infertility is a major clinical problem , affecting people medically and psychosocially . in recent years
oxidative stress results from either a decrease of natural cell antioxidant capacity or an increased amount of reactive oxygen species ( ros ) in organisms . however , the consumption of foods rich in antioxidant phytochemicals may help fight degenerative diseases caused by oxidative stress by improving body 's antioxidant status .  
high levels of fe play a crucial role in degenerative diseases by acting catalytically in the production of ros which have the potential to damage cellular lipids , nucleic acids , proteins , and carbohydrate resulting in wide - ranging impairment in cellular function and integrity .
ros can directly attack the polyunsaturated fatty acids of the cell membranes and induce lipid peroxidation .
malondialdehyde ( mda ) is the end - product of lipid peroxidation , which is a process where reactive oxygen species ( ros ) degrade polyunsaturated fatty acids .  
recently , much attention has been focused on the role of the antioxidative defense system to combat oxidative stress .
endogenous antioxidants in plants may play an important role in antioxidative defense against oxidative damage , possibly preserving the biological functions of cells .
there is an increasing interest in the protective biological function of natural antioxidants contained in dietary plants , which are candidates for the prevention of oxidative damage .
these antioxidants are polyphenolic compounds , which are found in all plants and in all parts of the plants ( tree bark , stalks , leaves , fruits , roots , flowers , pods , and seeds ) .  

cissus populnea is a plant associated with a myriad of medicinal uses in different parts of the world .
its extracts have been credited with antibacterial properties , as an antitrypanosomal plant and a source of gum powder   and as a component of a herbal antisickling nigerian formula . in benin republic , it is used for its diuretic properties while in ghana it is used as a postharvest ethnobotanical protectant .
the aqueous extract of its stem bark is associated with aphrodisiac / fertility potentials among the yoruba - speaking people of south west nigeria .
the use of c. populnea , as an aphrodisiac and fertility enhancer amongst the males , has been attributed to the declining fertility trend that has been established in this population over the years coupled with the attendant increasing levels of erectile dysfunction .
although c. populnea has been reportedly used in traditional medicine for the management / prevention of infertility diseases associated with oxidative stress , there is still limited information on some of the possible mechanisms by which they exert this effect .
hence , the objective of this study is to investigate the inhibitory effect of water extractible phytochemicals from stem bark of c. populnea on fe and snp - induced lipid peroxidation in rat 's testes in vitro .
fresh samples of stem bark of cissus populnea were purchased in a local market , in akure metropolis , nigeria .
authentication of the plant was carried out in the department of biology , federal university of technology , akure , nigeria .  
chemicals and reagents used such as thiobarbituric acid ( tba ) , 1,10-phenanthroline , deoxyribose , gallic acid , and folin - ciocalteau 's reagent were procured from sigma - aldrich , inc .
, ( st . louis , mo , usa ) , trichloroacetic acid ( tca ) was sourced from sigma - aldrich , chemie gmbh ( steinheim , germany ) , dinitrophenyl hydrazine ( dnph ) from acros organics ( nj , usa ) , hydrogen peroxide , methanol , acetic acid , and fecl3 were sourced from bdh chemicals ltd . , ( poole , england ) , thiourea , cuso45h2o , h2so4 , sodium carbonate , alcl3 , potassium acetate , tris - hcl buffer , sodium dodecyl sulphate , feso4 , and potassium ferricyanide were of analytical grade while the water was glass distilled .
the sample was washed under running water , air dried after which the dried sample was grinded to powdered form , and kept dry in an air - tight container prior to the extraction .
1  g of the powdered sample was weighed into 20  ml of distilled water and was left for 24 hours .
the mixture after 24 hours was filtered and the filtrate centrifuged at 805  g for 10 minutes .
the total phenolic content was determined using the method reported by singleton et al . .
appropriate dilutions of the extract were oxidized with 2.5  ml of 10% folin - ciocalteau 's reagent ( v / v ) and neutralized by 2.0  ml of 7.5% sodium carbonate .
the reaction mixture was incubated for 40 minutes at 45c , and the absorbance was measured at 765  nm in the spectrophotometer ( jenway 6305 , barloworld scientific , dunmow , uk ) .
the total phenolic content was subsequently calculated and expressed as mg gallic acid equivalent / g dry weight .  
the total flavonoid content of the sample was determined using a slightly modified method reported by meda et al . .
briefly 0.5  ml of appropriately diluted sample was mixed with 0.5  ml methanol , 50  l 10% alcl3 , 50  l 1  m potassium acetate , and 1.4  ml water , and allowed to incubate at room temperature for 30 minutes .
the absorbance of the reaction mixture was subsequently measured at 415  nm , and the total flavonoid content was subsequently calculated using quercetin as standard and expressed as mg / g quercetin equivalent dry weight .  
vitamin c content of the sample was determined using the method of benderitter et al . .
briefly , 75  l dnph ( 2  g dinitrophenyl hydrazine , 230  mg thiourea , and 270  mg cuso45h2o in 100  ml of 5  m h2so4 ) were added to 500  l reaction mixture ( 300  l of an appropriate dilution of the extract with 100  l 13.3% trichloroacetic acid ( tca ) and water ) .
the reaction mixtures were subsequently incubated for 3  h at 37c , then 0.5  ml of 65% h2so4 ( v / v ) was added to the medium ; their absorbance was measured at 520  nm and the vitamin c content of the sample was subsequently calculated using ascorbic acid as standard and expressed as mg / g ascorbic acid equivalent .
ten male wistar albino rats weighing between 190 and 250  g were purchased from the central animal house , department of biochemistry , university of ilorin , ilorin , nigeria .
they were housed in stainless steel cages under controlled conditions of a 12  hr light / dark cycle , 50% humidity , and 28c temperature .
the animals were used in accordance with the procedure approved by the animal ethics committee of the federal university of technology , akure , nigeria .
the rats were decapitated under mild diethyl ether anaesthesia , and the testes ( tissue ) were rapidly dissected and placed on ice and weighed .
this tissue was subsequently homogenized in cold saline ( 1/10  w / v ) with about 10-up- and downstrokes at approximately 1200  rev / min in a teflon glass homogenizer ( mexxcare , mc14 362 , aayushi design pvt . ltd .
co. , hong kong ) for 10 minutes at 3000  g to yield a pellet that was discarded and a low - speed supernatant ( si ) , which was kept for lipid peroxidation assay . 

fe - induced lipid peroxidationthe lipid peroxidation assay was carried out using the modified method of ohkawa et al . .
briefly 100  l of the si fraction was mixed with a reaction mixture containing 30  l of 0.1  m ph 7.4 tris - hcl buffer , extract ( 100  l ) , and 30  l of 250  m freshly prepared feso4 as the prooxidant .
the volume was made up to 300  l by water before incubation at 37c for 2  hrs .
the color reaction was developed by adding 300  l 8.1% sds ( sodium dodecyl sulphate ) to the reaction mixture , this was subsequently followed by the addition of 500  l of acetic acid / hcl ( ph 3.4 ) mixture and 500  l of 0.8% thiobarbituric acid ( tba ) .
thiobarbituric acid reactive species ( tbars ) produced were measured at 532  nm and expressed as malondialdehyde ( mda ) produced ( % control ) using mda standard curve ( 00.035  mm ) . 
 
the lipid peroxidation assay was carried out using the modified method of ohkawa et al . .
briefly 100  l of the si fraction was mixed with a reaction mixture containing 30  l of 0.1  m ph 7.4 tris - hcl buffer , extract ( 100  l ) , and 30  l of 250  m freshly prepared feso4 as the prooxidant .
the volume was made up to 300  l by water before incubation at 37c for 2  hrs .
the color reaction was developed by adding 300  l 8.1% sds ( sodium dodecyl sulphate ) to the reaction mixture , this was subsequently followed by the addition of 500  l of acetic acid / hcl ( ph 3.4 ) mixture and 500  l of 0.8% thiobarbituric acid ( tba ) .
thiobarbituric acid reactive species ( tbars ) produced were measured at 532  nm and expressed as malondialdehyde ( mda ) produced ( % control ) using mda standard curve ( 00.035  mm ) .
also , for sodium nitroprusside- ( snp- ) induced lipid peroxidation , the procedure was carried out as above using 5  mm sodium nitroprusside ( snp ) as the prooxidant .  
the free radical scavenging ability of the extracts against dpph ( 1,1-diphenyl-2 picrylhydrazyl ) free radical was evaluated as described by gyamfi et al . .
briefly , an appropriate dilution of the extract ( 1  ml ) was mixed with 1  ml of 0.4  mm methanolic solution containing dpph radicals , the mixture was left in the dark for 30  min , and the absorbance was measured at 516  nm .
the control was carried out using 2  ml dpph solution without the test samples .
the percentage ( % ) of dpph - free radical scavenging ability was subsequently calculated as follows : 
 ( 1)dpph  scavenging  ability(% )  = [ ( abscontrolabssamples)abscontrol]100 . the fe chelating ability of the extract was determined using a modified method of minotti and aust   with a slight modification by puntel et al . .
freshly prepared 500  m feso4 ( 150  l ) was added to a reaction mixture containing 168  l of 0.1  m tris - hcl ( ph 7.4 ) , 218  l saline and the extracts ( 025  l ) .
the reaction mixture was incubated for 5  min , before the addition of 13  l of 0.25% 1,10-phenanthroline ( w / v ) .
the fe ( ii ) chelating ability was subsequently calculated with respect to the reference ( which contains all the reagents without the test sample ) .
the reducing property of the extract was determined by assessing the ability of the extract to reduce fecl3 solution as described by oyaizu .
a 2.5  ml aliquot was mixed with 2.5  ml of 200  mm sodium phosphate buffer ( ph 6.6 ) and 2.5  ml of 1% potassium ferricyanide .
the mixture was incubated at 50c for 20  min , and then 2.5  ml of 10% trichloroacetic acid was added .
5  ml of the supernatant was mixed with an equal volume of water and 1  ml of 0.1% ferric chloride .
many plants are rich sources of phytochemicals , and intakes of these plant chemicals have protective potential against degenerative diseases . the total phenolic content and total flavonoid content of stem bark of c. populnea
the total phenolic content of the plant ( 17.33  mg / g ) is lower than what was reported for some hot peppers and green teas , but higher than some tropical leafy vegetables [ 25 , 26 ] .
phenolic compounds can protect the human body from free radicals , whose formation is associated with the normal metabolism of aerobic cells .
they are strong antioxidants capable of removing free radicals , chelate metal catalysts , activate antioxidant enzymes , reduce alpha - tocopherol radicals , and inhibit oxidases .  
the total flavonoid content of c. populnea ( 0.059  mg / g ) is lower than what was reported for some tropical green leafy vegetables .
the presence of derivatives of flavonoids has been found in many herbs ; moreover , numerous studies have conclusively shown that the majority of the antioxidant activity may be from compounds such as flavonoids , isoflavones , flavones , anthocyanins , catechin , and isocatechin rather than from vitamins c , e and -carotene [ 29 , 30 ] .
polyphenols are considered to be strong antioxidants due to the redox properties of their hydroxyl groups .  
the vitamin c content of stem bark of c. populnea is presented in table 1 .
ascorbic acid is a good reducing agent and exhibits its antioxidant activities by electron donation [ 25 , 26 ] .
it helps the immune system to fight off foreign invaders and tumor cells and supports the cardiovascular system by facilitating fat metabolism and protecting tissues from free radical damage , and it assists the nervous system by converting certain amino acids into neurotransmitters . as a water - soluble antioxidant
, vitamin c is in a unique position to  scavenge  aqueous peroxyl radicals before these destructive substances damage the lipids .
the vitamin c content of the plant is higher than that of related spices such as basil , bird pepper , black pepper , cinnamon , nutmeg , oregano , parsley and rosemary , and some commonly consumed green leafy vegetables in nigeria [ 25 , 26 ] , green and red pepper , and some commonly consumed and underutilized tropical legumes .
it is also worth noting that the vitamin c content of the plant is higher than its total phenol content .
the high vitamin c content of the plant will definitely contribute additively or synergistically to the observed antioxidant and medicinal properties of the plant .
lipids are considered to be the most susceptible macromolecules and are present in male reproductive organ .
one of the byproducts of lipid peroxidation is malondialdehyde , this byproduct has been used in various biochemical assays to monitor the degree of peroxidative damage sustained by spermatozoa [ 35 , 36 ] .
the finding that fe caused a significant increase in the mda content of the testis homogenate agreed with earlier report where fe was shown to be a potent initiator of lipid peroxidation .
the increased lipid peroxidation in the presence of fe could be attributed to the fact that fe can catalyze one - electron transfer reactions that generate reactive oxygen species , such as the reactive oh , which is formed from h2o2 through the fenton reaction .
iron also decomposes lipid peroxides , thus generating peroxyl and alkoxyl radicals , which favors the propagation of lipid oxidation . in the testis
, fe - induced lipid peroxidation destroys the structure of lipid matrix in the membranes of spermatozoa , and it is associated with loss of motility and impairment of spermatogenesis .
the ability of the water extractible phytochemicals from stem bark of c. populnea to inhibit fe - induced lipid peroxidation in the testes homogenate is presented in figure 1 .
the result revealed that the incubation of the testes homogenate in the presence of fe caused a significant ( p < 0.05 ) increase in the mda content ( 137.5% ) of the rat testes homogenate when compared with the basal ( 100% ) .
however , the aqueous extract of stem bark of c. populnea inhibited mda production in rat 's testes in a dose - dependent manner ( 00.63 mg / ml ) . nevertheless , judging by the ec50 ( extract concentration causing 50% inhibition ) values in table 2 , the plant ( ec50 = 0.027  mg / ml ) had a significantly ( p < 0.05 ) high inhibitory effect on fe - induced lipid peroxidation in the testes homogenate .
the decrease in the fe - induced lipid peroxidation in the rat testes homogenates in the presence of the extract could be as result of the ability of the extracts to chelate fe and/or scavenge free radicals produced by the fe - catalyzed production of reactive oxygen species ( ros ) in the rat testes .  
antioxidants carry out their protective role on cells either by preventing the production of free radicals or by neutralizing / scavenging free radicals produced in the body or by reducing / chelating the transition metal composition of foods [ 27 , 40 ] . in an attempt to explain the main mechanism through which the water extractable phytochemicals in the plant extract protect testes tissue against fe - induced lipid peroxidation ,
the prevention of the chain initiation step by scavenging various reactive species such as free radicals is considered to be an important antioxidant mode of action .
dpph is a free radical donor that accepts an electron or hydrogen to become a stable diamagnetic molecule .
the tendencies of electron or hydrogen donation are critical factors in characterizing antioxidant activity that involves free radical scavenging .
these antioxidants are polyphenolic compounds which have protective effect against diseases   and can be found in all plants and in all parts of the plants ( tree bark , stalks , leaves , fruits , roots , flowers , pods , and seeds ) .
the dpph radical scavenging ability of the aqueous extract from c. populnea as represented in figure 2 revealed that the extract scavenged dpph radicals in a dose - dependent pattern ( 03.33  mg / ml ) .  
this result , however , is in agreement with the fe - induced lipid peroxidation ( figure 1 ) , phenolic content ( table 1 ) , and antioxidant activity of the extracts , suggesting that fe chelation may be one of the possible mechanisms through which antioxidant phytochemicals from stem bark of c. populnea prevent lipid peroxidation in tissue by forming a complex with fe , thus preventing the initiation of lipid peroxidation .  
furthermore , the reducing power of the extractable phytochemicals from cissus populnea ( stem bark ) expressed as ascorbic acid equivalent ( aae ) is presented in table 3 .
the reducing power as typified by the ability of the plant extracts to reduce fe to fe is a potent antioxidation defense mechanism , and two mechanisms available to affect this reducing power is by electron transfer and hydrogen atom transfer .
reported that the reducing property can be a novel antioxidant defense mechanism , possibly through the ability of the antioxidant compound to reduce transition metals .
therefore , the higher reducing ability of cissus populnea extract may have contributed to the higher protective effect observed .   likewise , the incubation of rat 's testes tissue homogenates in presence of sodium nitroprusside also caused a significant increase ( p < 0.05 ) in the rat 's testes malondialdehyde ( mda ) content as shown in figure 1(b ) ; however , the extract inhibited mda production content in the tissue in a dose - dependent ( 00.63  mg / ml ) .
however , judging by the ec50 value in table 2 , the aqueous extract of c. populnea had a high inhibitory effect on sodium nitroprusside - induced lipid peroxidation in rat testes .
the protective properties of the plant extract against sodium nitroprusside - induced lipid peroxidation in the testes could be because of the ability of the antioxidant phytochemicals present in the aqueous extract to quench - scavenge the nitrous radical and fe produced from the decomposition of sodium nitroprusside .
the aqueous extract of stem bark of c. populnea was able to protect the testes homogenate from both fe- and snp - induced lipid peroxidation in vitro .
a part of the mechanisms through which the water extractable phytochemicals in the plant protect the testes from oxidative stress may be through their phenolic content and antioxidant activity ; dpph - scavenging ability , fe - chelating and -reducing power .
 
 cissus populnea are plants associated with a myriad of medicinal uses in different parts of the world and are good sources of carotenoids , triterpenoids , and ascorbic acid . 
 the antioxidant properties and inhibitory effect of water extractible phytochemicals from stem bark of c. populnea on feso4 and sodium nitroprusside- ( snp- ) induced lipid peroxidation in rat testes were investigated in vitro . 
 the results revealed that the extract was able to scavenge dpph radical , chelate fe2 + and also had a high reducing power . 
 furthermore , the incubation of the testes tissue homogenate in the presence of feso4 and snp , respectively , caused a significant increase in the malondialdehyde ( mda ) contents of the testes . 
 however , the aqueous extract of the stem bark of c. populnea caused a significant decrease in the mda contents of both fe2 + ( ec50  =  0.027  mg / ml ) and snp- ( ec50  =  0.22  mg / ml ) induced lipid peroxidation in the rat testes homogenates in a dose - dependent manner . the water extractible phytochemicals from c. populnea 
 protect the testes from oxidative stress and this could be attributed to their high antioxidant activity : dpph - scavenging ability , fe2 + -chelating and -reducing power . 
 therefore , oxidatively stress in testes could be potentially managed / prevented by this plant . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion