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Tetracarpidium conophorum (Mull.Arg) Hutch & Dalziel inhibits FeSO4 -induced lipid peroxidation in rat’s genitals
© Akomolafe et al.; licensee BioMed Central. 2015
Received: 9 October 2014
Accepted: 12 February 2015
Published: 12 March 2015
To search for new sources of safe and inexpensive antioxidant agents which can be used to treat various oxidative stress - related diseases, the phenolic contents of leaf of Tetracarpidium conophorum were characterized and its effect on pro-oxidant induced oxidative stress in rat’s genitals for the first time was investigated.
The aqueous extract of the plant was prepared, the antioxidant activities of the extract were then evaluated using spectrophotometric method.
The result revealed that the introduction of aqueous extract of the plant caused significant concentration-dependent decrease (P < 0.05) in the MDA content of the Fe2+ -stressed testes and penis homogenates. The least MDA production occurred at the highest concentration of the extract (0.625 mg/mL). However, characterization of the extract with HPLC revealed that its major constituents were gallic acid, catechin, chlorogenic acid, caffeic acid, coumarin, rutin, quercitrin, quercetin, kaempferol and luteolin. Also, the result revealed that the ABTS* scavenging ability of the extract was 4.60 mmol/100 g while its vitamin C content was 23.49 mg/g which indicated that the plant is very rich in vitamin C. Furthermore, the extract scavenged DPPH, NO, OH* radicals and chelated Fe2+ in a dose-dependent manner.
The inhibitory effect of Tetracarpidium conophorum leaves could be attributed to the high levels of quercitrin, quercetin and luteolin and the mechanism through which these compounds possibly do this, could be by their radical scavenging abilities.
Infertility is a major clinical problem, affecting people medically and psychosocially. It has been established that 15% of all couples in the United States are infertile, and that the male factor is responsible for 25% of these cases . In Nigeria, this problem affects one out of five men. Oxidative stress (OS) has been identified as one of the major factors that affects fertility status and its role in male infertility has been well documented in recent years. Oxygen is essential to sustain life, at physiological levels, reactive oxygen species (ROS) are necessary to maintain normal cell functions . Conversely, the breakdown products of oxygen such as ROS can be detrimental to cell function and survival .
Although iron is necessary physiologically as component of many enzymes and proteins, free iron in the cytosol and mitochondria could cause considerable oxidative damage 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 .
The mechanism by which iron causes this deleterious effect is that Fe (II) reacts with hydrogen peroxide (H2O2) to produce the hydroxyl radical (OH) via the Fenton reaction, whereas superoxide can react with iron (III) to regenerate iron (II) that participates in the Fenton reaction . The overproduction of ROS can lead to direct attack on the polyunsaturated fatty acids of the cell membranes and induce lipid peroxidation. Spermatozoa, like any other aerobic cells, are constantly facing the oxygen-paradox  because they are rich in polyunsaturated fatty acids (PUFA) and thus, they are very susceptible to ROS attack which results in decrease sperm motility .
Malondialdehyde (MDA) is the end product of lipid peroxidation. It is a process where ROS degrade polyunsaturated fatty acids. MDA is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism .
However, the most likely and practical way to fight degenerative diseases is to improve body antioxidant status, which could be achieved by higher consumption of fruits and vegetables. Phenolic compounds are important group of secondary metabolites which are synthesized by plants.
Tetracarpidium conophorum (Mull.Arg) Hutch & Dalziel commonly called African walnut is a climbing shrub in the family Euphorbiaceae. The plant is locally cultivated mainly for the nuts which are cooked and consumed as snacks . It is locally used by the elderly people for the treatment of constipation. The amino and fatty acids components of the nut are used for the treatment of prolonged and constant hiccups . The barks are used in coffee as laxative and also chewed to reduce toothache. The leaves, bark and fruit of the plant are used medicinally and their uses include giddiness, toothache, eczema, pruritus, psoriasis, common cold and prostate cancer . Also, in West Africa the leaves are used as male fertility agent and in the treatment of dysentery. T. conophoum seed have been shown to have signifcant effect (P ≤ 0.05) on testosterone and estradiol levels, sperm motility (progresive motile sperm and non progresive motile sperm), testes and epididymides weight of the rats treated with various doses of the seed powder while it increased the level of LH, FSH, sperm viability and sperm count [12,13].
Although a lot have been reported on the chemical characterization of the phytoconstituents and antioxidant properties of T. conophorum seeds, there is still limited information on the potential of its leaves in the management and prevention of infertility diseases associated with oxidative stress. Hence, the objective of this study is to investigate the inhibitory effect of aqueous extract of T. conophorum leaves on FeSO4 -induced lipid peroxidation in rat testes and penis in vitro.
Collection and identification of plant samples
Fresh samples of T. conophorum leaves were obtained from a farm land near Akure metropolis, Nigeria. Authentication of the sample was carried out at the Department of Plant Science, Ekiti State University, Ado-Ekiti where voucher specimen (number UHAE 335) was deposited at the herbarium of the same Department.
Ten male Wistar albino rats weighing between 190 g and 250 g were purchased from the Central Animal House, Department of Biochemistry, University of Ilorin, Nigeria. They were housed in stainless steel cages under controlled conditions of a 12-hour light/dark cycle and 28°C temperature. The rats were allowed asses to food and water ad libitum. The cages were cleaned once daily. This study was carried out following approval from the ethical committee on the use and care of experimental animals of the Department of Biochemistry, Federal University of Technology, Akure, Nigeria. The research also adhered strictly to the Principles of Laboratory Animal Care (NIH Publication, No. 85–23).
Chemicals and reagents
Chemicals and reagents used such as thiobarbituric acid (TBA), 1,10-phenanthroline, deoxyribose, acetonitrile, acetic, gallic, chlorogenic and caffeic acids were purchased from Merck (Darmstadt, Germany). Catechin, coumarin, quercetin, quercitrin, rutin, luteolin and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteau’s reagent were procured from Sigma-Aldrich, Inc. (St. Louis, MO). Trichloroacetic acid (TCA) and MDA were sourced from Sigma-Aldrich, Chemie GmbH (Steinheim, Germany). Hydrogen peroxide, methanol, acetic acid, and HCl were sourced from BDH Chemicals Ltd. (Poole, England). Sodium carbonate, AlCl3, potassium acetate, Tris–HCl buffer, sodium dodecyl sulphate (SDS), FeSO4, potassium ferricyanide and ferric chloride were of analytical grade. A UV-visible spectrophotometer (Model 6305; Jenway, Barlo world Scientific, Dunmow, United Kingdom) was used to measure absorbance. High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.
Aqueous extract preparation
The fresh leaf samples were air dried, homogenized and kept in an air-tight container prior to the extraction. The powdered sample (1 g) was soaked in 100 mL of distilled water for 24 hours . The mixture was later filtered and the filtrate centrifuged at 400 g for 10 minutes. The clear supernatant was used for subsequent analysis.
2,2-azinobis(3-ethylbenzo-thiazoline-6-sulfonate) ABTS* scavenging ability
The ABTS* scavenging ability of the extract was determined according to the method of Re et al.  ABTS* was generated by reacting an ABTS aqueous solution (7 mmol L−1) with K2S2O8 (2.45 mmol L−1) in the dark for 16 hours and adjusting the absorbance with ethanol to 734–0.700 nm with ethanol. Then, 0.2 ml of the extract was added to 2.0 ml ABTS* solution and the absorbance was measured at 734 nm after 15 minutes. The result was calculated and reported as trolox equivalent antioxidant capacity.
Preparation of tissue homogenate
The rats were decapitated under mild diethyl ether anesthesia and the testes and penis tissues were rapidly dissected and placed on ice and weighed. This tissue was separately homogenized in cold saline (1:10) with about 10-up-and-down strokes at approximately 1200 rev/minutes in a Teflon-glass homogenizer. The homogenate was centrifuged for 10 minutes at 3000 g to yield a pellet that was discarded, and a low-speed supernatant (S1) containing mainly water, proteins and lipids was kept for the lipid peroxidation assay .
Lipid peroxidation and thiobarbibutric acid reactions
The lipid peroxidation assay was carried out using the method of Ohkawa et al.  Briefly, 100 μL S1 fraction was mixed with a reaction mixture containing 30 μL of 0.1 M Tris- HCl buffer (pH 7.4), aqueous extract of T. conophorum leaves (0–100 μL) and 30 μL of 250 mM freshly prepared FeSO4. The volume was made up to 300 μL with water before incubation at 37°C for 1 hour. The color reaction was developed by adding 300 μL of 8.1% SDS to the reaction mixture containing S1. This was subsequently followed by the addition of 500 μL of acetic acid/HCl (pH 3.4) and 500 μL 0.8% TBA. The mixture was incubated at 100°C for 1 hour. The absorbance of thiobarbituric acid reactive species produced was measured at 532 nm. MDA produced was expressed in percentage (%). The EC50 (extract concentration required to inhibit 50% of MDA produced) value were calculated using non-linear regression analysis.
DPPH free radical scavenging ability
The free radical scavenging ability of the extract against DPPH (1,1-diphenyl-2 picrylhydrazyl) free radical was evaluated as described by Gyamfi et al.  Briefly, an appropriate dilution of the extracts (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 minutes and the absorbance was measured at 516 nm. The control was carried out using 2 mL DPPH solution without the test samples. The DPPH free radical scavenging ability was subsequently calculated as follow: DPPH scavenging ability (%) = [(absorbance of control − absorbance of samples)/absorbance of control] × 100.
Degradation of deoxyribose (Fenton’s reaction)
where Absref = absorbance of the reference (reacting mixture without the test sample) and Abssample = absorbance of reacting mixture with the test sample.
Nitric oxide scavenging assay
Where Abs control is the absorbance of NO radicals; Abs sample is the absorbance of NO radical + sample or standard.
Fe2+ chelation assay
The Fe2+ chelating ability of the extract was determined using a modified method of Untel et al.  Freshly prepared 500 mM FeSO4 (150 μL) was added to a reaction mixture containing 168 μL of 0.1 M Tris–HCl (pH 7.4), 218 μL saline and the extracts (100–400 μL). The reaction mixture was incubated for 5 minutes, before the addition of 13 μL of 0.25% 1, 10-phenanthroline. The absorbance was subsequently measured at 510 nm in a spectrophotometer. The Fe (II) chelating ability was subsequently calculated with respect to the reference which contains all the reagents without the test sample.
Quantification of compounds by HPLC-DAD
Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm × 150 mm) packed with 5 μm diameter particles; the mobile phase was water containing 2% formic acid (A) and acetonitrile (B), and the composition gradient was: 17% of B until 10 minutes and changed to obtain 20%, 30%, 50%, 60%, 70%, 20% and10% B at 20, 30, 40, 50, 60, 70 and 80 minutes, respectively, following the method described by Kamdem et al.  with slight modifications. Tetracarpidium conophorum aqueous extract was analyzed at a concentration of 20 mg/mL. The presence of ten antioxidants compounds was investigated, namely, gallic acid, chlorogenic acid, caffeic acid, catechin, coumarin, quercetin, quercitrin, rutin, luteolin and kaempferol. Identification of these compounds was performed by comparing their retention time and UV absorption spectrum with those of the commercial standards. The flow rate was 0.7 mL/min, injection volume 40 μl and the wavelength were 270 nm for gallic acid, 276 nm for coumarin, 280 nm for catechin, 327 nm for caffeic and chlorogenic acids, and 365 nm for quercetin, quercitrin, rutin, kaempferol and luteolin. The samples and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.025 – 0.250 mg/ml for catechin, coumarin, quercetin, quercitrin, rutin, kaempferol and luteolin; and 0.030 – 0.350 mg/ml for caffeic, chlorogenic and gallic acids. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 600 nm). Calibration curve for catechin: Y = 13649x + 1257.8 (r = 0.9995); coumarin: Y = 11974x + 1309.5 (r = 0.9999); gallic acid: Y = 12890x + 1253.7 (r = 0.9998); caffeic acid: Y = 13078x + 1186.3 (r = 0.9991); chlorogenic acid: Y = 11952x + 1187.5 (r = 0.9996); rutin: Y = 12657x + 1238.9 (r = 0.9999); quercetin: Y = 13591x + 1318.7 (r = 0.9998); quercitrin: Y = 11783x + 1263.8 (r = 0.9994); kaempferol: Y = 11974x + 1271.0 (r = 0.9999) and luteolin Y = 13528x + 1367.2 (r = 0.9997). All chromatography operations were carried out at ambient temperature and in triplicate. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves, as defined by Kamdem et al.  LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve.
The results of the replicates were pooled and expressed as mean ± standard deviation. Analysis of variance (ANOVA), Tukey test, and student t-test were carried out where appropriate to analyze the result. Significance was accepted at P ≤ 0.05.
Results and discussion
Antioxidant capacity, vitamin C content and EC 50 values of inhibition of FeSO 4 -induced lipid peroxidation in rat’s testes and penis by aqueous extract of T. conophorum leaves
Antioxidant capacity(mmol. TEAC/100 g)
Vitamin C content(mg/g)
EC50value in testes(mg/ml)
EC50value in penis(mg/ml)
4.60 ± 0.23
23.49 ± 0.04
0.43 ± 0.12
0.72 ± 0.38
Components of Tetracarpidium conophorum aqueous leaf extract
2.47 ± 0.03a
1.93 ± 0.01b
6.71 ± 0.01c
3.85 ± 0.02d
6.79 ± 0.03c
1.90 ± 0.01b
10.47 ± 0.03e
10.28 ± 0.01e
1.89 ± 0.02b
12.56 ± 0.01f
Antioxidants carry out their protective role on cells either by preventing the production of free radicals or by neutralizing or scavenging free radicals produced in the body or by reducing or chelating the transition metal composition of foods [31,22]. In an attempt to explain the main mechanism through which the aqueous extract of the plant protect testes tissue against Fe2+ induced lipid peroxidation, the DPPH radical scavenging, ABTS radical scavenging and Fe2+ chelating abilities were assessed.
ABTS, a blue green chromophore is mostly reactive toward phenolics, thiols and other antioxidants . The ABTS* free radical scavenging ability model has the advantage of being more versatile due to the minimal spectral interference as the absorption maximum used is 760 nm, a wavelength not normally encountered with natural products . The result revealed that the ABTS* scavenging ability of the aqueous extract is 4.60 mmol/100 g while its vitamin C content is 23.49 mg/g which indicate that the plant is very rich in vitamin C (Table 1). The ABTS* free radical scavenging ability of the extract could be because of the hydrogen donating ability of the polyphenolics present in the extract.
The protection of testes and penis tissues from Fe2+ -induced lipid peroxidation by the aqueous extract of Tetracarpidium conophorum leaves could be attributed to very high level of quercitrin, quercetin and luteolin and the mechanism through which they possibly do this could be by their radical scavenging abilities.
This research was supported by grants from Govan Mbeki Research and Development Centre, University of Fort Hare, South Africa. The authors appreciate the effort of Aline Augusti Boligon, João Batista Teixeira da Rocha and Margareth Linde Athayde of Phytochemical Research Laboratory, Department of Industrial Pharmacy, Federal University of Santa Maria, Build 26, room 1115, Santa Maria, CEP 97105–900, Brazil, who helped in carry out the HPLC analysis.
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