Doxorubicin induced neuro- and cardiotoxicities in experimental rats: Protection against oxidative damage by Theobroma cacao Stem bark.

80 rats, randomly selected, were divided into 3 treatment groups: pre-, co- and post-treatment; consisting of 6 sub-groups each (5 rats per sub-group): baseline, normal saline (2 mL), α-lipoic acid (20 mg/kg body weight), 200 mg/kg, 400 mg/kg or 800 mg/kg body weight Theobroma cacao stem bark aqueous extract (TCAE). All rats except for baseline group were intoxicated with 20 mg/kg body weight doxorubicin (DOX) intraperitoneally. The animals in pre- or post-treatment group received a single dose of DOX (20 mg/kg body weight) intraperitoneally 24 h before or after 7 days' oral administration with TCAE respectively while those in co-treatment group were co-administered 2.86 mg/kg body weight of DOX with either normal saline, α- lipoic acid or TCAE orally for 7 days. Animals were sacrificed (pre- and post- treatment groups were sacrificed on the ninth day while the co-treatment group sacrificed on the 8th day). Brain and heart tissue samples were harvested for enzyme markers of toxicity, oxidative stress and histopathological examinations. DOX intoxication caused significant decrease in activities of LDH and ACP, and increase in γGT and ALP activities in brain tissues while causing a significant increase in LDH, ACP, γGT activities and decrease in ALP activity in the cardiac tissues. DOX intoxication caused a significant increase in concentrations of H2O2 generated, MDA and PC, XO, MPx and NOX activities with concomitant decrease in CAT, SOD, GPx and GST activities, and in concentrations of GSH, AsA and α-Toc in brain and cardiac tissues. Pre-, co- and post-treatment with TCAE at either 200 mg/kg, 400 mg/kg or 800 mg/kg body weight significantly reversed the oxidative damage to the organs induced by DOX-intoxication. The result affirmed that T. cacao stem bark aqueous extract protected against DOX induced oxidative damage in brain and cardiac tissues of experimental rats.

Introduction

Doxorubicin (DOX) obtained from soil actinomycetes Streptococcus peucetius is a powerful drug used for the treatment of solid tumors such as those arising in the breast, bile ducts, endometrial tissue, esophagus and liver, osteosarcomas, soft-tissue sarcomas and non-Hodgkin's lymphoma [47]. DOX is known as a powerful anthracycline antibiotic widely used to treat many human cancers, but significant cardiotoxicity and brain damage [24], hepatotoxicity [37], nephrotoxicity [32] and testicular toxicity [48] limits its clinical application. A number of studies were conducted for antioxidants screening from the natural medicine aiming to minimize oxidative injury by DOX. Several natural antioxidants have been shown to alleviate the DOX-induced cell damage without compromising its anti-tumor efficacy in the animal studies [52]. Over the past few years, the antioxidant and health-promoting properties of cocoa () and cocoa-related products have been thoroughly investigated. Polyphenols, widely distributed in plant foods, are the main antioxidant-active fraction of cocoa, and within polyphenols, flavanols and procyanidins have been identified as the active antioxidant agents of cocoa and dark plain chocolate [28]. More than 200 studies have reported that various parts of the cocoa plant, e.g., cocoa beans (prepared as chocolate), the bark, flower, pulp, and leaf, and cocoa butter have been used for medicinal purposes. The phenolic compounds in cocoa contain bioactive compounds that have potential health benefits for chronic diseases such as inflammation, cardiovascular illness, neurodegenerative disorders, and cancer [43]. α-Lipoic acid (ALA) also known as thioctic acid (TA) and 1,2 dithiolane −3-pentanoic acid, is a naturally occurring substance, that is essential for the function of different enzymes of oxidative metabolism. It is believed that ALA or its reduced form, dihydrolipoic acid (DHLA) have many biochemical functions acting as biological antioxidants, as metal chelators, reducing the oxidized forms of other antioxidant agents such as vitamin C and E and glutathione (GSH), and modulating the signaling transduction of several pathways, like insulin and nuclear factor kappa B (NF-kB) [15]. Brain is the main organ of the human nervous system. It is located in the head, protected by the skull. It has the same general structure as the brains of other mammals, but with a more developed cerebral cortex. Despite being protected by the thick bones of the skull, suspended in cerebrospinal fluid, and isolated from the bloodstream by the blood–brain barrier, the human brain is susceptible to damage and disease [9]. Heart is a muscular organ in humans and other animals, which pumps blood through the blood vessels of the circulatory system and also assists in the removal of metabolic wastes. The heart is located in the middle compartment of the mediastinum in the chest. The heart pumps blood through both circulatory systems. In addition, the blood carries nutrients from the liver and gastrointestinal tract to various organs of the body, while transporting waste to the liver and kidneys [30]. The aim of the study is to investigate the protective potential of Theobroma cacao stem bark aqueous extract against DOX-induced oxidative damage in the brain and heart in experimental rats.

Materials and methods

Chemicals and reagent

Sodium hydroxide, sodium chloride, doxorubicin, α-lipoic acid, formalin, potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Sigma Chemical Co., Saint Louis, MO, USA. Diethylether, ethanol, xylene, paraffin wax, haemotoxylin and eosin were purchased from Sigma Chemical Co., (St Louis, Mo USA). All other chemicals were supplied by Zayo Company, Jos, Nigeria, which is an accredited supplier of Sigma and BDH chemicals in Nigeria. All reagents and chemicals used were of analytical grade (greater than or equal to 99.7%).

Preparation of extract

Freshly peeled stem barks of Theobroma cacao tree were collected in a village farm at Ekiti, Ekiti state southwest Nigeria. The plant part was identified and authenticated at the Department of Botany, University of Ibadan, Nigeria. The fresh stem bark of Theobroma cacao was allowed to air-dry to a constant weight at room temperature in a well-ventilated room for a period of four weeks. Conventional extraction process described by [22] was adopted.

Animals

Eighty (80) Inbred male Wistar rats, weighing between 100 and 210 g were purchased from the Animal House of the Institute for Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Nigeria. The animals were kept in well-ventilated cages in the departmental animal house at room temperature (28–30 °C) and under controlled light cycles (12 h light:12 h dark) for two weeks acclimatization before the commencement of the experiment. They were maintained on normal laboratory chow (Ladokun Feeds, Ibadan, Nigeria) and water ad libitum. Rats handling and treatments conform to guidelines of the National Institute of Health (NIH publication 85–23, 1985) for laboratory animal care and use. The study was approved by the College of Biosciences, Federal University of Agriculture Abeokuta Animal Ethics Committee.

Experimental design

Animals were randomly selected, after acclimatization, and distributed into four (4) groups, viz; baseline, pre-treatment, co- treatment and post-treatment groups, with each group except the baseline group further sub-divided into five different sub-groups of five rats per sub-group as follows: normal saline, α-lipoic acid, 200TCAE, 400TCAE or 800TCAE groups.

Pre-treatment group

This group comprises of 25 rats divided into five sub-groups of five rats each. All the rats were administered single dose of 20 mg/kg body weight DOX intraperitoneally on the first day. After 24 h, oral treatment with either normal saline (negative control), 20 mg/kg body weight α-lipoic acid (positive control), 200 mg/kg body weight TCAE, 400 mg/kg body weight TCAE or 800 mg/kg body weight TCAE respectively in each group was conducted for seven days. The rats were fasted overnight and sacrificed 24 h after the last treatment.

Co-treatment group

This group comprises of 25 rats divided into five sub-groups of five rats each. A dose of 2.86 mg/kg body weight doxorubicin was co-administered intraperitoneally with either normal saline (negative control), 20 mg/kg body weight α-lipoic acid (positive control), 200 mg/kg body weight TCAE, 400 mg/kg body weight TCAE or 800 mg/kg body weight TCAE respectively in each group for seven days orally. The rats were fasted overnight and sacrificed 24 h after the last administration.

Post-treatment group

This group comprises of 25 rats divided into five sub-groups of five rats each. The rats were first treated with normal saline (negative control), 20 mg/kg body weight α-lipoic acid (positive control), 200 mg/kg body weight TCAE, 400 mg/kg body weight TCAE or 800 mg/kg body weight TCAE orally respectively in each group for seven days. Single dose of 20 mg/kg body weight DOX was administered intraperitoneally on the eight day, the rats fasted overnight and sacrificed 24 h after the last intoxication.

Baseline group

This group comprises of five rats administered normal saline orally per day for seven days, fasted overnight and sacrificed 24 h after the last administration.

Preparation of tissues

Rats were fasted overnight and sacrificed 24 h after the last treatment. Brain and heart tissue samples were quickly removed and washed in ice-cold 1.15% KCl solution to remove blood stain, dried and weighed. Part of these tissues were fixed in 10% formalin solution and used for histopathology. The remaining tissues were homogenized separately in 4 volumes of 50 mM phosphate buffer, pH 7.4 and centrifuged at 10,000g for 15 min to obtain post-mitochondrial fraction (PMF). Procedures were carried out at temperature of 4 °C.

Hydrogen peroxide scavenging assay

Plant extract (4 mL) prepared in distilled water at various concentration was mixed with 0.6 mL of 4 mM H2O2 solution prepared in phosphate buffer (0.1 M pH 7.4) and incubated for 10 min. The absorbance of the solution was taken at 230 nm. Ascorbic acid was used as a positive control compound. The percentage of inhibition was calculated by comparing the absorbance values of the control and test samples using following equation [35].

Cupric ion reducing capacity assay (CUPRAC)

1 mL 10 mM cupric chloride, 1 mL 7.5 mM neocuproine and 1 mL 1 M ammonium acetate buffer of pH 7 solutions were added to test tubes containing 2 mL of distilled water. The plant extract in different concentration were added to each test tube separately. These mixtures were incubated for half an hour at room temperature and measured against blank at 450 nm. Ascorbic acid was used as positive reference standard [2].

Metal ion chelating activity

The plant extract in different concentration were added to each test tube separately (150 μL), 0.25 mM FeCl2 solution (50 μL) was added. After 5 min, the reaction was initiated by adding 1.0 mM ferrozine solution (100 μL). Absorbance at 545 nm was recorded after 10 min of incubation at room temperature. A reaction mixture containing methanol (150 μL) instead of substance solution served as a control. Ascorbic acid was used as the chelating standard. Chelating activity was calculated using Acont (absorbance of the negative control, e.g., blank solution without test compound) and Asample (absorbance of the substance solution). Chelating activity was expressed as EC50, the concentration that chelates 50% of Fe2+ ions [11].

Superoxide radical scavenging activity

The non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system generates superoxide radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The 1 mL reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT (50 μM), PMS (15 μM) and various extract concentrations (0–20 μg/mL) of sample solution. After incubation for 5 min at ambient temperature, the absorbance at 562 nm was measured against an appropriate blank to determine the quantity of formazan generated. All tests were performed six times. Ascorbic acid was used as positive control [27].

Peroxynitrite scavenging assay

An acidic solution (0.6 M HCl) of 5 mL H2O2 (0.7 M) was mixed with 5 mL 0.6 M KNO2 on an ice bath for 1 s and 5 mL of ice-cold 1.2 M NaOH was added. Excess H2O2 was removed by treatment with granular MnO2 prewashed with 1.2 M NaOH and the reaction mixture was left overnight at −20 °C. Peroxynitrite solution was collected from the top of the frozen mixture and the concentration was measured spectrophotometrically at 302 nm (ε=1670 M−1 cm−1). An Evans Blue bleaching assay was used to measure peroxynitrite scavenging activity. The reaction mixture contained 50 mM phosphate buffer (pH 7.4), 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM Evans Blue, various doses of plant extract (1000–4000 μg/mL) and 1 mM peroxynitrite in a final volume of 1 mL. After incubation at 25 °C for 30 min the absorbance was measured at 611 nm. The percentage scavenging of ONOO- was calculated by comparing the results of the test and blank samples. All tests were performed six times. Gallic acid was used as the reference compound [4].

Total antioxidant capacity

The plant extract in different concentration ranging from 1000 to 4000 μg/mL were added to each test tube individually containing 3 mL of distilled water and 1 mL of Molybdate reagent solution. These tubes were kept incubated at 95 °C for 90 min. After incubation, these tubes were normalized to room temperature for 20–30 min and the absorbance of the reaction mixture was measured at 695 nm. Mean values from three independent samples were calculated for each extract. Ascorbic acid was used as positive reference standard [17].

Reducing power ability

The extract (0.75 mL) at various concentrations was mixed with 0.75 mL of phosphate buffer (0.2 M, pH 6.6) and 0.75 mL of potassium hexacyanoferrate [K3Fe(CN)6] (1%, w/v), followed by incubating at 50 °C in a water bath for 20 min. The reaction was stopped by adding 0.75 mL of trichloroacetic acid (TCA) solution (10%) and then centrifuged at 3000 r/min for 10 min 1.5 mL of the supernatant was mixed with 1.5 mL of distilled water and 0.1 mL of ferric chloride (FeCl3) solution (0.1%, w/v) for 10 min. The absorbance at 700 nm was measured as the reducing power. Higher absorbance of the reaction mixture indicated greater reducing power [29]. Ascorbic acid was used as positive reference standard.

Biochemical assays

Brain and cardiac alkaline phosphatase (ALP) activity was determined according to the method described by Bassey et al., [3] and as modified by Wright et al., [51] using Randox kits, gamma-glutamyl transferase (γ-GT) activity was monitored according to the method described by Szasz [46], acid phosphatase (ACP) activity was determined according to the method described by Brandt et al. [7] while lactate dehydrogenase (LDH) activity was determined according to the method described by Bower (1963). Tissues hydrogen peroxide (H2O2) concentration was quantified based on [50], protein carbonyl (PC) concentration was carried out by following method described by [25], malondialdehyde (MDA) concentration was determined by measuring the thiobarbituric acid reactive substances (TBARS) produced during lipid peroxidation. This was measured using method of Moore and Roberts [33], myeloperoxidase (MPX) activity was determined using method of [20], NADPH oxidase (NOX) activity was measured by the method of Reusch and Burger [39], xanthine oxidase (XO) activity was determined according to the method of Bergmeyer et al., [5], glutathione-S-transferase (GST) activity was determined according to Habig et al., [16], enzymatic assay of glutathione peroxidase (GPX) activity was determined following the method described by Rotruck et al. [40], catalase (CAT) activity was determined according to the method of [44], the activity of superoxide dismutase (SOD) was determined by the method of Misra and Fridovich [31], the method of [6] was followed for the determination of reduced glutathione (GSH) concentration, ascorbic acid (AsA) concentration was quantified according to the method of Omaye et al. [36] and concentration of α–tocopherol (α-toc) was carried out following the procedure of Kayden et al., [19].

Histopathological examination of brain and heart sections

The tissues were excised and immediately fixed in 10% buffered formalin at the end of the experiment. The tissue specimens were embedded in paraffin after being dehydrated in alcohol and subsequently cleared with xylene. Four micrometer (4 µm) thick serial histological sections were obtained from the paraffin blocks and stained with hematoxylin and eosin. The sections were examined under light microscope by a histopathologist (who was ignorant of the treatment groups) to evaluate pathological changes and photomicrographs were taken [23].

Statistical analysis of data

Values were expressed as mean±standard deviation of five animals per group. Data were analysed using one-way ANOVA followed by the post-hoc Duncan multiple range test using SPSS (V20.0). Values were considered statistically significant at p<0.05.

Results

Hydrogen peroxide scavenging activity, Cupric ion reducing capacity activity, Metal ion chelating activity, Superoxide radical scavenging activity, Peroxynitrite scavenging activity, Total antioxidant capacity, Reducing power ability and effects of Theobroma cacao on relative organ weights of DOX-exposed rats

Table 3.1.1 revealed that DOX intoxication caused a significant decrease in brain and heart weights of experimental rats relative to baseline (p<0.05). Pre-, co- or post-treatment of experimental animals with 200 mg/kg body weight T. cacao caused a further decrease in brain weight of experimental rats. Pre-, co- or post-treatment of experimental animals with 400 mg/kg or 800 mg/kg body weight T. cacao caused an insignificant change in brain weight of experimental rats. Pre-, co- or post-treatment with 200 mg/kg, 400 mg/kg or 800 mg/kg body weight T. cacao caused a significant increase in heart weight of experimental rats (p<0.05). Theobroma cacao stem bark aqueous extract (TCAE) showed significant dose dependent increase in hydrogen peroxide scavenging activity, cupric ion reducing activity, metal ion chelating activity, superoxide radical scavenging activity, peroxynitrite scavenging activity, total antioxidant capacity and reducing power ability (p<0.05) (Figs. 3.1.1, 3.1.2, 3.1.3, 3.1.4, 3.1.5, 3.1.6, Fig. 3.1.7, Fig. 3.1.8 respectively). However, the activities of the standards were significantly higher in the assays than TCAE.

Table 3.1.1

Relative organ weights (g) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	1.65±0.04αγ	1.59±0.06α	1.65±0.11αγ	–	3.672±0.703αγ	4.788±0.435α	4.651±0.754αβγ	–
α-LIPOIC ACID	1.23±0.21β	1.60±0.04	1.41±0.90 β	–	3.347±0.433β	4.930±0.537	5.122±0.206αβ	–
200TCAE	1.40±0.33 βγ	1.48±0.16 βγ	1.40±0.26 β	–	4.452±0.066αβγ	4.221±0.223αβγ	5.756±0.712αβγ	–
400TCAE	1.71±0.13γ	1.55±0.11γ	1.15±0.31αβγ	–	4.965±0.623αβγ	5.643±0.365αβγ	5.438±0.212αβγ	–
800TCAE	1.68 ± 0.01γ	1.60±0.28	1.62 ± 0.38	–	4.753±0.002αβγ	5.556±0.702αβγ	5.231±0.501αβ	–
BASELINE	–	–	–	1.73 ± 0.26	–	–	–	7.63±0.459

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05

α=significant difference compared with baseline.

β=significant difference compared with normal saline

γ=significant difference compared with α – lipoic acid

Fig. 3.1.1

Hydrogen peroxide scavenging activity of TCAE.

Fig. 3.1.2

Cupric ion reducing capacity activity of TCAE.

Fig. 3.1.3

Metal ion chelating activity of TCAE.

Fig. 3.1.4

Superoxide radical scavenging activity of TCAE.

Fig. 3.1.5

Superoxide radical scavenging activity of TCAE.

Fig. 3.1.6

Peroxynitrite scavenging activity of TCAE.

Fig. 3.1.7

Total antioxidant capacity of TCAE.

Fig. 3.1.8

Reducing power ability of TCAE.

Effects of Theobroma cacao on brain and cardiac alkaline phosphatase, acid phosphatase, lactate dehydrogenase and γ-glutamyl transferase activities in DOX-exposed rats

From Tables 3.2.1, 3.2.2, Table 3.2.3, Table 3.2.4, doxorubicin intoxication induced a significant changes and perturbation in brain and cardiac ALP, ACP, LDH and γ-GT activities respectively in experimental rats relative to the baseline group. Pre-, co- or post-treatment with Theobroma cacao stem bark aqueous extract caused a significant apparent dose-dependent resolution to normalcy by the intoxication (comparable to baseline group) in the activities of brain and cardiac toxicity marker enzymes across the three modes of treatments compared with DOX-intoxicated groups (p<0.05).

Table 3.2.1

Alkaline phosphatase (ALP) activity (IU/L) in DOX-induced toxicity and protective role of Theobroma cacao stem bark aqueous extract (TCAE).

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	21.66 ±1.16αγ	21.59 ±0.92αγ	21.31±1.57α	–	0.944±0.016 αγ	3.927±0.079 αγ	10.15 ±0.312 αβγ	–
α-LIPOIC ACID	4.53±0.33αβ	4.63 ± 0.27αβ	10.73±1.14β	–	6.614±0.311 αβ	8.990±0.926 αβ	16.01 ±0.374 αβγ	–
200TCAE	3.86±0.49αβ	4.28 ±0.43αβ	11.32±2.75βγ	–	22.64±0.122 αβγ	28.94 ±0.341 αβγ	31.44 ±1.113 αβγ	–
400TCAE	5.19±0.35αβ	5.55±0.54αβ	11.10±1.15β	–	29.84±0.178 αβγ	34.98 ±0.091 αβγ	36.97 ±0.377 αβγ	–
800TCAE	4.99±0.52αβ	5.27±0.29αβ	10.75±0.95β	–	32.99±0.056 αβγ	37.97 ±0.734 αβγ	39.47 ±0.060 αβγ	–
BASELINE	–	–	–	10.53 ±1.16	–	–	–	44.53±1.327

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ=significant difference compared with α-lipoic –acid. 200TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.2.2

Acid phosphatase (ACP) activity (IU/L) in DOX-induced toxicity and the protective properties of Theobroma cacao stem bark aqueous extract (TCAE).

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	2.06±0.05αγ	2.90±0.78α	2.36±0.02αγ	–	31.60±0.647 αγ	21.14±0.955 αγ	28.18 ±0.458 αγ	–
α-LIPOIC ACID	3.10±0.48β	2.75±0.28α	2.90±0.56αβ	–	16.84±0.164 αβ	12.24±0.534 αβ	19.66±0.374 αβ	–
200TCAE	2.36±0.16αβγ	2.96±0.11α	2.24±0.16αγ	–	11.74±0.562 αβ	8.23±1.037 αβγ	8.435 ±0.898 αβγ	–
400TCAE	2.55±0.14αβγ	2.48±0.31αβγ	2.78±0.23αβ	–	11.54±0.362 αβ	5.423 ±0.435 αβγ	8.937 ±0.638 αβγ	–
800TCAE	2.54±0.22 βγ	2.08±0.16βγ	2.54±0.31	–	7.34±0.586 αβγ	4.778 ±0.857 αβγ	7.348 ±0.467 αβγ	–
BASELINE	–	–	–	3.44 ± 0.18	–	–	–	2.632±0.453

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ=significant difference compared with α-lipoic –acid. 200TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.2.3

Lactate dehydrogenase (LDH) activity (IU/L) in doxorubicin-induced toxicity and the protective properties of Theobroma cacao stem bark aqueous extract (TCAE).

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	1.62±0.01αγ	1.63±0.04αγ	1.62±0.02αγ	–	180.23±1.142 αγ	141.32±1.423 αγ	136.02±1.273 αγ	–
α-LIPOIC ACID	3.32±0.07β	3.35 ±0.04β	3.16±0.56β	–	153.32±0.347 αβ	121.22±1.082 αβ	116.54±1.838 αβγ	–
200TCAE	3.21±0.01β	3.28±0.16β	3.59±0.16αβ	–	94.11±0.380 αβγ	79.56 ±0.802 αβγ	80.39 ±3.320 αβγ	–
400TCAE	3.19±0.04β	3.43±0.11β	3.41±0.23β	–	79.05 ±2.913 αβγ	76.11 ±0.432 αβγ	78.01 ±0.717 αβγ	–
800TCAE	3.62±0.14αβγ	3.51±0.28β	3.41±0.31αβγ	–	72.01 ±1.005 αβγ	69.84 ±0.738 αβγ	68.48 ±1.021 αβγ	–
BASELINE	–	–	–	3.29 ± 0.01	–	–	–	63.01±0.893

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ=significant difference compared with α-lipoic –acid. 200TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.2.4

Gamma-glutamyl-transferase (γ-GT) activity (IU/L) in doxorubicin-induced toxicity and protective properties of Theobroma cacao stem bark aqueos extract (TCAE).

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	5.65±0.27αγ	5.80±0.30αγ	5.66±0.25αγ	–	83.12±0.672 αβγ	79.71 ±0.058 αβγ	81.93 ± 0.393 αγ	–
α-LIPOIC ACID	2.83±0.61αβ	2.69 ±0.44β	2.86±0.49β	–	68.32±0.987 αβ	65.77 ±0.982 αβ	63.34 ± 0.943 αβ	–
200TCAE	3.38±0.28βγ	2.47±0.23αβ	2.30±0.40αβγ	–	57.94±0.091 αβγ	56.12 ±0.899 αβγ	58.53 ±0.602 αβγ	–
400TCAE	3.44±0.17βγ	3.55±0.21αβγ	3.00±0.11β	–	56.24±0.982 αβγ	55.41 ±0.623 αβγ	56.84 ±0.731 αβγ	–
800TCAE	3.37±0.08αβγ	2.35±0.25αβ	2.99±0.48β	–	53.73±0.589 αβγ	52.53 ±0.216 αβγ	49.84 ±0.644 αβγ	–
BASELINE	–	–	–	3.05 ± 0.44	–	–	–	45.22±0.881

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ=significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Effects of Theobroma cacao on antioxidant parameters, and histology of tissues of DOX-exposed rats

From Tables 3.3.1, Table 3.3.2, Table 3.3.3, doxorubicin intoxication caused a significant elevation in hydrogen peroxide, malondialdehyde and protein carbonyl concentrations in the heart and cardiac tissues of the experimental rats compared with baseline group (p<0.05). Pre-, co- or post-treatment with either 200 mg/kg, 400 mg/kg or 800 mg/kg body weight Theobroma cacao stem bark aqueous extract caused a significant dose dependent reduction in hydrogen peroxide, malondialdehyde and protein carbonyl concentrations in the tissues of experimental rats relative to DOX-exposed group (p<0.05). Tables 3.3.4, Table 3.3.5, Table 3.3.6 revealed a significant elevation in brain and cardiac myeloperoxidase, NADPH oxidase and xanthine oxidase activities of experimental rats following DOX intoxication compared with baseline group (p<0.05). A dose-dependent reduction in these enzymes’ activities were observed following treatment with either 200, 400 or 800 mg/kg body weight in the three (3) modes of treatment with Theobroma cacao stem back aqueous extract relative to the DOX-intoxicated group (p<0.05). The result in Table 3.3.7, Table 3.3.8 also indicated that doxorubicin administration caused a significant decrease in catalase and superoxide dismutase activities in studied tissues of experimental rats compared with baseline group (p<0.05). There was significant increase in these enzymes’ activities following pre-, co- and post-treatment with 200 mg/kg, 400 mg/kg or 800 mg/kg body weight T. cacao compared with DOX-intoxicated group (p<0.05). The result in Tables 3.3.9, Table 3.3.10, Table 3.3.11 revealed that doxorubicin intoxication caused a significant reduction in the activities of glutathione peroxidase and glutathione S-transferase with concomitant decline in reduced glutathione concentration in the tissues of experimental rats compared with baseline group. A significant dose dependent elevation in the studied glutathione metabolism markers were observed following pre-, co- and post-treatment with 200 mg/kg, 400 mg/kg or 800 mg/kg body weight T. cacao groups. The result in Table 3.3.12, Table 3.3.13 reported a significant decrease in brain and cardiac α-tocopherol and ascorbic acid concentrations among DOX-intoxicated rats relative to the baseline group (p<0.05). A significant increase in brain and cardiac α-tocopherol and ascorbic acid concentrations were observed following pre-, co- and post-treatment with 200 mg/kg, 400 mg/kg or 800 mg/kg body weight T. cacao relative to DOX-intoxicated group (p<0.05).

Table 3.3.1

Hydrogen peroxide (H2O2) concentration (µmol/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	11.79±0.29 α	7.89 ±0.61 α	5.63±0.34 α	–	9.715±0.441 αγ	6.432±0.293 αγ	4.584±0.151 αβγ	–
α-LIPOIC ACID	3.42±0.39 αβ	2.34 ±0.17 αβ	1.62±0.29 αβ	–	2.793±0.310 αβ	1.934±0.205 αβ	1.321± 0.132 αβγ	–
200TCAE	2.09±0.09 αβ	1.44 ± 0.24 αβ	1.01±0.78 αβ	–	1.707±0.076 αβγ	1.117±0.141 αβγ	0.823±0.036 αβγ	–
400TCAE	1.29±0.24 αβ	0.98±0.09 αβ	0.41±0.03 αβ	–	1.123±0.047 αβγ	0.767±0.039 αβγ	0.551±0.027 αβγ	–
800TCAE	0.84±0.04 αβ	0.56±0.07 αβ	0.41±0.02 β	–	0.679±0.017 αβγ	0.464±0.017 αβγ	0.333±0.007 αβγ	–
BASELINE	–	–	–	0.18 ± 0.01	–	–	–	0.15 ± 0.01

Table 3.3.2

Malondialdehyde (MDA) concentration (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	19.32±0.29 αβγ	13.28±0.61 αγ	9.49±0.34 αγ	–	16.22±0.572 αγ	10.82±0.494 αγ	7.712± 0.252 αγ	–
α-LIPOIC ACID	5.77± 0.69 αβ	4.11±0.37 αβ	2.97±0.29 αβ	–	4.69±0.521 αβ	3.24 ±0.344 αβγ	2.223± 0.222 αβ	–
200TCAE	3.54±0.17 αβγ	2.42±0.24 β	1.01±0.78 β	–	2.87±0.128 αβγ	1.982±0.237 αβγ	1.385±0.061 αβγ	–
400TCAE	2.29±0.09 βγ	1.58±0.09 βγ	1.14±0.03 β	–	1.892±0.081 αβγ	1.294±0.432 αβγ	0.925±0.046 αβγ	–
800TCAE	1.19±0.04 αβ	0.96±0.07 βγ	0.68±0.02 β	–	1.142±0.021 αβγ	0.789±0.029 αβγ	0.561±0.012 αβγ	–
BASELINE	–	–	–	0.31 ± 0.01	–	–	–	0.252±0.007

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.3

Protein carbonyl (PC) concentration (nmol/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	21.22±0.14 αγ	14.45±1.66 αγ	10.25±0.34 αγ	–	17.56±0.507 αγ	11.77±0.537 αγ	8.388±0.277 αγ	–
α-LIPOIC ACID	6.27±0.69 αβ	4.35±0.33 αβ	2.97±0.34 αβ	–	5.111±0.567 αβ	3.532±0.375 αβ	2.419±0.242 αβ	–
200TCAE	3.84±0.17 αβγ	2.64±0.32 αβγ	2.97±0.29 αβ	–	3.124±0.139 αβγ	2.154±0.258 αβγ	1.507±0.067 αβγ	–
400TCAE	2.51±0.09 αβγ	1.73±0.09 αβγ	2.24±0.11 αβγ	–	2.062±0.087 αβγ	1.404±0.072 αβγ	0.877±0.044 αβγ	–
800TCAE	1.55±0.04 αβγ	1.04±0.07 αβγ	0.76±0.02 βγ	–	1.242±0.031 αβγ	0.849±0.032 αβγ	0.531±0.011 αβγ	–
BASELINE	–	–	–	0.31 ± 0.01	–	–	–	0.274±0.008

Table 3.3.4

Myeloperoxidase (MPX) activity (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	35.24±0.22 αγ	24.17±1.19 αγ	17.22±0.64 αγ	–	30.54±0.992 αγ	21.37±0.976 αγ	15.23±0.503 αγ	–
α-LIPOIC ACID	10.49±1.17 αβ	7.27±0.83 αβ	4.97±0.49 αβ	–	8.084±1.032 αβ	6.414±0.681 αβ	4.391±0.432 αβ	–
200TCAE	6.42±0.29 αβγ	4.42±0.58 αβγ	3.09±0.15 αβγ	–	5.673±0.254 αβγ	3.915±0.472 αβγ	2.737±0.121 αβγ	–
400TCAE	4.02±0.07 αβγ	2.88±0.18 αβγ	2.07±0.11 αβγ	–	3.742±0.158 αβγ	2.552±0.131 αβγ	1.829±0.092 αβγ	–
800TCAE	2.77±0.07 αβγ	1.74±0.07 αβγ	1.27±0.06 βγ	–	1.965±0.057 αβγ	1.542±0.059 αβγ	1.108±0.023 αβγ	–
BASELINE	–	–	–	0.61 ± 0.08	–	–	–	0.497±0.014

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.5

NADPH oxidase (NOX) activity (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	38.59±0.44 αγ	26.27±1.19 α	18.69±0.64 αγ	–	28.31±0.677 αγ	19.69±0.899 αγ	14.03±0.463 αγ	–
α-LIPOIC ACID	11.39±1.27 αβ	7.87±0.83 αβ	5.37±0.49 αβ	–	8.56 ±0.949 αβ	5.908±0.627 αβ	4.046±0.405 αβ	–
200TCAE	6.96±0.31 αβγ	4.82±0.58 αβγ	3.36±0.15 αβγ	–	5.23±0.234 αβγ	3.626±0.432 αβγ	2.523±0.111 αβγ	–
400TCAE	4.59±0.19 αβγ	3.13±0.18 αβγ	2.24±0.11 αβγ	–	3.45±0.147 αβγ	2.342±0.121 αβγ	1.684±0.085 αβγ	–
800TCAE	2.55±0.17 αβγ	1.89±0.07 αβγ	1.36±0.06 βγ	–	2.07±0.053 αβγ	1.423±0.047 αβγ	1.028±0.222 αβγ	–
BASELINE	–	–	–	0.61 ± 0.08	–	–	–	0.458±0.013

Table 3.3.6

Xanthine oxidase (XO) activity (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	37.88±0.53 αγ	24.17±1.19 αγ	18.69±0.64 αγ	–	31.09±0.219 αγ	21.37±0.976 αγ	15.23±0.503 αγ	–
α-LIPOIC ACID	11.25±1.26 αβ	7.79±0.83 αβ	5.39±0.53 αβ	–	9.284±1.031 αβ	6.416±0.681 αβ	4.392±0.439 αβ	–
200TCAE	6.89±0.67 αβγ	4.75±0.58 αβγ	3.36±0.15 αβγ	–	5.674±0.254 αβγ	3.912±0.470 αβγ	2.731±0.121 αβγ	–
400TCAE	4.02±0.27 αβγ	2.88±0.18 αβγ	2.24±0.11 αβγ	–	3.745±0.158 αβγ	2.551±0.131 αβγ	1.829±0.092 αβγ	–
800TCAE	2.55±0.07 αβγ	1.87±0.07 αβγ	1.37±0.06 βγ	–	2.256±0.057 αβγ	1.542±0.059 αβγ	1.108±0.024 αβγ	–
BASELINE	–	–	–	0.31 ± 0.06	–	–	–	0.498±0.014

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.7

Catalase (CAT) activity (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	8.88±0.13 αγ	17.46±1.54 α	18.69±1.84 αγ	–	7.114±1.352 αγ	14.33±1.162 αγ	69.62±5.322 αγ	–
α-LIPOIC ACID	15.39±0.89 α	27.42±6.26 α	85.45±6.53 αβ	–	12.42±0.269 αβ	22.34±4.842 αβ	90.57±6.926 αβ	–
200TCAE	8.18±0.67 αγ	35.65±7.75 αβγ	151.11±8.48 αβγ	–	6.426±0.655 αβγ	29.04±6.292 αβγ	118.30±9.051 αβγ	–
400TCAE	11.46±0.43 αγ	45.62±4.18 αβ	151.81±0.11 αβγ	–	9.296±0.351 αβγ	37.17±3.402 αβγ	147.20±11.94αβγ	–
800TCAE	13.89±0.17 αγ	64.08±9.57 βγ	151.81±0.06 αβγ	–	10.50±0.129 αβγ	52.21±7.802 αβγ	110.30±0.023αβγ	–
BASELINE	–	–	–	72.00 ± 1.66	–	–	–	58.67±1.351

Table 3.3.8

Superoxide dismutase (SOD) activity (units/mg protein) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	9.17±0.28 αγ	18.45±0.54 α	34.35±1.84 αγ	–	7.52±0.183 αγ	15.01±1.212 αγ	28.24±1.458 αγ	–
α-LIPOIC ACID	15.99±0.99 α	28.87 ± 6.26 α	89.56 ± 6.53 αβ	–	13.01±0.765 αβ	23.40±5.07 αβ	65.98±5.57 αβ	–
200TCAE	15.94±0.74 αγ	37.38±8.58 α	111.21±8.88 αβγ	–	6.981±0.569 αβγ	30.43±6.594 αβγ	94.82±7.24 αβγ	–
400TCAE	11.92±0.49 αγ	47.79±4.85	151.52±11.64αβγ	–	9.731±0.368 αβγ	38.93±3.572 αβγ	124.00±9.487αβγ	–
800TCAE	13.36±0.22 αγ	67.13±10.57 β	181.85±56.91αβγ	–	11.00±0.135 αβγ	54.69±8.185 αβγ	155.10±14.28αβγ	–
BASELINE	–	–	–	75.43 ± 1.73	–	–	–	61.46±1.423

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α= significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.9

Glutathione peroxidase (GSH consumed/ mg protein) activity in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	9.77±0.01 α	19.46±1.54 α	36.35±1.84 αγ	–	10.79 ± 0.013 αγ	15.83 ± 1.282 αγ	29.77±1.532 αγ	–
α-LIPOIC ACID	16.79±0.99 αβ	30.27±6.26 α	94.27±7.22 αβ	–	15.41 ± 3.654 αβ	24.67 ± 5.346 αβ	76.89±5.882 αβ	–
200TCAE	9.04±0.76 α	39.38±6.58 α	151.00±7.68 αβγ	–	7.364±0.600 αβγ	25.29±6.953 αβ	99.96±7.648 αβγ	–
400TCAE	12.62±0.49 αβ	50.38±4.65 αβγ	153.42±10.00αβγ	–	10.26±0.388 αγ	41.04±3.762 αβγ	130.72±9.992αβγ	–
800TCAE	16.66±0.22 αβ	70.76±10.57 βγ	192.45±49.91αβγ	–	11.60±0.142 αγ	57.65±8.864 αβγ	162.60±13.19αβγ	–
BASELINE	–	–	–	70.43 ± 1.63	–	–	–	64.79±1.493

Table 3.3.10

Glutathione S-transferase (µmol/min/mg protein) activity in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	9.77±0.08 αγ	19.46±1.54 α	36.30±1.84 αγ	–	7.905 ± 0.701 αγ	15.59±1.263 αγ	29.33±1.571 αγ	–
α-LIPOIC ACID	16.79 ± 0.99 α	29.87±6.56 α	92.36±7.22 αβ	–	13.51 ± 0.794 αβ	24.31 ± 5.267 αβ	75.75±5.792 αβ	–
200TCAE	9.04±0.76 αγ	39.38±6.58 α	121.21±9.28 αβγ	–	7.253±0.594 αβγ	31.60±6.846 αβγ	98.18±7.512 αβγ	–
400TCAE	12.62 ±0.49 αγ	38.79±6.85 α	151.42±11.04αβγ	–	10.11±0.387 αβγ	40.44±3.703 αβγ	128.79±9.842αβγ	–
800TCAE	14.06±0.22 αγ	67.13±10.57 β	181.85±56.91αβγ	–	11.43±0.140 αβγ	56.80±8.492 αβγ	160.00±13.02αβγ	–
BASELINE	–	–	–	78.34 ± 1.83	–	–	–	63.83±1.475

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.11

Reduced glutathione (µg/mL) concentration in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	1.86±0.12 α	3.82±3.31 α	7.19±0.37 α	–	1.588±0.007 αγ	3.115±0.252 αγ	8.863±0.301 αγ	–
α-LIPOIC ACID	3.31±0.19 α	5.96±1.29 α	18.57±1.42 αβ	–	2.703±3.651 αβ	4.852±1.052 αβ	15.13±1.152 αβ	–
200TCAE	1.78±0.15 α	7.75±1.68 α	24.17±1.85 αβ	–	1.443±0.600 α	6.315±1.364 αβγ	19.67±1.502 αβγ	–
400TCAE	2.48±0.09 α	9.91±0.91 α	31.57±2.41 αβ	–	2.023±0.388 αβγ	8.072±2.903 αβγ	25.72±1.964 αβγ	–
800TCAE	2.82±0.34 α	13.93±2.08 β	39.28±3.19 αβ	–	2.287 ± 0.142 αβ	11.34±1.692 αβγ	32.00±2.515 αβγ	–
BASELINE	–	–	–	42.35 ± 0.97	–	–	–	34.50±0.797

Table 3.3.12

α-tocopherol concentration (µmol/L) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	8.77±0.01 α	11.66±0.14 α	29.32±1.54 α	–	6.61±0.014 αγ	12.96±1.052 αγ	24.38±1.255 αγ	–
α-LIPOIC ACID	13.79±0.81 α	11.66±5.81 α	77.26±5.91 αβ	–	11.23±0.661 αβ	20.20±4.373 αβ	62.95±4.812 αβ	–
200TCAE	7.39±0.60 α	32.32±7.56 αβ	101.00±7.70 αβ	–	6.029±0.491 αγ	26.26±5.679 αβγ	81.84±6.252 αβγ	–
400TCAE	10.32±0.39 α	41.25±1.03 αβ	131.42±1.00 αβ	–	8.406±0.317 αβγ	33.60±3.082 αβγ	107.00±8.182αβγ	–
800TCAE	11.66±0.41 α	57.94±9.37 αβ	162.50±49.90 αβ	–	9.499±0.116 αβγ	47.20±7.058 αβγ	47.20 ±7.058 αβγ	–
BASELINE	–	–	–	70.42 ± 1.63	–	–	–	53.04±1.226

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ= significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE=800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Table 3.3.13

Ascorbic acid concentration (µmol/L) in doxorubicin-induced toxicity & the ameliorative role of TCAE.

	BRAIN				HEART
	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE	PRE-TREATMENT	CO-TREATMENT	POST-TREATMENT	BASELINE
NORMAL SALINE	8.49±0.39 α	17.54±1.14 α	32.67±1.84 α	–	7.03±0.155 αγ	14.02±1.136 αγ	26.37±1.352 αγ	–
α-LIPOIC ACID	14.91±0.88 α	26.82±5.81 α	83.53±6.39 αβ	–	12.15±0.714 αβ	21.85±4.735 αβ	68.10±5.308 αβ	–
200TCAE	8.00±0.65 α	34.87±7.56 αβ	101.09±8.30 αβ	–	6.525±0.531 αγ	28. 41±6.152αβγ	88.53±6.765 αβγ	–
400TCAE	11.16±0.42 α	46.62±1.03 αβ	141.40±10.90 αβ	–	9.098±0.343 αβγ	36.35±3.333 αβγ	115.70±8.853αβγ	–
800TCAE	12.61±0.16 α	62.67±9.37 αβ	171.90±14.30 αβ	–	10.27±0.126 αβγ	51.06±7.365 αβγ	144.00±43.47αβγ	–
BASELINE	–	–	–	70.42 ± 1.63	–	–	–	57.38±1.324

Values are expressed as mean±standard deviation (n=5). Significant at p<0.05. α=significant difference compared with baseline group. β=significant difference compared with normal saline group. γ=significant difference compared with α-lipoic acid. 200 TCAE=200 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 400 TCAE=400 mg/kg body weight of Theobroma cacao stem bark aqueous extract. 800 TCAE =800 mg/kg body weight of Theobroma cacao stem bark aqueous extract

Discussion

Doxorubicin (DOX) is a widely used chemotherapeutic agent in the treatment of tumors with a major side effect on the brain and most especially cardiac toxicity. High concentration of DOX leads to a high redox reactivity in these tissues. DOX-increased ROS generation resulted in the oxidation of lipids, proteins, and signaling molecules [24]. The principal mechanism of DOX is chelating DNA, inhibiting topoisomerase II and producing free radicals to kill cancer cells [10]. α-Lipoic acid (ALA) and its reduced form DHLA, are considered as powerful natural antioxidant agents with a scavenging capacity for many reactive oxygen species (ROS). ALA/DHLA have some important advantages over other antioxidant agents such as vitamin E and C, because they have amphiphilic properties that confer their antioxidant actions in the membrane as well as in the cytosol. ALA/DHLA can also regenerate other antioxidant substances such as vitamin C, vitamin E and the ratio of reduced/oxidized glutathione (GSH/GSSG) [34]. The present result showed that treating animal with α-lipoic acid improved and reversed the biochemical changes induced in the heart and brain tissues by DOX intoxication. This result correlates with the findings of Li et al. [26] where it was reported that α-lipoic acid ameliorates oxidative stress by increasing aldehyde dehydrogenase-2 activity in the heart and brain and also caused a significant fall in the lipid peroxide concentration. (Fig. 3.2.1, Fig. 3.2.2).

Fig. 3.2.1

Histopathology of the brain sections.

Fig. 3.2.2

Histopathology of the heart sections.

The phenolic compounds in T. cacao stem bark contain bioactive compounds that have potential health benefits for chronic diseases such as inflammation, cardiovascular illness, neurodegenerative disorders, and cancer [43]. This present study revelaed the protective potential of Theobroma cacao stem back aqueous extract (TCAE) on brain and cardiac enzymes and oxidative damages caused by doxorubicin induced toxicity. These findings also correlate with Zainal et al. [53] where it was stated that consumption of cocoa T. cacao stem bark and which have high antioxidant activity, could be beneficial in decreasing the damage from genotoxic and epigenetic carcinogens, and inhibiting the complex processes leading to cancer. T. cacao stem bark because of its polyphenolic compounds has become an important potential chemopreventive and therapeutic natural agent. Cocoa flavonoids influenced several important biological activities in vitro and in vivo by their free radical scavenging ability or through the regulation of signal transduction pathways to stimulate apoptosis, inhibit inflammation, cellular proliferation, apoptosis, angiogenesis and metastasis [53].

LDH catalyzes the conversion of pyruvate to lactate and back, as it converts NADH to NAD+. LDH is found extensively in body tissues, such as blood cells and heart muscle [13]. The result of the present study revealed that DOX intoxication caused a significant reduction and increase in LDH activity in brain and heart tissues respectively while pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reversal in LDH activity compared with DOX-intoxicated untreated rats. This was supported by the work of Koti et al. [21] where a significant modulation in LDH activity was observed during tissue damage. ACPs have had considerable impact as tools of clinical investigation and intervention. One particular example is tartrate resistant acid phosphatase, which is detected in the serum in raised amounts [42]. The result of the present study revealed that DOX intoxication caused a significant decrease and increase in ACP activity in brain and heart tissues respectively while pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reversal in ACP activity compared with DOX-intoxicated untreated rats. This experimental result correlate with the work of Koti et al. [21] where it was stated that a modulation in the activities of cardiac and brain enzymes (LDH, GGT and ACP) was observed as a result of cardiac and brain damage caused by DOX. ALP activity on endothelial cell is responsible, in part, for the conversion of adenosine nucleotides to adenosine, a potent vasodilator and anti-inflammatory mediator that can protect tissues from the ischemic damage that results from injury [45]. The result of the present study revealed that DOX intoxication caused a significant increase and reduction in ALP activity in brain and heart tissues respectively while pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reversal in ALP activity compared with DOX-intoxicated untreated rats. This was supported by the work of [1] where modulation in ALP activity in heart and brain were reported as an indicator of cardiac and brain damage. Gamma-glutamyl transferase (GGT) is a cell-surface protein contributing to the extracellular catabolism of glutathione (GSH). The enzyme is produced in many tissues. High levels of GGT have been associated in populations with increased risk of atherosclerotic cardiovascular disease (CVD) and brain damage [18]. In the present study, we revealed that DOX intoxication caused a significant increase in GGT activity in cardiac and brain tissues while pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reduction in GGT activity in these tissues. This was supported by the work of Ragavendran et al. [38] where similar elevations in cardiac and brain enzymes activities in rats following challenge with a single cumulative dose of DOX was reported. According to Vijay et al. [49], GGT was reported as an important marker of tissue injury especially during clinical follow-up of DOX therapy.

DOX administration induced oxidative stress on these tissues as manifested by the alterations observed in both enzymatic and non-enzymatic cardiac antioxidant defense systems. From the present study, it was clear that DOX intoxication significantly increased concentrations of hydrogen peroxide generated, malondialdehyde (MDA) and protein carbonyl (PC). This is in agreement with the findings of Brett et al. [8] where it was reported that cells exposed to increasing concentration of DOX had an increase in concentrations of hydrogen peroxide generated, malondialdehyde (MDA) and protein carbonyl (PC) due to metabolic reductive activation of DOX to a semiquinone. Pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reduction in H2O2, MDA and PC concentrations. This also agrees with the work of Zainal et al. [53] where it was reported that cocoa flavonoids influenced several important biological activities in vitro and in vivo by their free radical scavenging ability. In this study, DOX intoxication caused a significant increase in the activities of enzymes implicated in free radical generation: myeloperoxidase (MPX), NADPH oxidase (NOX) and xanthine oxidase (XO). This agrees with the work of Daniel et al. [14] where exposure of rats to DOX led to an increase in the activities of MPX, NOX and XO due to ability of DOX to bioactivate mitomycin C to generate oxygen radicals. Pre-, co-, and post-treatment with TCAE caused a significant dose-dependent reduction in these enzymes activities. This correlate with the work of Crozier et al. [12] as a result of its ability to inhibit the complex processes leading to cancer. Administration of TCAE reversed the DOX induced oxidative damage and significantly increased the antioxidant enzymes (catalase, superoxide dismutase, glutathione S-transferase and glutathione peroxidase). This is in agreement with the findings of Saratchandran and Cherupally [41] where it was stated that reduction in these enzymic antioxidant activities is associated with a marked increase in cardiac and brain lipid peroxidation as manifested by increased MDA level. This study also revealed that DOX intoxication significantly decreased the concentration of non-enzymatic antioxidant (reduced glutathione, ascorbic acid and α-tocopherol). This is in agreement with [20] where it was reported that high concentration of DOX leads to a high redox reactivity in the heart and brain. Pre-, co-, and post-treatment with TCAE significantly reversed the decrease observed in the concentrations of these non-enzymic markers caused by DOX intoxication. This finding correlates with the work of Golbidi et al., [15], where both cocoa and ALA was reported to act as biological antioxidants, as metal chelators, reducing the oxidized forms of other antioxidant agents such as vitamins C and E and reduced glutathione. The tissue histology showed no visible lesions. This is in agreement with Blanco et al. (2012) who stated that the neurotoxicity and cardiotoxicity of DOX remains difficult to predict and is often not detected until years after the completion of chemotherapy.

Conclusion

Thus, the result of the present study affirmed that T. cacao stem bark aqueous extract protected against DOX induced oxidative damage in the brain and heart of experimental rats.