Research On Identification And Isolation Effective Components Of Cordyceps Militaris Anti Chronic Renal Failure

CRF, chronic renal failure, means nephron lesion and renalfunction damage, resulted from primary or secondary renal diseases. Itinduces a series of metabolic disorder and a group of clinic symptoms.Hemodialysis and kidney transplantation play a very important role inthe terminal treatment of CRF, while not in the early and middle stageprevention and cure. The renal diseases are relatively frequent; nomatter what kind of pathogeny, whether are induced by immunologicmechanisms or not, if can not be treated timely and effectively, when thelesions of renal parenchyma accumulate to certain extent, they are allprogressive and irreversible as CRF. Thus, in the world there are manypatients of early CRF, several time of hemodialysis ones, urgently needtherapies to delay or stop the progression of CRF.It has been proved that Cordyceps sinensis(Berk.) Sacc and itspreparations not only help to delay the progression of CRF, but alsoalleviate the side effect of hemodialysis, accordingly improve theexistent qualification of CRF patients. But Cordyceps sinensis is tooprecious to apply widely, because of its limited natural sources anddifficult to ferment. Cordyceps militaris (L.ex Fr) Link is similar to C.sinensis on life history, chemical components, pharmacological actions,and so on. But it is only developed as a kind of common hygienical food.Moreover there is no report on whether C. militaris can anti the CRF yet.Objective: to study whether C. militaris has anti CRF function, andconfirm what is the effective component of it.Methods: mensurated the Median Lethal Dose and MaximumTolerance Dose of raw material fruit body of C. militaris on Kunmingmice by i.g. Induced Wistar rats into CRF model by feeding adulterated0.75% adenine food in 4 weeks. Then treated them 4 weeksrespectively with uremic clearance granule, two deferent doses of rawmaterial fruit body of C. militaris, aqueous extract from the fruit body ofC. militaris, and polysaccharide deposited by ethanol from the aqueousextract. Before and after the treatment, examined and compared thedifference of avoirdupois, the quantity of diet, drink and urine within 24h;routine analysis of blood, urine, kidney and liver function in all groups. Ttest analyzed the results of all the groups; routine examined thepathological sections of kidney samples. Cup method mensurated theinhibiting bacterial growth activity of C. militaris polysaccharide used inthe experiment. Hot water extracted polysaccharide from fruit body of C.militaris; coarsely isolated it by deposited it in different concentrations ofethanol from the aqueous extracts. Protein removed using Sevagmethod. Phenol-Sulfuric acid method determined the totalcarbohydrate content of sugar; carbazole method measured glucuronicacid content; Bradford method examined the quantity of been removedprotein. Applied Sephacryl S-300 gel column to further isolate and purifypolysaccharide, and determined their molecular weight. DNS methodmeasured reducing sugar content of coarse polysaccharidecomponents; Pyrogallol autooxidation method and Fenton reaction todetect the antioxidant activity of them, to speculate about their anti CRFmechanism.Results: the LD50 of C. militaris can't be check out, its MTD is rawmaterial 17k/kg, whose dose is equal to the 34 times clinical quantityused for a human being. The physiological and biochemistry outcomesindicated that the CRF model rats had come into being. After 4-week i.g.administration, those belong to treatment groups recuperated withincreasing avoirdupois and diets; while negative control group died a lot.Blood examination: after treated by C. militaris the amount ofalanine aminotransferase did not change obviously. The values of SCrand BUN in negative control group and low dose model group were farhigher than those in normal control group; and there also existedsignificant (P < 0.01) difference on Ccr between them; while the highdose model group, aqueous extract group, and the positive controlgroup were different from the normal control group(P<0.05). The threeitems of polysaccharide group had no difference from normal controlgroup(P>0.05).Urine examination: the 24h urine volume of treatment groups waslower than that of negative control group; but still more than that ofnormal control group. The TP of negative control group, positive controlgroup, and low dose model group exists no difference(P>0.05); Whilethat of high dose model group, aqueous extract group was different fromthat of negative control group(P < 0.05). However, the TP ofpolysaccharide group was similar to the normal control group, andremarkable lower than those of the negative control group (P<0.01).Pathologic anatomy examination: The kidney of normal controlgroup had no pathologic change; while that of negative control groupwith an increasing in weight, was hypertrophic, full of pallid stains andrough in the surface. The kidney of treatment groups was relativesmaller, lighter, darker, and smoother.Histological examination: the pathological sections of negativecontrol group kidney samples showed that glomerulus appearedpathologic atrophy, glasslike lesion; renal tubule epithelial tissuepresented degeneration and necrosis; renal mesenchyme could beobserved serious inflammation and a great number of lymphocytesinfiltration; adenine metabolites accumulated. The pathological status oftreatment groups was much better than that of negative control group,but could not reach the level of normal control group.C. militaris polysaccharide had strong inhibiting Gram-positivebacteria growth activity. It is mainly composed of neutral polysaccharidecomponents, while lack of acid ones. Sevag method could removeprotein ingredient effectively. Isolated normal distribution polysaccharidepeaks. Their average molecular weight ranged from 70KDa to 200KDa.Reducing sugar content could be ignored. Whether Pyrogallolautooxidation method or Fenton reaction, the antioxidant activity ofthem were quite high.Conclusions: the toxicity of fruit body of C. militaris is extremely low.It's safety for the normal mice. After 4-week administration, there was noapparently effect on the liver function of CRF rats. The groupsadministrated with C. militaris, especially the one with its polysaccharide,were improved their existent qualification remarkably. Their death ratewas low, body weight and diet increased. The function of their kidneysrecovered partially. Although did not reach the normal level, but TP,BUN, SCr decreased evidently, Ccr raised. Pathological sectionsrevealed it's relatively much lighter that the damages of glomerulus,renal tubule and renal mesenchyme in those groups. C. militarispolysaccharide inhibited the growth of Gram-positive bacteria. It'smainly composed of neutral polysaccharide components, while lack ofacid ones. Their average molecular weight ranged from 70KDa-200KDa.All of them showed quite high antioxidant activity.