Characterization and assessment of antifungal activity of chemically modified berberine

Keywords: alkaloid, Alternaria, antifungal activity, berberine, Colletotrichum, Fusarium, Rhyzoctonia Doi: 10.4038/jnsfsr.v28i3.2663 J.Natn.Sci.Foundation Sri Lanka 2000 28 (3): 215-223


INTRODUCTION
There are a number of report,s in the literature1 on the isolation of berberine allraloids from plants of the Annonaceae, Berberidaceae, Convolvulaceae, Lauraceae, Menispermaceae, Papaveraceae, Ranunculaceae and Rutaceae. Pharnlacological ~n v e s t l g n t l o~~s~ of berberine alkaloids have shown them to have antimicrobial, antileukemic, antiulcerous, gastrlc antisecretary activities and uterine stimulating effects.
Coscintu,in ferl,cstratz~nz (venivel) is a woody climber which belongs to the Family Menispermaceae. The waterextract of the stem of this plant is widely u.sed in Ayurvedic medicine in Sri Lanka. The major constituents of the plant have been identified as all~aloids:' of which berberine is the principal component.
A closer look at the berberine skeleton ind~cates the possibility of its easy conversion lnto a quinolizidine nucleus. Quinolizidine alkaloids4 are lrnown to be polsons in small concentrations. The structural analogues of quinolizidine; jndolizidine and pyrolizidine alkaloids have interesting biological properties.The present study is part of an investigation into the structure-activity relationsl~ip of tetrahydroberberirle derivatives which could be produced using the readily available berberine. Antifungal activity was the chose11 bioassay due to the literature precedence of antifungal activity shown by the isoquinoline~lktlloids. Our previous work has shown that 8-amino-8(1H)-berberine is active against
An attempt to convert compound 3 into the tetrahydroderivative by hydrogenation (H,/Pdj and reduction (NaBH,)' failed. The hydrogenation resulted in compound. 6 while reduction gave 2. The conversion of the nitrile group in compound. 3 into a carboxylic acid moiety using NaOH and H,SO, also failed. Treatment of compound 4 with NaBH, and HJPd gave tetrahydroberberine.

Measuremelzt of an,tifungal activity:
The f~lngus was first grown on potato dextrose agar (200g potato, 20g glucose, 30g agar and 1000ml distilled water) for 7 days a t 29'C. The fungus forms sclerotia readi1.y on the agar medium.
Stoclr solutions of 10,000 ppm were made from the test compounds. The required concentrations of the compounds (gl100 ml) were obtained by mixing the amount of stock solutlon shown with 15 ml of molten agar. The agar was then poured mto petri dishes and allowed to set. A single sclerotium of the fungus was placed a t the centre of the agar medium :~n d the experiment was triplicated for each concentration. The diameter of the fimgal mat was measured after 4 days at 28C.
Compound 2 was characterised using NMR spectroscopy. The assignments of protons and carbons are tabulated in Table 1 and Table 2    OCI-I,.
The decomposition temperatures of Compounds 3, 4, and 5 were noted as 169-171TC, 160-162" and 105-107% respectively. Compounds 3,4 and 5 were studied using NMR methods and the protons and carbons were assigned as in Table  3 and 4. However there is an ambiguity in the assignment of quaternary carbons. lH NMR data of Compound 6 was comparable to that reported for oxyberberine."    The chemical shift assignments of protons and carbon were made using the 2DXHCORR and COSY pulse sequences. The downfield chemical shift of proton attached t o C-8 in compound 4 and the observation of two triplet,^ for protons attached to C-5 and C-6 confirms that the azide is not attached to C-8 in compound 4.
Mass spectra1 analys~s of the colnpoullds 3,4, 5 under FAI3 gave a base peak at 33G. which corresponds to the stable berberine cation showi~ig that the berberine struct,ure is very stable and the products prefer to acquire the flat berberjne skeleton even under mild ionisation technique. The base peak under electrospray for a11 three compoulids was a t 336, with the molecular ion being noted for conlpoulld 5 alone a t 352.2.
This Inass spectral results indirectly confirms the results obtained from the trent,lrlellt of' 8-cyano-(8Hbberberine with NaBI-I,. I t is clear that nucleopl~ilic reactions on carbon 8 make the berberine skeleton to be slightly deviated from the otherwise flat structure. Hence it always prefers to revert back to the starting berberinc: cat,ion. I t is very clear t h a t the reduction of 13.14 double bond in 8-cyano-(8H)-berberille is not a favourable way to obtain the quinolizidine nu-c1eu.s. Bexber:jne.HCl. (I), tet,rahydroberberjne (2) arid 8-oxyberberine (6) did not have prom-;.sing nct,i.vity against R/hizocton.ia, soln.7l.i. Compounds 3 and 4 showed very good actjvit,y against, the s a n e fungus. Bo+,h compouncls indicated almost 100% inl~ibitioll at or above O. 15(% concentration. They compl.et,ely inhibited the growt,h of Altcr.,xarin, Colletotrich.z,,r,~, and Fusnrtunt al; or below 0.025% concentration. I t is not clear whether the activity of con~pouncl 3 is due to the compound it,self or due to the release of' cyanid.e into the rriediunl resultj.ng in a stable herberjne cation.