IBCEW'16 conference paper

1. Effects of extract of Drymaria cordata
on isolated rat liver mitochondrial
membrane permeability transition
(MMPT) pore
By
Olowofolahan, A.O.1 , Adeoye, O.A. 1, Offor, G.N. 2 and
Olorunsogo, O.O. 1
1Dept. of Biochemistry, University of Ibadan; 2Dept. Of Biological Sciences Covenant
University, Ota.
June, 2016.

2. Outline
• Background
• Statement of problem
• Justification
• Aim and objectives
• Materials and methods
• Results and discussion
• Conclusion
• References
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3. Background
• Mitochondria are organelles with many intriguing
aspects; they play critical roles in both the life and
death of cells (Goodsell, 2010).
• Mitochondria are involved in cell death (Apoptosis)
through the permeabilization of mitochondrial
membranes.
• Opening of MMPT pore leads to the release of
apoptogenic proteins such as cytochrome c, which in
turn can trigger caspase activation and ultimately
execution of apoptosis (Sun et al., 2004).
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4. • Drymaria cordata (tropical chickweed)
belong to Caryophyllaceae.
• a weak creeping annual or less commonly
perennial herb.
• Extracts of Drymaria cordata have
previously been reported to possess
• antitussive (Mukherjee et al., 1997)
• anti-inflammatory (Adeyemi et al., 2008)
• anxiolytic (Barua et al., 2009)
• cytotoxic (Sowemimo et al., 2009)
• antioxidant (Bilma et al., 2011)
• analgesic and antipyretic activity (Akindele
et al., 2012).
• A combination of Drymaria cordata and
Dissotis rotundifolia is used in the
traditional treatment of fibroid (Adebisi,
2010).
Figure 1: Drymaria cordata
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5. Statement of problem
• Apoptosis dysregulation contributes to half of all
human diseases.
• Excessive apoptosis occurs in neurodegenerative
disorders such as Alzheimers, Parkinsons,
autoimmune disorders, heart disease and
infectious diseases, including AIDS while failure to
initiate apoptosis can induce many diseased states
such as arthritis and cancer.
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6. Justification
• Various studies have shown that the permeabilization
of the outer mitochondrial membrane is a major
event in the induction of mitochondrial pathway of
apoptosis (Crompton, 1999; Wang, 2001).
• Therefore, MMPT pore serves as a target for the
design of novel strategies for blocking pathological
cell loss or for killing unwanted cells.
• It has been discovered that certain bioactive agents
present in medicinal plants elicit their chemo
protective and therapeutic effects through the
induction or inhibition of the opening of MMPT pore
(Martin, 2006).
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7. Aim/objectives
• This study seeks to ascertain if D. cordata is a
potent inducer of the MMPT pore opening.
• The specific objectives include:
– To isolate low ionic strength liver mitochondria from male albino
rats.
– To estimate mitochondrial protein concentration.
– To investigate the effects of chloroform and aqueous fractions of
the methanol extract of Drymaria cordata on mitochondrial
swelling in the presence and absence of calcium.
– To evaluate the effects of these fractions on mitochondrial
ATPase activity and lipid peroxidation.
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8. Materials/methods
• Experimental animals
– Male albino rats (Wister strain) weighing between 120 and150g
were used for the study.
• Plant material
– Collection of plant material
– Preparation of extract
• Preparation of low ionic strength liver
mitochondria
– Low-ionic-strength liver mitochondria were isolated from male
albino rats using a modification of the procedure described by
Johnson and Lardy (1967)23/05/2017 8

9. • Protein determination
– The Mitochondrial protein was estimated according to the
method of Lowry et al., (1951) using Bovine serum Albumin
(BSA) as standard.
• Assessment of mitochondrial membrane
permeability transition in rat liver mitochondria
– Mitochondrial swelling were measured quantitatively at 540 nm
in a Camspec M105 spectrophotometer based on the
procedure of Lapidus and Sokolove (1993).
• Determination of mitochondrial ATPase activity
– ATPase activity was determined by the method of Lardy and
Wellman (1953) as modified by Olorunsogo and Bababunmi
(1979). Concentration of inorganic phosphate released was
determined as described by Bassir (1963).
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10. • Determination of lipid peroxidation
– A modified thiobarbituric acid reactive species (TBARS) assay was
used to measure the lipid peroxide formed using liver
homogenates as lipid-rich media, as described by Ruberto et al.,
(2000).
• All reagents used for the study were of analytical
standard grade.
• Statistical analysis of data
– Experiments were repeated for at least three times.
– Results were represented as data expressed as Mean ± S.D, n=5:
otherwise representative data are presented.
– ʻpʼ values< 0.05 were considered significant.
– Statistical package for the social sciences (SPSS) version 15.0 was
used for the statistical analysis .23/05/2017 10

11. Results/discussion
-0.8
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0
CHANGEINABSORBANCE(540nm)
TIME (MINUTES)
NTA
TA
SPERMINE
Figure 2: calcium-induced mitochondrial membrane permeability transition pore opening in
normal rat liver mitochondria and its reversal by spermine.
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12. -1
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CHANGEINABSORBANCE(540nm)
TIME (MINUTES)
NTA
TA
SPERMINE
10µg/ml
30µg/ml
50µg/ml
70µg/ml
90µg/ml
Figure 3: Effect of varying concentrations of chloroform fraction of methanol extract of
Drymaria cordata (CFDC) on the mitochondrial membrane permeability transition pore in the
absence of calcium.
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13. -0.9
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CHANGEINABSORBANCE(540nm) TIME (MINUTES)
NTA
TA
SPERMINE
10µg/ml
30µg/ml
50µg/ml
70µg/ml
90µg/ml
Figure 4: Effect of varying concentrations of chloroform fraction of methanol extract of Drymaria
cordata (CFDC) on the mitochondrial membrane permeability transition pore in the presence of
calcium.
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14. -0.8
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CHANGEINABSORBANCE(540nm)
TIME (MINUTES)
NTA
TA
SPERMINE
10µg/ml
30µg/ml
50µg/ml
70µg/ml
90µg/ml
Figure 5: Effect of varying concentrations of aqueous fraction of methanol extract of Drymaria
cordata (AFDC) on the mitochondrial membrane permeability transition pore in the absence of
calcium.
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15. -0.9
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CHANGEINABSORBANCE(540nm)
TIME (MINUTES)
NTA
TA
SPERMINE
10µg/ml
30µg/ml
50µg/ml
70µg/ml
90µg/ml
Figure 6: Effect of varying concentrations of aqueous fraction of methanol extract of Drymaria
cordata (AFDC) on the mitochondrial membrane permeability transition pore in the presence of
calcium.
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16. Table 1: Effect of Chloroform fraction of methanol extract of Drymaria
cordata on mitochondrial ATPase activity (pH 7.4)
Chloroform fraction (10-90µg/ml) Concentration of phosphate released
(µmoles Pi/mg protein/min)
Control 3.26±0.18
10 7.32±0.12
30 7.96±0.16
50 9.83±0.14
70 11.14±0.17
90 7.51±0.12
25µM 2,4-Dinitrophenol 12.08±0.13
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17. Table 2: Effect of Aqueous fraction of methanol extract of Drymaria cordata
on mitochondrial ATPase activity (pH 7.4)
Aqueous fraction (10-90µg/ml) Concentration of phosphate released
(µmoles Pi/mg protein/min)
Control 1.66±0.06
10 2.21±0.06
30 2.34±0.04
50 2.54±0.05
70 2.58±0.03
90 2.76±0.05
25µM 2,4-Dinitrophenol 11.06±0.17
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18. 0
10
20
30
40
50
60
70
80
50 100 200 400 800
%inhibition
conc(µg/ml)
Figure 7: Effect of varying concentrations of chloroform fraction of methanol extract of Drymaria
cordata (CFDC) on Fe2+-induced lipid peroxidation in normal rat liver mitochondria.
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19. 0
10
20
30
40
50
60
70
50 100 200 400 800
%inhibition
conc (µg/ml)
Figure 8: Effect of varying concentrations of aqueous fraction of methanol extract of Drymaria
cordata (AFDC) on Fe2+-induced lipid peroxidation in normal rat liver mitochondria.
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20. Conclusion
• This study shows the potency of the whole plant
extract of Drymaria cordata in the induction of MMPT
pore opening.
• The inductive fold is in the order CFDC ˃AFDC, thus
indicating that the chloroform fraction is the more
potent fraction and therefore contains the putative
agent in the plant.
• However, further studies to isolate and characterize
components responsible for the observed activities
and also the effect of the various fractions on
cytochrome c release and mechanisms associated
with apoptosis are necessary.
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21. References
• Adeyemi, O. O., Akindele, A. J. & Ndubuisi, N. 2008. Anti-inflammatory activity of Drymaria cordata extract. J.
Nat.Rem. 8.1: 93-100.
• Akindele, A., Ibe, I. and Adeyemi, O. 2012. Analgesic and Antipyretic activities of drymaria cordata (Linn) Wlld
(Caryophyllaceae) extract”. Afr J Tradit Complement Altern Med. 9:25-35.
• Barua, C. C., Roy, J. D., Buragohain, B., Barua, A. G., Borah, P. et. al. 2009. Anxiolytic effect of hydroethanolic
extract of Drymaria cordata L Willd. Indian J. Exp. Biol. 47.12: 969- 73.
• Crompton, M. 1999. The mitochondrial permeability transition pore and its role in in cell death. Biochem. J.
341: 233–249.
• Goodsell, D. 2010. Illustrating the machinery of life: Mitochondria. Biochem and Mol. Biol. Edu. 38: 134-40.
• Johnson, D. & Lardy, H. 1967. Methods Enzymol. 10: 94-96.
• Lardy, H. A. & Wellman, H. 1953. The catalyst effects of 2,4-dinitrophenol on adenosinetriphosphatase
hydrolysis by cell particles and soluble enzymes. J. Biol. Chem. 201: 357-370.
• Lapidus, R. G. & Sokolove, P. M. 1993. Spermine inhibition of the permeability transition of isolated rat liver
mitochondria: An investigation of mechanism. Journal of Biochemical and Biophysical Methods 64: 246−253.
• Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 1951. Protein measurement with the Folin phenol
reagent. Journal of Biological Chemistry 193: 262−275.
• Martin, K. R. 2006. Targeting Apoptosis with Dietary Bioactive Agents: society for Experimental Biology and
Medicine. Minireview
• Mukherjee, P. K., Saha, K., Bhattacharya, S., Giri, S.N., Pal, M. et. al. 1997. Studies on antitussive activity of
Drymaria cordata Willd (Caryophyllaceae). J. Ethnopharmacol. 56.1: 77-80.
• Ruberto, G., Baratta, M. T., Deans, S. G. & Dorman, H. J. D. 2000. Antioxidant and antimicrobial activity of
Foeniculum vulgare and Crithmum maritimum essential oils. Planta. Med. 66: 687-693.
• Sowemimo, A., van de Venter, M., Baatjies, L. & Koekemoer, T. 2009. Cytotoxic activity of selected Nigerian
plants. Afr. J. Tradit. Complement. Altern. Med. 6.4: 526-28.
• Sun, S., Hail, N. & Lotan, R. 2004. Apoptosis as a novel target for cancer chemoprevention. Journal of the
National Cancer Institute 96: 662−672.
• Wang, X. 2001. The expanding role of mitochondria in apoptosis. Genes Dev. 15: 2922-2933.23/05/2017 21

22. THANK
YOU
FOR LISTENING
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