ISOLATION OF STIGMASTEROL FROM Piptadeniastrum africanum (HOOK. F.) AND ITS MODULATORY EFFECT ON MITOCHONDRIAL-MEDIATED CELL DEATH IN LIVER AND COLONIC TOXICITY IN MICE

Abstract

Hepatocellular and colonic damage are fatal outcomes arising from liver and colon toxicity. Modulation of mitochondrial-mediated cell death is a strategy in these diseases management. The use of synthetic drugs in the treatment of these diseases presents several side effects. In folklore, Piptadeniastrum africanum (PA) is used for the treatment of these disorders but the mechanism has not been explored. This study was designed to investigate the role of bioactive compound(s) purified from PA on liver and colon damage via the modulation of mitochondrial-dependent cell death. The istem ibark iof iPiptadeniastrum iafricanum iwas icollected ifrom ia iforest iat iIbadan, identified iat ithe idepartment iof iBotany, iUniversity iof iIbadan i(UIH-22562) iair-dried, pulverised iand isoaked iin iabsolute imethanol ito iobtain iits iextract i(CMEPA). i iThe iCMEPA was ifractionated iusing in-hexane, ichloroform, iethyl iacetate iand imethanol ito iobtain itheir respective ifractions i(HFPA, iCFPA, iEFPA iand iMFPA) iwhich iwere itested ion imitochondrial permeability itransition i(mPT) ipore iopening iand iATPase iactivity i(in ivitro). i iThe ieffects iof the ifractions iof iPA ion iliver iand icolon itissues iwere ievaluated iin istudy i1. iTwenty imice (20±2g; in=5) iwere igrouped iand itreated ias ifollows, igroups i1 i(vehicle), i2, i3 iand i4 iwere treated iintraperitoneally iwith i25, i50 iand i100 img/kg iof iEFPA ionce idaily ifor i14 idays iand liver imPT iassayed. iThe iEFPA iwas ipurified iusing icolumn ichromatography ito iobtain ia ipure compound iwhich iwas icharacterised ias istigmasterol iby ispectroscopic itechniques. iIn istudy i2, forty-two imice iwere igrouped i(n=7), itreated iwith iDextran iSulphate iSodium i(DSS) iand Benzo{a}Pyrene i(BaP) ifor i10 days as follows, groups 1 (vehicle), 2 (oral 4% DSS), 3 (125mg/kg BaP), 4 (DSS+BaP), 5 (DSS+BaP and 200mg/kg of stigmasterol) and 6 (DSS+BaP and 400mg/kg of stigmasterol). The mice were sacrificed and colon samples prepared. Tumor Necrosis Factor-α (TNF-α), Interleukin 6 (IL-6), p53, Caspases 9 (C9), Bax, Bcl-2, were performed on the colon using immunohistochemistry. All data were analysed using descriptive statistics and ANOVA at α0.05. In vitro, EFPA had the highest induction of mPT pore opening (3.80, 5.60, 6.40, 8.10 and 8.90 folds) at 20, 60, 100, 140 and 180µg/ml, respectively and enhanced ATPase activity (0.20±0.01, 0.35±0.10, 0.40±0.10, 0.45±0.20 and 5.20±0.80µmole/Pi/mg/protein/min), relative to the vehicle (0.05µmole/Pi/mg/protein/min), respectively. In vivo, EFPA caused mPT induction of 2.50, 4.90 and 6.90 folds at 25, 50 and 100mg/kg, respectively. The toxicant groups (DSS, BaP and DSS+BaP), relative to the vehicle significantly increased TNF-α (40.00±1.20%, 30.00±0.90%, 34.00±1.10% vs 15.00±0.70%), IL-6 (160.00±3.50%, 110.00±2.20%, 120.00±1.50% vs 60.00±1.50%) and p53 (80.00±2.30%, 70.00±1.10%, 85.00±2.20% vs 25.00±0.90%) However, stigmasterol (200 and 400mg/kg), relative to DSS+BaP significantly attenuated TNF-α (12.00±0.09%, 13.00±0.10% vs 34.00±1.10%), IL-6 (53.00±1.30%, 50.00±1.20% vs 120.00±1.50%), p53 (40.00±2.20%, 50.00±1.70% vs 85.00±2.20%). The DSS, BaP and DSS+BaP, relative to the vehicle increased C9 (1.10±0.10, 0.90±0.01, 1.10±0.10ng/ml vs 0.01ng/ml), Bax (120.00%, 100.00%, 90.00% vs 110.00%) and reduced Bcl-2 (80.00%, 75.00%, 75.00% vs 90.00%) which were modulated by stigmasterol. Stigmasterol isolated from the Piptadeniastrum africanum modulated mitochondrial-mediated cell death via a decrease in pro-inflammatory cytokines and levels of pro-apoptotic proteins.