effect mechanisms against allergic inflammation.Conclusion:1,The research of optimum preparation of CFS would provide experimental evidence for production in large scale.The results of pharmacological experiments indicated that CFS by gavage could significantly inhibite eosinophils accumulation in airway of the sensitized mice in a dose-dependent manner.It also indicated that CFS had active contents which could be absorbed by stomach.2,CP had stronger anti-allergic inflammation action than alkaloids and organic acids separated from Cryptoporous volvatus fermentation.So Cryptoporus polysaccharide might be the main bioactive ingredient which had special anti-inflammatory action.3,Cryptoporus polysaccharide futherly purified has stronger anti-allergic inflammation action than CFS.4,Inhibitory effects of CP on eotaxin and TNF-αmRNA expression might be one of effect mechanisms against allergic inflammation.