Studies On Anti-ad Mechanism Of Biruloquinone From Secondary Metabolites Of Cladonia Macilenta

Cladonia macilenta (Hoffm.) is a saprophytic lichen, belong to the Cladonia genus,Cladoniaceae family, Ascolichens class, Lichenes phylum. In this paper, the fermentation brothof lichen-forming fungi Cladonia macilenta was extracted with an equal volume of ethyl acetate.The extract was then further purified by silica gel column chromatography and recrystallizationwith chloroform-methanol to give the dark purple crystals of biruloquinone. Biruloquinonebelongs to the ortho-phenanthraquinone compounds, which are extremely rare compoundsamong natural quinones. These compounds have been found that have a variety of importantpharmacological activity. In this study the anti-AD mechanism of biruloquinone was researchedby following four aspects: AChE inhibitor activity, M1receptor agonist activity, M2receptorantagonist activity and anti-oxidative stress activity.1. The AChE inhibitor activity test was performed using a96-microtiter well plate assaybased on Ellman’s reaction. The results showed that the inhibition activity was positivelycorrelated with concentration, the inhibition percentage increased rapidly with the increase ofbiruloquinone concentration from0to100μg/mL. The concentration that was required for the50%enzyme inhibition (IC50) was27.1μg/mL (83.1μM). When the concentration was100μg/mL, the inhibition rate reached the maximum75.39%. The enzymatic reaction kineticsanalysis results indicated that biruloquinone was a mixed-II inhibitor of AChE, and theequilibrium constants for the inhibitor binding with enzyme, KI, and with enzyme substratecomplex, KISwas204μM and12.5μM, respectively.2. Establishing a stable cell line with M1receptor gene, response elements (CRE, MRE,SRE) and luciferase reporter gene for testing the M1receptor agonist activity of biruloquinone.When sample binds to M1receptor, it will activate M1receptor and the signal pathway tochange the expression of the second messenger in the cell. Response elements respond to thischange and effect the expression of reporter gene. The M1receptor agonist activity of thesample could be evaluated after the expression quantity of the luciferase reporter gene wasmeasured. The results showed that the agonist effect of biruloquinone to M1cell lines was notobvious, and biruloquinone made the luciferase of negative control cell lines had a certainamount of expression, this suggested that biruloquinone might be activating report gene notthrough M1receptor, caused false positives.3. Establishing a stable cell line with M2receptor gene, response elements (CRE, MRE,SRE) and luciferase reporter gene for testing the M2receptor antagonist activity of biruloquinone. When sample binds to M2receptor, it will inhibit M2receptor and the signalpathway to change the expression of the second messenger in the cell. Response elementsrespond to this change and effect the expression of reporter gene. The M2receptor antagonistactivity of the sample could be evaluated after the expression quantity of the luciferase reportergene was measured. The results show that biruloquinone could not to bring down luciferaseexpression of M2cell lines in the case of existing M2receptor antagonist, this indicatedbiruloquinone didn’t have obviousas M2receptor antagonist activity.4. Targeted active oxygen and free radicals, the anti-oxidative stress activity was detectedwith DPPH and ABTS method. The results showed that biruloquinone exhibited high ability toscavenge DPPH and ABTS free radicals in vitro, and the activity was concentration-dependentin the test concentration range. In100μg/mL, DPPH free radical clearance rate was90.83%with an EC50value of24.91μg/mL. When the concentration was1.2mM, ABTS free radicalclearance rate was75.39%with an EC50value of0.488mM. The total antioxidant capacity ofbiruloquinone was2.5mM Trolox measured by ABTS method.