Hepatic Oxidative Stress and Haematological Parameters of Wistar Rats Following Infusion of Carbon Tetrachloride and Treated with Raw and Hydrothermal Processed Herbs

Ojiako, Chikezie, Ukairo, Ibegbulem, and Nwaoguikpe: Hepatic Oxidative Stress and Haematological Parameters of Wistar Rats Following Infusion of Carbon Tetrachloride and Treated with Raw and Hydrothermal Processed Herbs

Authors

INTRODUCTION

Oxidative stress is elicited following persistent generation of reactive oxygen and nitrogen species (RONS) such that the antioxidant capacity of the cell is overwhelmed.1,2,3,4,5 Major generators of RONS are the mitochondrial electron transfer chain, cytochrome P450 family and peroxisomes as well as activities of lipoxygenase, xanthine oxidase and membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase (NADPH oxidase).6,7,8 For instance, the superoxide (O2•−) radicals are largely generated from complexes I and III of the mitochondrial electron transfer chain and, for the most part, responsible for the cellular oxidative damage that underlie degenerative diseases, inflammation, mutation, carcinogenesis and senescence.9,10 The pathophysiology of degenerative diseases revealed that oxidative tissue damage is mediated by activating a number of cellular stress-sensitive pathways, which include nuclear factor-ĸB (NF-ĸB), p38 mitogen-activated protein kinase (MAPK), NH2-terminal Jun kinases/stress-activated protein kinases (JNK/SAPK) and hexosamines.1,11,12

Superoxide dismutase (SOD) activity in concert with spontaneous dismutation of O2•− are the major sources of cellular hydrogen peroxide (H2O2).9,13 The oxidizing potential of H2O2, by its capability to act as substrate to produce other oxidizing species like hydroxyl (OH•−) radicals and hypochlorous acid (HClO),14,15 is largely responsible for the deleterious actions of H2O2. Furthermore, H2O2 in the presence of metallic ions like Fe2+ and Cu2+, in the so-called Fenton reaction, is converted to reactive OH•−, whereas in the Haber-Weiss reaction, O2•− and H2O2 generates more OH•−.9,10 The H2O2 is capable of degrading haem proteins, inactivate enzymes, oxidize DNA, lipids and sulphydryl groups (-SH).13,15 Specifically, OH•− reacts with membrane lipid components yielding alkoxyl (RO2•−) radicals, which undergo auto-oxidation in a self-propagating chain reaction to form lipid peroxides (LOO•−) and hydroperoxides (LOOH).9 In the presence of O2•−, nitric oxide (NO) is converted to highly potent oxidant molecule- peroxynitrite (¯ONOO).1,9,16 The pro-radicals like peroxynitrite (¯ONOO) are relatively stable compared to free radicals. However, protonated ¯ONOO forms the highly reactive peroxynitrous acid (ONOOH).17 Generation of excess ¯ONOO leads to oxidative damage and tissue lesion.9

Toxicological evaluation showed that the metabolism of certain xenobiotics elicits the production of overwhelming levels of RONS.9,18 Carbon tetrachloride (CCl4) is a toxic lipophilic molecule that easily absorbs into lipid structural compartments of the cell and is often used to provoke organ lesion in animal models by virtue of its capability to elicit microsomal CYP2E1–induced formation of trichloromethyl (CCl3•−) and trichloromethylperoxyl (Cl3COO•−) radicals.19,20,21,22 In a concerted manner, CCl3•− binds to -SH group of glutathione and thiol entities of proteins, whereas Cl3COO•− initiates a chain reaction of lipid peroxidation, and thereby alters the permeability of mitochondria, endoplasmic reticulum and the plasma membrane.23 Additionally, CYP2E1-derived RONS, like H2O2, LOOH, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), may diffuse from the hepatocytes to other cell types where fibrotic collagen Type I and III molecules are activated, eliciting fibrotic changes in the affected tissues.24 These alterations in homeostasis lead to tissue lesion, necrosis and ultimately apoptosis.25

Blood is a tissue comprised of the corpuscles (erythrocytes, leucocytes and platelets) suspended in fluid medium called plasma and circulating within a close system in vertebrates referred to as vascular system. The plasma mostly consists of water with dissolved metabolites, proteins, lipoproteins, hormones, electrolytes and nutrients. By virtue of the fact that the polyunsaturated fatty acids (PUFAs), which are potent targets of RONS cytotoxicity, account for the majority of the erythrocyte membrane structural lipid components, the erythrocyte is highly vulnerable to the vagaries of oxidative stress.26,27 Erythrocytes in particular, are further predisposed to oxidative damage as a result of their continual exposure to oxygen in the presence of transition metal catalysts, especially in the form of free iron and haemin.26,28,29,30 Oxidative damage to erythrocyte structural components is exacerbated in the event of exposure of the vascular system to xenobiotics that promote or elicit the generation of RONS,31,32 and thereby cause profound perturbation of blood homeostasis.

Protective mechanisms have evolved to limit overwhelming levels of RONS and their detrimental outcome.1,9,33,34,35,36,37 Nevertheless, endogenous antioxidant systems may not confer absolute protection in the event of overwhelming levels of RONS. Therefore, additional protective mechanisms in the form of dietary antioxidants supplementation and intake of herbal products are required to stave off oxidative stress-induced pathologic conditions.37,38 The use of herbs, which are rich in antioxidant phytochemicals and vitamins, has been suggested to be an effective therapeutic strategy for the management and amelioration of pathologic conditions link with oxidative stress.37,38,39 Many Nigerian indigenous herbs such as Monodora myristica, Chromolaena odorata (Linn), Buccholzia coriacea and Sphenostylis stenocarpa are commonly used among traditional herbal medicine practitioners as remedies for pathologic conditions and disorders. The phytochemical contents and medicinal usefulness of these Nigerian indigenous herbs have been reported elsewhere.40,41,42,43,44,45,46,47 In traditional herbal medicine practice, these herbs are either administered raw or subjected to hydrothermal processing prior their application.

Previous studies have shown that raised levels of products of lipid peroxidation and alterations in antioxidant enzymes activities in plasma are diagnostic of oxidative stress and serve as biomarkers for evaluating therapeutic interventions.33,36 The present study evaluated the differentials in phytochemical contents of raw and hydrothermal processed Nigerian indigenous herbs, namely, M. myristica, C. odorata, B. coriacea and S. stenocarpa as well as their capacity to ameliorate CCl4-induced oxidative stress in hepatic tissues of Wistar rats, in which the levels of products of lipid peroxidation and antioxidant enzymes activities served as measure of therapeutic interventions. Furthermore, the levels of adjustments of some haematological parameters of the rats were also investigated following the administration of the herbs.

MATERIALS AND METHODS

Collection and preparation of samples

High-grade raw seeds of M. myristica, B. coriacea and S. stenocarpa were purchased from Relief and Obazu-Mbieri Markets located in Owerri Capital Territory, Imo State, Nigeria. Fresh leaves of C. odorata were harvested from a private garden in Amakihia, Owerri-North Local Government Area, Imo State, Nigeria. The samples were transported to the laboratory, identified and authenticated by Dr. E.S. Willie at the Herbarium of the Department of Agronomy, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. All samples were collected between the months of February and March, 2015. Voucher specimens were deposited at the Herbarium for reference purposes.

The various samples were washed separately in a continuous flow of distilled water for 15 min and allowed to dry at laboratory ambient temperature (T = 24 ± 5°C). The samples were divided into two portions on an equal weight basis and designated as follows:

Group R: Raw samples

Group H: Hydrothermal processed samples

Appropriate separate quantities of Group R samples were pulverized using Thomas-Willey milling machine (ASTM D-3182, India). The ground samples were transferred into corresponding vacuum desiccators and allowed to dry at laboratory ambient temperature until a constant weight was achieved. Appropriate separate quantities of Group H samples were boiled in distilled water in corresponding conical flasks (sample/water ratio = 1:4 w/v). According to local traditional medicine practice, hydrothermal processing of the seed samples was in the following durations: M. myristica = 10 min, B. coriacea = 1 h and S. stenocarpa = 1.5 h, whereas leaves of C. odorata were subjected to hydrothermal processing for 5 min. Next, the Group H samples were dried separately in an oven (Gallenkamp Oven 300 plus series, England) at 50°C until a constant weight was achieved. Finally, Group H samples were ground using the Thomas-Willey milling machine (ASTM D-3182; India), after which the samples were stored in air-tight plastic bottles with screw caps pending extraction.

Extraction of samples

Extraction of Group R and Group H samples was according to the methods previously described.48 Portion of 10 g each of the ground and dried Group R and Group H samples were subjected to repeated soxhlet extraction cycles for 2 h using 96% CH3OH (BDH, U.K) as solvent to obtain final volume of 250 mL of corresponding extracts. The volumes of the extracts were concentrated and recovered in a rotary evaporator (Büch Rotavapor R-200) for 12 h at 50°C under reduced pressure. The extracts were dried in vacuum desiccators for 24 h, wrapped in aluminum foil and stored in air-tight plastic bottles with screw caps at ≤ 4 °C. The yields were calculated to be as follows:

  • Extract R1; Raw seeds of M. myristica = 8.94% (w/w).

  • Extract R2; Raw leaves of C. odorata = 6.22% (w/w).

  • Extract R3; Raw seeds of B. coriacea = 8.07% (w/w).

  • Extract R4; Raw seeds of S. stenocarpa = 14.02% (w/w).

  • Extract H1; Hydrothermal processed seeds of M. myristica = 6.41% (w/w).

  • Extract H2; Hydrothermal processed leaves of C. odorata = 4.39% (w/w).

  • Extract H3; Hydrothermal processed seeds of B. coriacea = 7.12% (w/w).

  • Extract H4; Hydrothermal processed seeds of S. stenocarpa = 13.51% (w/w).

A portion of the each extract was measured for phytochemical contents. Also, each extract was reconstituted in phosphate buffered saline (PBS) solution that was osmotically equivalent to 100 g/L PBS (90.0 g NaCl, 17.0 Na2HPO4.2H2O and 2.43 g NaH2PO4.2H2O) and an appropriate dose was administered to corresponding experimental animals.

Phytochemicals

Quantitative compositions of alkaloids, flavonoids, tannins and saponins were measured using standard methods. The concentration of alkaloids and saponins were measured using the methods of Harborne.49 The flavonoids content was according the methods of Boham and Kocipal.50 The concentration of tannins was measured using the methods of Van-Burden and Robinson,51 as reported by Belonwu et al.52

Experimental animals

Healthy male Wistar rats (90 days old) weighing between 150-260 g were maintained at laboratory ambient temperature of 30–55% relative humidity on a 12-h light/12-h dark cycle, with access to water and standard commercial feeds (SCF) (Ewu Feed Mill, Edo State, Nigeria) ad libitum, for 2 weeks acclimatization period. The Institutional Review Board of the Department of Biochemistry, Federal University of Technology, Owerri, Nigeria, granted approval for this study. The care and handling of the animals conformed to the standard principles of laboratory animal care of the United States National Institutes of Health (NIH, 1978).

Carbon tetrachloride infusion/experimental design

A total of 72 male Wistar rats were allotted into 12 groups of 6 rats each. Fibrosis was induced in the Wistar rats by single dose intra-peritoneal (i.p) injection of CCl4 in paraffin oil as vehicle {1:1 v/v; dose = 1.0 mL/kg body weight (b.wt.)} for 2 consecutive days.53 The animals were deprived of feed only for an additional 16 h before commencement of treatment as described.54,55 The animal groups were randomly designated on the bases of diets and treatments (dose = 250 mg/kg b.wt.; i.p. of the extracts and silymarin or otherwise 1.0 mL/kg b.wt.; i.p. of PBS, paraffin oil and CCl4/paraffin oil mixture) received for 28 consecutive days. Silymarin (Medical Union Pharmaceuticals Company) was used as the standard drug for reference treatment of fibrotic rats.54

  • Group 1: Normal rats received SCF + water ad libitum + PBS.

  • Group 2: Normal rats received SCF + water ad libitum + paraffin oil.

  • Group 3: Fibrotic rats received SCF + water ad libitum + CCl4/paraffin oil mixture.

  • Group 4: Fibrotic rats received SCF + water ad libitum + silymarin.

  • Group 5: Fibrotic rats received SCF + water ad libitum + Extract R1.

  • Group 6: Fibrotic rats received SCF + water ad libitum + Extract R2.

  • Group 7: Fibrotic rats received SCF + water ad libitum + Extract R3.

  • Group 8: Fibrotic rats received SCF + water ad libitum + Extract R4.

  • Group 9: Fibrotic rats received SCF + water ad libitum + Extract H1.

  • Group 10: Fibrotic rats received SCF + water ad libitum + Extract H2.

  • Group 11: Fibrotic rats received SCF + water ad libitum + Extract H3.

  • Group 12: Fibrotic rats received SCF + water ad libitum + Extract H4.

At the end of the feeding and treatment period, the rats were subjected to fasting for 12 h, after which time the animals were sacrificed and autopsy samples of the liver were excised for assessment of oxidative stress parameters. Blood samples were drawn from the orbital sinus.56 for measurement of haematological parameters.

Preparation of liver homogenates

Organ homogenate was prepared according the procedures of Adekunle et al.57 as previously described.58 Briefly, the liver was excised and placed between blotting papers to remove accompanying blood. Next, the organ was rinsed in 1.15% KCl solution to obliterate residual hemoglobin molecules. The sample was homogenized using a Teflon homogenizer in aqueous K2PO4/KHPO4 buffer (0.1 M; pH = 7.4); in 4:1 volume of buffer to organ weight. Subsequently, the homogenate was centrifuged at 10,000 ×g for 20 min at 4°C to obtain the post mitochondrial supernatant (PMS) fraction and collected into sample bottles. The PMS fraction was stored at 4°C until further analysis.59 The homogenate was used to assay the following oxidative stress parameters: MDA content, CAT activity and SOD activity. Protein concentration was measured at maximum wavelength (λmax) = 595 nm by the methods of Bradford,60 using bovine serum albumin as standard.

Malondialdehyde

Measurement MDA content of PMS fraction was according to the methods of Tjahjani et al.,61 as described.62 A mixture of 20% trichloroacetic acid (TCA) and 0.67% thiobarbituric acid (TBA) in a ratio of 2:1 was added into a test tube. A volume of 0.2 mL of PMS fraction was introduced in the mixture and boiled for 10 min in a water bath. After cooling to 24°C, the mixture was centrifuged at 3,000 ×g for 10 min. The absorbance of supernatant was read with a spectrophotometer (SPECTRONIC 20, Labtech–Digital Blood Analyzer®) at λmax = 532 nm. The absorbance of the samples was converted to MDA concentrations using the MDA standard curve.63

Catalase

Measurement CAT activity of PMS fraction was according to the methods of Luck.64 The final reaction volume of 3.0 mL contained 0.05 M Tris-buffer, 5 mM EDTA (pH = 7), and 10 mM H2O2 (in 0.1 M K2PO4/KHPO4 buffer; pH = 7). A hundred micro milliliter (100 µL) aliquot of the PMS fraction was added to the above mixture. The rate of change of absorbance per min at λmax = 240 nm was recorded for 5 min. CAT activity was calculated using Σ = 43.6 M-1cm-1 and expressed in terms of mole H2O2 consumed/min/mg protein (U/mg protein).

Superoxide dismutase

SOD activity of PMS fraction was estimated according to the methods of Kono,65 as previously reported.58 Briefly, the reaction mixture contained solution A (50 mM Na2CO3, 0.1 mM EDTA, pH = 10), solution B (96 µM nitroblue tetrazolium [NBT] in solution A), and solution C (0.6% Triton X-100 in solution A) were incubated at 37°C for 10 min. The reaction was started by introducing 100 µL of solution D (20 mM hydroxylamine hydrochloride, pH = 6) to the reaction. The rate of NBT dye reduction by O2•− generated due to photo-activation of hydroxylamine hydrochloride was measured at λmax = 560 nm in the absence of PMS fraction. Next, a 10 µL aliquot of PMS were added to the reaction mixture and 50% inhibition in the rate of NBT reduction by SOD present in the enzyme source was measured. A unit (U) of SOD activity was defined by the 50% inhibition of NBT. SOD activity was expressed in U/mg protein.

Haematology

Measurement of plasma haemoglobin concentration was the cyanomethaemoglobin method.66 Red blood cells count (RBC), white blood cells count (WBC) and platelets count (PC) were measured by direct enumeration using a haemocytometer with improved neubauer slide.67

Statistical analysis

The data collected were analyzed by the analysis of variance procedure while treatment means were separated by the least significance difference (LSD) incorporated in the statistical analysis system (SAS) package of 9.1 version, (2006).

RESULTS

Table 1 shows the levels of disparities in some phytochemical contents between the raw and hydrothermal processed herbal extracts. Extract R1 gave the highest concentration of alkaloids, but showed 35.84% loss in alkaloids in corresponding hydrothermal processed herbal extract (Extract H1). Likewise, hydrothermal processed herbal extracts (Extract H2, Extract H3 and Extract H4) represented 55.58%, 78.51% and 55.07% loss in alkaloids compared with their corresponding raw extracts (Extract R2, Extract R3 and Extract R4); p < 0.05. Similarly, the hydrothermal processed herbal extracts exhibited lower concentrations of flavonoids compared with their corresponding raw herbal extracts. However, the loss in flavonoid contents in Extract H3 and Extract H4 was not significantly different (p > 0.05) from their corresponding raw herbal extracts (Extract R3 and Extract R4). The concentrations of tannins in the four hydrothermal processed herbal extracts (Extracts H1-H4) were significantly lower (p < 0.05) than their corresponding raw herbal extracts (Extracts R1-R4). For instance, concentration of tannins in Extract H1 was 21.1 fold lower than that of Extract R1. The concentrations of saponins in all the hydrothermal processed herbal extracts were not significantly different (p > 0.05) from their corresponding raw herbal extracts.

Figure 1 shows that hepatic MDA concentration of Group 1 was not significantly different (p > 0.05) from that of Group 2. Hepatic tissue of Group 3 gave the highest MDA concentration, which was significantly different (p < 0.05) from those of Group 1 and Group 2. However, hepatic MDA concentration of Group 3 was not significantly (p > 0.05) from those of Group 6, Group 7, Group 10 and Group 11. Hepatic tissue of Group 4 accounted for the lowest MDA concentration amongst the CCl4 treated groups (Groups 3-12). In terms of the herbal treated experimental rat groups (Groups 5-12), hepatic MDA concentrations of Group 5 and Group 6 were not significantly different (p > 0.05) from those of corresponding Group 9 and Group 10. Conversely, hepatic MDA concentrations of Group 7 and Group 8 were not significantly different (p > 0.05) from those of corresponding Group 11 and Group 12.

Table 1

Some phytochemical contents of raw and hydrothermal processed herbal extracts.

 Concentration (mg/100 g dry sample) 

ExtractAlkaloidsFlavonoidsTanninsSaponins*
R131.33 ± 0.29a14.17 ± 0.10b13.28 ± 0.11a1.67 ± 0.04
R216.50 ± 0.10b,c21.00 ± 0.18a12.63 ± 0.09a,b2.23 ± 0.02
R37.00 ± 0.09d,e6.33 ± 0.08d,e7.39 ± 0.07c,d2.33 ± 0.03
R44.83 ± 0.03d,e,f6.23 ± 0.07d,e,f8.10 ± 0.12c2.83 ± 0.03
H120.01 ± 0.21b6.50 ± 0.07d0.62 ± 0.01e1.33 ± 0.09
H27.33 ± 0.07d12.33 ± 0.09b,c0.42 ± 0.01e,f,g,h1.00 ± 0.01
H31.50 ± 0.01e,f,g,h4.33 ± 0.03d,e,f,g0.46 ± 0.01e,f,g1.67 ± 0.02
H42.17 ± 0.03e,f,g3.17 ± 0.03d,e,f,g,h0.52 ± 0.01e,f2.33 ± 0.02

The mean (X) ± S.D of six (n = 6) determinations. Means in the column with the same letter are not significantly different at p > 0.05 according to LSD.

* Concentrations of saponins showed no significant difference p > 0.05 according to LSD.

Figure 1

Hepatic malondialdehyde concentrations of experimental rat groups

#: MDA concentrations are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2)

*: MDA concentrations are not significantly different (p > 0.05) from untreated fibrotic rats (Group 3).

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Figure 2

Hepatic catalase and superoxide dismutase activities of experimental rat groups

#: SOD activities are significantly different (p < 0.05) from untreated fibrotic rats (Group 3)

*: CAT activities are significantly different (p < 0.05) from untreated fibrotic rats (Group 3)

£: SOD activities are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2)

¥: CAT activities are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2).

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Figure 2 shows that the hepatic SOD and CAT activities of Group 1 and Group 2 were not significantly different (p > 0.05). The lowest hepatic SOD and CAT activities were observed in Group 3 and were significantly different (p < 0.05) from those of Group 1 and Group 2. Furthermore, hepatic SOD and CAT activities of Group 4 were significantly increased (p < 0.05) compared with that of Group 3. Hepatic CAT activities of Group 5, Group 6 and Group 7 were not significantly different (p > 0.05) from those of corresponding Group 9, Group 10 and Group 11, whereas hepatic CAT activity of Group 8 was significantly higher (p < 0.05) than that of corresponding Group 9. Conversely, hepatic SOD activities of Group 9, Group 10 and Group 11 were not significantly higher (p > 0.05) than those of corresponding Group 5, Group 6 and Group 7, whereas hepatic SOD activities of Group 8 and correspond Group 12 showed no significant difference (p > 0.05).

Plasma haemoglobin concentration of Group 1 was not significantly different (p > 0.05) from that of Group 2. Figure 3 showed that Group 3 gave the lowest plasma haemoglobin concentration. Furthermore, plasma haemoglobin concentrations of Group 1 and Group 2 were significantly higher (p < 0.05) than that of group 4. Similarly, plasma haemoglobin concentrations of groups (5-8) were not significantly different (p > 0.05) from those of corresponding Groups (9-12) (Figure 3).

Figure 4 shows that RBC of Group 1 was not significantly different (p > 0.05) from that of Group 2. Group 3 exhibited the lowest RBC amongst the experimental rat groups and was significantly different (p < 0.05) from those of Groups 4-12. Additionally, RBC of Group 4 was not significantly different (p > 0.05) from those of Groups 5-12. However, RBCs of Group 9 and Group 10 were significantly higher (p < 0.05) to those of corresponding Group 5 and Group 6.

The WBC of Group 1 was significantly different (p < 0.05) from that of Group 2 (Figure 5). Group 3 gave the highest WBC compared with other experimental rat groups (p < 0.05). Additionally, WBCs of Group 5, Group 8, Group 9, Group 11 and Group 12 were not significantly different (p > 0.05) from that of Group 2. Likewise, Figure 6 shows that the PCs amongst the experimental rat groups (Groups 1-12) varied within the range of (6.69 ± 0.06 - 3.81 ± 0.06) x 102 cells/µL. The PCs of the experimental rat groups (Groups 4-12) showed no significant difference (p > 0.05). Conversely, PCs of Group 1 and Group 2 were significantly lower (p < 0.05) than those of Groups (3-12).

Figure 3

Plasma haemoglobin concentrations of experimental rat groups

#: Haemoglobin concentrations are significantly different (p < 0.05) from untreated fibrotic rats (Group 3)

¥: Haemoglobin concentrations are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2).

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Figure 4

Plasma red blood cell counts of experimental rat groups.

#: RBCs are significantly different (p < 0.05) from untreated fibrotic rats (Group 3)

¥: RBCs are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2)

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Figure 5

Plasma white blood cell counts of experimental rat groups

#: WBCs are not significantly different (p > 0.05) from Group 2

*: WBCs are significantly different (p < 0.05) from control rat group (Group 1).

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Figure 6

Plasma platelet counts of experimental rat groups

*: RBCs are significantly different (p < 0.05) from control rat groups (Group 1 and Group 2).

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DISCUSSION

Bioactive principles from plants, namely the antioxidant phytochemicals, are of paramount importance in the diet because of their nutritional and medicinal benefits.68,69 The impacts of hydrothermal processing of medicinal plants and vegetables on their therapeutic and nutritional benefits have been controversially discussed.70,71,72,73,74 However, previous reports had noted that heat processing of animal feedstuffs and raw vegetables altered their phytochemical contents such that their nutraceutical properties were compromised.75,76,77 For instance, Zhang and Hamauzu,72 reported that ascorbic acid content in broccoli declined dramatically following cooking, whereas contrary reports revealed that heat treatment could actually improve the availability of polyphenols of frozen broccoli.78 Furthermore, boiling of peas caused marginal losses in their water and lipid-soluble antioxidant activities.79 The present study showed that hydrothermal processing of the experimental herbal extracts caused varying reductions in their phytochemical contents, of which the saponins contents were not affected.

The raised level of hepatic MDA of Group 3 was above baseline concentrations of those of Group 1 and Group 2, which was an obvious indication of exacerbated oxidative damage of the hepatocytes following CCl4 intoxication as previously reported.54,80,81,82,83 Furthermore, the raw and hydrothermal processed herbal extracts exhibited equivalent capacities to lower the severity of hepatic oxidative damage in the fibrotic rats but did not provide full therapeutic benefits. By implication, hydrothermal processing of the herbs did not alter their capacities to lower the severity of CCl4-induced hepatic damage. Group 4 exhibited lower MDA concentrations than those of fibrotic rats treated with raw and hydrothermal processed herbal extracts, which were indications that silymarin offered better therapeutic benefits against hepatic oxidative injury than the experimental herbal extracts. Reports from a related study showed that bark extract of Bathysa cuspidate lowered hepatic MDA concentrations in male Wistar rats, which was a demonstration of the efficacy of the extract to scavenge RONS and conferred protection against biomembranes peroxidation.84 Additionally, these findings corroborated the outcomes of previous studies using extracts of Solanum spp. (false “Jurubeba), Artemisia capillaries, and Decalepis hamiltonii.85,86,87

The present study revealed that fibrotic rats exhibited relatively lower hepatic CAT and SOD activities, which concurred with earlier reports.58,59,82,88 According to Avti et al.,59 overwhelming levels of RONS caused the inhibition of redox enzymes such as CAT and glutathione peroxidase activities as typified by relatively low CAT and SOD activities of Group 3. Therefore, reversion of hepatic CAT and SOD activities towards baseline levels of the control rat groups was diagnostic of the capacities of raw and hydrothermal herbal extracts as well as silymarin to neutralize overwhelming levels of RONS and ameliorate oxidative stress in the fibrotic rats. Furthermore, the present study showed that the hydrothermal processed herbal extracts exhibited greater capacities than the raw herbal extracts to improve SOD activity in the fibrotic rats. Conversely, the raw herbal extracts showed greater capacities than the hydrothermal processed herbal extracts to improve CAT activity in the fibrotic rats. However, the raw and hydrothermal processed herbal extracts did not exert full therapeutic benefits since hepatic CAT and SOD activities were comparatively lower than those of the control groups at the end of 28 consecutive days of treatment.

The present study showed that CCl4 intoxication perturbed haematological parameters of the rats and the pattern of alterations in serum haemoglobin concentrations paralleled that of RBCs. These findings concurred with previous reports.89,90 and appeared to suggest that CCl3•− and Cl3COO•− from hepatic metabolism of CCl4 diffused into systemic circulation and rendered the erythrocyte vulnerable to oxidative damage. Accordingly, infusion of the experimental rats with CCl4 provoked massive haemolysis with attendant anaemia. Additionally, the results of the present study showed that raw and hydrothermal processed herbal extracts exhibited equivalent limited capacities to protect the erythrocytes against to oxidative damage. By implication, hydrothermal processing of the raw herbs did not affect their capacities to offer limited protection against CCl4-induced erythrocytes oxidative damage. Previous studies had shown that potent antioxidants such as melatonin, ascorbic acid and α-tocopherol as well as plant extracts that exhibited high antioxidant activity protected rats against experimentally-induced tissue oxidative damage.88,89,90,91

The elevations of plasma WBCs of the experimental rat groups following CCl4 intoxication were consistent with previous findings,92,93 in which they noted that increased plasma WBC paralleled the levels of tumor necrosis factor-α (TNF-α) and interferon (INF-γ) in CCl4-induced fibrotic rats. Furthermore, acute oxidative stress had been widely reported to provoke increased plasma WBC, neutrophil count as well as the ratio of neutrophil to lymphocyte in experimental animals.94 Previous studies had shown that antioxidant-containing diets caused significant reversion of elevated WBC to normal levels by inhibiting downstream pro-inflammatory factors associated with acute and chronic hepatic damages in animal models.92,95 Additionally, elevated plasma WBC could also be diagnostic of the presence of pathogenic antigens in systemic circulation. The elevation of plasma PC in untreated fibrotic rats may not be unconnected with intravascular coagulation associated with CCl4-induced acute hepatic injury as previously reported elsewhere.96

CONCLUSION

Hydrothermal processing of the raw herbs did not adversely affect their capacities to ameliorate hepatic oxidative stress and haematological disorders in the fibrotic rats.

PICTORIAL ABSTRACT

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SUMMARY

  • Hydrothermal processing of the experimental herbal extracts caused varying reductions in their phytochemical contents, of which the saponins contents were not affected.

  • Hydrothermal processing of the raw herbs did not adversely affect their capacities to ameliorate hepatic oxidative stress and haematological disorders in the fibrotic rats.

  • The raised level of hepatic MDA was an obvious indication of exacerbated oxidative damage of the hepatocytes following CCl4 intoxication.

  • Reversion of hepatic CAT and SOD activities towards baseline levels of the control rat groups was diagnostic of the capacities of raw and hydrothermal herbal extracts to neutralize overwhelming levels of RONS and ameliorate oxidative stress in the fibrotic rats.

  • The raw herbal extracts showed greater capacities than the hydrothermal processed herbal extracts to improve CAT activity in the fibrotic rats.

  • The raw and hydrothermal processed herbal extracts exhibited equivalent limited capacities to protect the erythrocytes against to oxidative damage. By implication, hydrothermal processing of the raw herbs did not affect their capacities to offer limited protection against CCl4-induced erythrocytes oxidative damage.

  • The raw and hydrothermal processed herbal extracts exhibited equivalent capacities to lower the severity of hepatic oxidative damage in the fibrotic rats but did not provide full therapeutic benefits.

ACKNOWLEDGEMENT

The authors are grateful for the technical assistance offered by Dr. E.S. Willie of the Department of Agronomy, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

ABOUT AUTHORS

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Professor Okey A. OJIAKO Ph.D; Medical Biochemistry Department of Biochemistry, Federal University of Technology, Owerri, Nigeria.

Dr. Paul C. CHIKEZIE Ph.D; Medical Biochemistry Department of Biochemistry, Imo State University, Owerri, Nigeria.

Dr. Doris I. UKAIRO Ph.D; Medical Biochemistry Department of Biochemistry, Federal University of Technology, Owerri, Nigeria.

Dr. Chiedozie O. IBEGBULEM Ph.D; Nutritional Biochemistry/Toxicology Department of Biochemistry, Federal University of Technology, Owerri, Nigeria.

Professor Reginald N. NWAOGUIKPE Ph.D; Biochemical Pharmacology/Toxicology Department of Biochemistry, Federal University of Technology, Owerri, Nigeria.

Notes

[3] Conflicts of interest COMPETING INTERESTS The authors declare that there is no conflict of interest regarding the publication of this article.

ABBREVIATIONS USED

CCl4

Carbon tetrachloride

CH3OH

Methanol

NaCl

Sodium chloride

Na2HPO4.2H2O

Diso-dium hydrogen phosphate dihydrate

NaH2PO4.2H2O

Sodium dihydrogen phosphate dihydrate

K2PO4

Potassium phosphate

KHPO4

Potassium hydrogen phosphate

Na2CO3

Sodium carbonate

KCl

Potassium chloride.

REFERENCES

1. 

Abraham WM, Bourdelais AJ, Ahmed A, Serebriakov I, Baden DG , authors. Effects of inhaled brevetoxins in allergic airways: Toxin/allergen interactions and pharmacologic intervention. Environ Health Perspect. 2005;113(5):632–7

2. 

Adekunle AS, Adedeji AL, Oyewo EO, Adedosu OT, Omotoso AT , authors. Hyperlipidemia induced by atherogenic diet enhanced oxidative stress in the kidney and inflammatory responses: an in-vivo study. Asian J Nat Appl Sci. 2013;2(1):82–93

3. 

Adeneye AA, Awodele O, Aiyeola SA, Benebo AS , authors. Modulatory potentials of the aqueous stem bark extract of Mangifera indica on carbon tetrachloride-induced hepatotoxicity in rats. J Tradit Compl Med. 2015;5(5):106–15

4. 

Adewole SO, Salako AA, Doherty OW, Naicker T , authors. Effect of melatonin on carbon tetrachloride-induced kidney injury in Wistar rats. Afr J Biomed Res. 2007;10(2):153–64

5. 

Akinwunmi KF, Oyedapo OO , authors. Evaluation of antioxidant potentials of Monodora myristica (Gaertn) dunel seeds. Afr J Food Sci. 2013;7(9):317–24

6. 

Albokhadaim I , author. Effect of aqueous extract of green tea (Camellia sinensis) on hematology and oxidative stress biomarkers in rats intoxicated with carbon tetrachloride. J Biol Sci. 2016;16(3):49–57

7. 

Alqasoumi S , author. Carbon tetrachloride-induced hepatotoxicity: Protective effect of ‘Rocket’ Eruca sativa L. in rats. Am J Chinese Med. 2010;38(1):75–88

8. 

Althnaian T, Albokhadaim I, El-Bahr SM , authors. Biochemical and histopathological study in rats intoxicated with carbon tetrachloride and treated with camel milk. Spring Open J Res. 2013;2(1):57

9. 

Avti PK, Kumar S, Pathak CM, Vaiphei K, Khanduja KL , authors. Smokeless tobacco impairs the antioxidant defense in liver, lung, and kidney of rats. Toxicol Sci. 2006;89(2):547–53

10. 

Bahadoran Z, Mirmiran P, Azizi F , authors. Dietary polyphenols as potential nutraceuticals in management of diabetes: A review. J Diabetes Metab Disord. 2013;12(1):43

11. 

Baker MS, Gebicki JM , authors. The effect of pH on yields of hydroxyl radicals produced from superoxide by potential biological iron chelators. Arch Biochem Biophys. 1986;246(2):581–8

12. 

Balavoine CG, Gelehi YV , authors. Peroxyntrite scavenging by different antioxidants. Part 1: Convenient study. Nitric Oxide. 1999;3(1):40–54

13. 

Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL , authors. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem. 2009;390(3):191–214

14. 

Baure JD , author. Laboratory Investigation of Hemoglobin. Gradwohl’s Clinical Laboratory Methods and Diagnosis. Sonnenwirth AC, Jarett L , editors. St. Louis, MO; Mosby: 1980

15. 

Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA , authors. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci. 1990;87(4):1620–4

16. 

Belonwu DC, Ibegbulem CO, Nwokocha MN, Chikezie PC , authors. Some phytochemicals and hydrophilic vitamins of Anacardium occidentale. Res J Phytochem. 2014;8(3):78–91

17. 

Benhusein GM, Mutch E, Aburawi S, Williams FM , authors. Genotoxic effect induced by hydrogen peroxide in human hepatoma cells using comet assay. Libyan J Med. 2010;5(1):1–5

18. 

Boham AB, Kocipal AC , authors. Flavonoid and condensed tannins from leaves of Hawaiian vaccininum, vaticulum and vicalycinium. Pac Sci. 1994;48:458–63

19. 

Bradford MM , author. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Ann Biochem. 1976;72(1-2):248–54

20. 

Brattin WJ, Glende EA Jr, Recknagel RO , authors. Pathological mechanisms in carbon tetrachloride hepatotoxicity. J Free Radic Biol Med. 1985;1(1):27–38

21. 

Brito NJN, López JA, Nascimento MA, Macêdo JBM, Silva GA, Oliveira CN, et al. , authors. Antioxidant activity and protective effect of Turnera ulmifolia Linn. var. elegans against carbon tetrachloride-induced oxidative damage in rats. Food Chem Toxicol. 2012;50(12):4340–7

22. 

Chakraborty AK, Ranbhade S, Patil UK , authors. Chromolaena odorata (L): An overview. J Pharm Res. 2011;4:573

23. 

Chempe PC, Harvey RA, Ferrier DR , authors. Lippincott’s Illustrated Reviews: Biochemistry. 3rd. Jaypce Brother Medical Publisher (P) Ltd; 2005

24. 

Chikezie PC, Uwakwe AA , authors. Protective effect of Allium Sativa extract against carbon tetrachloride-induced hepatic oxidative stress and hyperlipidemia in rats. Afr J Biotechnol. 2014;13(15):1671–8

25. 

Chikezie PC, Ibegbulem CO, Mbagwu FN , authors. Bioactive principles from medicinal plants. Res J Phytochem. 2015;9:88–115

26. 

Chikezie PC , author. Oxidative stress indicators of human sickle erythrocytes incubated in aqueous extracts of three medicinal plants. Asian J Biochem. 2011;6(5):373–83

27. 

Dani C, Pasquali MA, Oliveira MR, Umezu FM, Salvador M, Henriques JA, et al. , authors. Protective effects of purple grape juice on carbon tetrachloride induced oxidative stress in brains of adult Wistar rats. J Med Food. 2008;11(1):55–6

28. 

Dey A, De JN , authors. Neuroprotective therapeutics from botanicals and phytochemicals against Huntington’s disease and related neurodegenerative disorders. J Herbal Med. 2015;5(1):1–19

29. 

Douglas W, Harold T , authors. The Complete Blood Count and Bone Marrow Examination: General Comments and Selected Techniques. 4th. Saunders and Elsevier Publisher; Philadelphia, Pennsylvania, USA: 2004. p. 32–33

30. 

Elbakry KA, Malak CAA, Howas MM , authors. Immunomodulatory role of honey and propolis on carbon tetrachloride (CCl4) injected rats. Int J Pharm Pharm Sci. 2015;7:259–62

31. 

El-Missiry MA, Fayed TA, El-Sawy MR, El-Sayed AA , authors. Ameliorative effect of melatonin against gamma-irradiation-induced oxidative stress and tissue injury. Ecotoxicol Environ Saf. 2007;66(2):278–86

32. 

Evans JL, Goldfine ID, Maddux BA, Grodsky GM , authors. Are oxidative stress-activated signaling pathways mediators of insulin resistance and β-cell dysfunction? Diabetes. 2003;52(1):1–8

33. 

Ferreira ALA, Machado PEA, Matsubara LS , authors. Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro. Braz J Med Biol Res. 1999;32(6):689–94

34. 

Fujiwara K, Ogata I, Ohta Y, Hirata K, Oka Y, Yamada S, et al. , authors. Intravascular coagulation in acute liver failure in rats and its treatment with antithrombin III. Gut. 1988;29(8):1103–8

35. 

Gahler S, Otto K, Bohm V , authors. Alterations of vitamin C, total phenolics, and antioxidant capacity as affected by processing tomatoes to different products. J Agricult Food Chem. 2003;51(27):7962–8

36. 

Gandhimathi R, Kumar AS , authors. Evaluation of antioxidant activity of Cordia Subcordata Lam. against carbon tetrachloride (CCl4) induced erythrocyte damage in rats. Pharmacol. 2009;2:720–7

37. 

Gawlik-Dziki U , author. Changes in the antioxidant activities of vegetables as a consequence of interactions between active compounds. J Funct Foods. 2012;4(4):872–82

38. 

Gonçalves RV, da Matta SLP, Novaes RD, Leite JPV, Peluzio MCG, Vilela EF , authors. Bark extract of Bathysa cuspidata in the treatment of liver injury induced by carbon tetrachloride in rats. Braz Arch Biol Technol. 2014;57(4):504–13

39. 

Grattagliano I, Caraceni P, Calamita G, Ferri D, Gena P, Carreras FI, et al. , authors. Severe liver steatosis correlates with nitrosative and oxidative stress in rats. Eur J Clin Invest. 2008;38(7):523–30

40. 

Halliwell B, Gutteridge JMC , authors. Free Radicals in Biology and Medicine. 3rd. Oxford, UK: Oxford University Press; 1999

41. 

Harborne JB , author. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 1st. London: Chapman and Hall Ltd; 1973. p. 278

42. 

Hartley DP, Kolaja KL, Reichard J, Petersen DR , authors. 4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: immunochemical detection and lobular localization. Toxicol Appl Pharmacol. 1999;161(1):23–33

43. 

Hermann PB, Henneberg R, Nascimento AJ, Frigeri HR, Furman AEF, Leonart MSS , authors. Deferoxamine and deferasirox action in erythrocytes of patients with transfusion-dependent anemia, under “in vitro” oxidative stress. J Biotechnol Biodivers. 2014;5(2):121–9

44. 

Hoff N , author. Methods of blood collection in the mouse. Lab Ani. 2000;29:47–53

45. 

Huang Q, Zhang S, Zhang L, He M, Huang R, Lin X , authors. Hepatoprotective effects of total saponins isolated from Turaphochlamys affiris against carbon tetrachloride-induced liver injury in rats. Food Chem Toxicol. 2012;50(3):713–8

46. 

Huff GR, Huff WE, Balog JM, Rath NC, Anthony NB, Nestor KE , authors. Stress response differences and disease susceptibility reflected by heterophil to lymphocyte ratio in turkeys selected for increased body weight. Poult Sci. 2005;84(5):709–17

47. 

Hunter KJ, Fletcher JM , authors. The antioxidant activity and composition of fresh, frozen, jarred and canned vegetables. Innov Food Sci Emerg Technol. 2002;3(4):399–406

48. 

Hye-Lin H, Hye-Jun S, Mark AF, Dae-Yeul Y , authors. Oxidative stress and antioxidants in hepatic pathogenesis. World J Gastroenterol. 2010;16(48):6035–43

49. 

Ibegbulem CO, Chikezie PC , authors. Hypoglycemic properties of ethanolic extracts of Gongronema latifolium, Aloe perryi, Viscum album and Allium sativum administered to alloxan-induced diabetic albino rats (Rattus norvegicus). Pharmacog Commun. 2013;3(2):12–6

50. 

Ibrahim TA, Fagboun ED , authors. Phytochemical and nutritive quality of dried seed of Buchholzia coricea. Greener J Phys Sci. 2012;2(5):185–91

51. 

Ismail A, Marjan ZM, Foong CW , authors. Total antioxidant activity and phenolic content in selected vegetables. Food Chem. 2004;87(4):581–6

52. 

Jaramillo-Juárez F, Rodríguez-Vázquez ML, Rincón-Sánchez AR, Martínez MC, Ortiz GG, Llamas J , authors. Acute renal failure induced by carbon tetrachloride in rats with hepatic cirrhosis. Ann Hepatol. 2008;7(4):331–8

53. 

Kaur C, Kapoor HC , authors. Anti-oxidant activity and total phenolic content of some Asian vegetables. Int J Food Sci Technol. 2002;37(2):153–61

54. 

Kaur P, Bains K, Kaur H , authors. Effect of hydrothermal treatment on free radical scavenging potential of selected green vegetables. Indian J Nat Prod Resour. 2012;3:563–9

55. 

Khan AA, Alzohairy M , authors. Hepatoprotective effects of camel milk against CCl4-induced hepatotoxicity in rats. Asian J Biochem. 2011;6(2):171–81

56. 

Klein BP, Kurilich AC , authors. Processing effects on dietary antioxidants from plant foods. Hortsci. 2000;35(4):580–4

57. 

Klings ES, Farber HW , authors. Role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease. Respir Res. 2001;2(5):280–5

58. 

Kohen R, Nyska A , authors. Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol Pathol. 2002;30(60):620–50

59. 

Kono Y , author. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys. 1978;186(1):189–95

60. 

Kuo DH, Kang WH, Shieh PC, Chen FA, Chang CD, Tsai ML, et al. , authors. Protective effect of Pracparatum mungo extract on carbon tetrachloride-induced hepatotoxicity in rats. Food Chem. 2010;123(4):1007–12

61. 

Luck H , author. Catalase. Methods of enzymatic analysis. Bergmeyer HW , editor. Section 3. Academic Press; New York: 1963. p. 885–94

62. 

Mate´s JM , author. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicol. 2000;153(1):83–104

63. 

Mathew JE, Mantri A, Vachala SD, Srinivasan KK, Movaliya V , authors. Effect of Sphaeranthus indicus ethanol extract on tissue antioxidant activity in gentamicin induced nephrotoxic rats. Herba Polonica. 2009;55(4):86–95

64. 

Matough FA, Budin SB, Hamid ZA, Alwahaibi N, Mohamed J , authors. The role of oxidative stress and antioxidants in diabetic complications. Sultan Qaboos Univ Med J. 2012;12(1):5–18

65. 

Middleton E, Kandaswami C, Theohardes TC , authors. The effects of plant flavonoids on mammalian cells, implication for inflammation, heart disease and cancer. Pharmacol Rev. 2000;52(4):673–751

66. 

Mordi JC, Awire IE, Awhin PE , authors. Effects of the aqueous extract of Hibiscus sabdariffa L petal (zobo) drink on ethanol–induced changes in plasma superoxide dismutase and lipid peroxidation levels in brain and hepatic tissues of rats. J Biol Sci Bioconserv. 2011;3:35–41

67. 

Ndidi US, Ndidi CU, Olagunju A, Muhammad A, Billy FG, Okpe O , authors. Proximate, anti-nutrition, and mineral composition of raw and processed (boiled and roasted) Sphenostylis stenocarpa seeds from Southern Kaduna, Northwest Nigeria. ISRN Nutr. 2014. 2014;9

68. 

Nikolaids MG, Jamurtas AZ , authors. Blood as a reactive species generator and redox status regulator during exercise. Arch Biochem Biophys. 2009;490(2):77–84

69. 

Nwaehujor CO, Ode OJ, Nwinyi FC, Udeh NE , authors. Effect of methanol extract of Buchholzia coriacea fruits on streptozotocin-induced diabetic rats. J Pharmacol Toxicol. 2012;7:181–91

70. 

Ojiako AO, Chikezie PC, Ogbuji CA , authors. Histopathological studies of renal and hepatic tissues of hyperglycemic rats administered with traditional herbal formulations. Int J Green Pharm. 2015;9(3):184–91

71. 

Ojo OO, Kabutu FR, Bell M, Babayo U , authors. Inhibition of paracetamol induced oxidative stress in rats by extract of Cymbopogon citracus (green tea). Afr J Biotech. 2006;5(12):12

72. 

Okonkwo CC, Njoku OU, Ikevude CT, Odo CE , authors. Hepatoprotective effect of methanol extract of Sphenostylis stenocarpa (Hoschst ex. A. Rich. Harms) against carbon tetrachloride-induced liver toxicity in Wistar rats. J Pharm Res. 2013;6:293–8

73. 

Paik Y-H, Kim J, Aoyama T, De Minicis S, Bataller R, Brenner DA , authors. Role of NADPH oxidases in liver fibrosis. Antioxid Redox Signal. 2014;20(17):2854–72

74. 

Pandith H, Zhang X, Liggert J, Min K, Gritsanaptan W, Baek SJ , authors. Hemostatic and wound healing properties of Chromolaena odorata leaf extract. ISRN Dermatol. 2013;8

75. 

Pisoschi AM, Negulescu GP , authors. Methods for total antioxidant activity determination: A Review. Biochem Anal Biochem. 2011;1(1):1–12

76. 

Poljšak B, Fink R , authors. The protective role of antioxidants in the defense against ROS/RNS-mediated environmental pollution. Oxid Med Cell Longev. 2014. 2014;22

77. 

Raškovic A, Pavlovic N, Kvrgic M, Sudji J, Mitic G, Capo I, et al. , authors. Effects of pharmaceutical formulations containing thyme on carbon tetrachloride-induced liver injury in rats. BMC Compl Altern Med. 2015;15(1):442

78. 

Roopha PD, Padmalatha C , authors. Effect of herbal preparation on heavy metal (Cadmium) induced antioxidant system in female Wistar rats. J Med Toxicol. 2012;8(2):101–7

79. 

Rosa DP, Bona S, Simonetto D, Zettler C, Marroni CA, Marroni NP , authors. Melatonin protects the liver and erythrocytes against oxidative stress in cirrhotic rats. Melatonin protects the liver and erythrocytes against oxidative stress in cirrhotic rats. Arq Gastroenterol. 2010;47(1):72–8

80. 

Saba AB, Oyagbemi AA, Azeez OI , authors. Amelioration of carbon tetrachloride-induced hepatotoxicity and haemotoxicity by aqueous leaf extract of Cnidoscolus aconitifolius in rats. Nig J Physiol Sci. 2010;25(2):139–47

81. 

Sahreen S, Khan MR, Khan RA, Shah NA , authors. Effect of Carissa opaca leaves extract on lipid peroxidation, antioxidant activity and reproductive hormones in male rats. Lipids Health Dis. 2013;12(1):90

82. 

Schmuck G, Roehrdanz E, Hayes RK, Kahl R , authors. Neurotoxic mode of action of artemisinin. Antimicrob Agents Chemother. 2002;46(3):821–7

83. 

Sellamuthu PS, Arulselvan P, Kamalraj S, Fakurazi S, Kandasamy M , authors. Protective nature of Mangiferin on oxidative stress and antioxidant status in tissues of streptozotocin-induced diabetic rats. ISRN Pharmacol. 2013;10

84. 

Shaker E, Mahmoud H, Mnaa S , authors. Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food and Chem Toxicol. 2010;48(3):803–6

85. 

Sharma I, Laware SL , authors. Khadiranrishta retrains lipid peroxidation in human erythrocytes. J Pharmceut Sci Biosci Res. 2015;5:342–6

86. 

Singal AK, Jampana SC, Weinman SA , authors. Antioxidants as therapeutic agents for liver disease. Liver Int. 2011;31(10):1432–48

87. 

Singh A, Bhat TK, Sharma OP , authors. Clinical biochemistry of hepatotoxicity. J Clin Toxicol. 2011;4(0001):1–9

88. 

Styskal JL, Van Remmen H, Richardson A, Salmon AB , authors. Oxidative stress and diabetes: what can we learn about insulin resistance from antioxidant mutant mouse models? Free Radic Biol Med. 2012;52(1):46–58

89. 

Sur A, Chakraborty H, Basu A, Chakraborty S , authors. Oxidative stress induced carbonyl group incorporation to human RBC membrane: Role in vivo senescence of erythrocyte. Global J Med Res: C Microbiol Pathol. 2014;14:29–47

90. 

Thi ND, Hwang E-S , authors. Effects of drying methods on contents of bioactive compounds and antioxidant activities of black chokeberries (Aronia melanocarpa). Food Sci Technol. 2016;25(1):55–61

91. 

Tjahjani S, Puji BSA, Syafruddin D, Agoes R, Hanggono T, Immaculata M , authors. Oxidative stress in Plasmodium falciparum culture incubated with artemisinin. Proc ASEAN Congr Trop Med Parasitol. 2008;3:47–50

92. 

Turkman N, Sari F, Velioglu YS , authors. The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chem. 2005;93(4):713–8

93. 

Van-Burden TP, Robinson WC , authors. Formation of complex between protein and tannic acid. J Agric Food Chem. 1981;17(4):772–7

94. 

Vignais PV , author. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci. 2002;59(9):1428–59

95. 

Wang X, Hai CX, Liang X, Yu SX, Zhang W, Li YL , authors. The protective effects of Acanthopanax senticosus Harms aqueous extracts against oxidative stress: Rolem of Nrf2 and antioxidant enzymes. J Ethnopharmacol. 2010;127(2):424–32

96. 

Zhang D, Hamauzu Y , authors. Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. Food Chem. 2004;88(4):503–9