Cardio-protective and antioxidant properties of caffeic acid and chlorogenic acid: Mechanistic role of angiotensin converting enzyme, cholinesterase and arginase activities in cyclosporine induced hypertensive rats

Biomedicine & Pharmacotherapy 109 (2019) 450–458

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Original article

Cardio-protective and antioxidant properties of caffeic acid and chlorogenic acid: Mechanistic role of angiotensin converting enzyme, cholinesterase and arginase activities in cyclosporine induced hypertensive rats

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Odunayo Michael Agunloyea, , Ganiyu Oboha, Adedayo Oluwaseun Ademiluyia, Ayokunle Olubode Ademosuna, Akintunde Afolabi Akindahunsia, Ademola Adetokunbo Oyagbemib, Temidayo Olutayo Omobowalec, Temitayo Olabisi Ajibadeb, Adeolu Alex Adedapod a

Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, P.M.B 704, Akure 340001, Nigeria Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University of Ibadan, Nigeria c Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Ibadan, Nigeria d Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cyclosporine Caffeic acid Chlorogenic acid Blood pressure Nitric oxide

Caffeic acid (CAA) and chlorogenic acid (CHA) are important members of hydroxycinnamic acid with natural antioxidant and cardio-protective properties. The present study aimed to determine the effect of CAA and CHA on systolic blood pressure, heart rates (HR) as well as on the activity of the angiotensin-1-converting enzyme (ACE), acetylcholinesterase (AChE), butrylcholinesterase (BChE) and arginase in cyclosporine-induced hypertensive rats. Experimental rats were distributed into 7 groups (n = 6): normotensive control rats; hypertensive rats (induced rats) as well as hypertensive- treated groups with captopril (10 mg/kg/day), CAA (10 and 15 mg/kg/day) and CHA (10 and 15 mg/kg/day), respectively. The experiment lasted for 7 days and the systolic blood pressure (SBP) and heart rates were recorded using tail-cuff method. Oral administration of captopril, caffeic acid and chlorogenic acid normalized hypertensive effect caused by cyclosporine administration. CAA and CHA significantly (P < 0.05) reduced SBP and HR, activity of ACE, AChE, BChE and arginase in the treated hypertensive rats compared with cyclosporine induced-hypertensive rats. Likewise, CAA and CHA improved nitric oxide (NO) bioavailability, increased catalase activity and reduced glutathione content while malondialdehyde (MDA) level was reduced compared with cyclosporine hypertensive rats. Findings from this study shows that CAA and CHA exhibited blood pressure lowering properties and reduced activities of key enzymes linked to the pathogenesis of hypertension in cyclosporine-induced rats. These might be some of the possible mechanisms of action by which their cardio-protective properties are exhibited.

1. Introduction Hypertension commonly refers to as a persistent increase in the systolic and diastolic blood pressure, thus, identified as a common risk factor in cardiovascular diseases. Hypertension is asymptomatic in nature and often refers to as a silent killer responsible for around 9.4 million deaths worldwide [1]. The pathogenesis of hypertension involves some enzymes system such as renin-angiotensin converting enzymes system (RAS) [2], arginase [3], cholinesterase [4] as well as oxidative stress [5]. The involvement of ACE activity in the pathogenesis of hypertension central around production and vasoconstrictive

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effect of angiotensin II (ANG II) coupled with free radical generation as well as degradation of bradykinin, a potent vasodilator [6]. The Elevated level of ANG II triggers activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which in turn increase the generation of free radicals. Thus, it is important for an antihypertensive therapy to possess a high degree of radical scavenging properties [7]. Inhibitors of ACE activity such as captopril and enalapril have been used as antihypertensive drugs [8] and are known to have some unpleasant side effects. Also, arginase plays a significant role in the development of cardiovascular disorders such as hypertension [3]. Arginase has been involved in the metabolism of arginine to urea and

Corresponding author. E-mail address: [email protected] (O.M. Agunloye).

https://doi.org/10.1016/j.biopha.2018.10.044 Received 24 July 2018; Received in revised form 8 October 2018; Accepted 9 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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USA), cyclosporine A (Sandimun Neoral, 25 mg, Novartis). All the chemicals used in this experiment were of analytical grade.

ornithine and competes directly with endothelial nitric oxide synthase (eNOS) for same substrate (L-arginine), thereby limiting nitric oxide (NO) availability from eNOS for vasodilatory processes. The pathological changes observed as a result of reduction in the bioavailability of NO due to little or no L-arginine for NO production might occur as a result of over expression of arginase activity [9]. Also, metabolites from the hydrolytic effect arginase are interconnected to vascular thickening and stiffness [10,11]. The cholinergic induced vasodilation is mediated mainly through activation of muscarinic receptors by ACh which in turn cause the release of NO from endothelial cell leading to relaxation of vascular smooth muscle, vasodilation and a recordable increase in blood flow rate in the myocardium [12,13]. It is noteworthy that ACh is prone to the hydrolytic effect of cholinesterase (acetylcholinesterase and butyrylcholinestrase) leading to the formation of acetate and choline. This hydrolytic effect of cholinesterase reduces ACh availability, hence, the pharmacological interaction between ACh and muscarinic receptor is then halted. In this context, holistic management of hypertension could be achieved if the activity of these key enzymes are reduced or halted in the hypertensive and healthy individual. Cyclosporine A (CSA) is an immunosuppressant drug used in organ transplantation to prevent incidence of graft rejection and for the treatment of autoimmune are thereby reduced. CSA administration has been linked with severe cardiovascular disorders such as hypertension [14]. However, vascular abnormalities [15,16] and up-regulation of RAS activity [17,18], elevated activity of ACE, over expression of angiotensin AT1 receptor expression, inhibition of eNOS, as well as promoting oxidative stress have been linked to CSA administration. These factors might account for the hypertensive effect of CSA. Research finding has shown that consumption of polyphenols or polyphenol-rich foods have an inverse relationship to the manifestation of disease state [19]. Dietary phenolic acids have been shown to offer a protective role against cardiovascular diseases, possess high antioxidant property in vitro [20] and in vivo [21], anti-inflammatory as well as improve endothelial function [22,23]. It has been reported that polyphenol can promote the production of vasoprotective factors such as NO and endothelium-derived hyperpolarizing factor (EDHF) which could improve vascular smooth muscle function as well as checkmating vascular oxidative stress associated with many cardiovascular risk factors [24,22]. About one-third of polyphenol compounds present in plants are phenolic acid. Hydroxylbenzoic and hydroxycinnamic acids are the major class of phenolic acid available in plants [25], while caffeic acid and chlorogenic acid are prominent members of hydroxylcinnamic acid, which are widely abundant in the foods such as fruits, spices, vegetables, wine, olive oil, and coffee [25]. However, dose of CAA and CHA less than 2437 mg/kg and 1250 mg/kg respectively has been reported non-toxic. Therefore, due to the differences in foods phenolic acid constituent, a lower dose ranges from 10 to 15 mg/ kg body weight is achievable in food with lower polyphenol content. Research findings have shown that caffeic acid and chlorogenic acid inhibits DNA damage [26], together with anti-inflammation [27], antiAlzheimer’s disease [28,21], Anti-diabetic [29], hypocholesterolemic effects [30]. However, owing to health implication of hypertension as well as associated endothelial dysfunction, up regulation of RAS and eminent generation of reactive oxygen species (ROS). It is important to explore therapeutic benefits of major phenolic acids bioavailable in foods such as CAA and CHA. It is expedient to evaluate and compared blood pressure lowering property of CAA and CHA as well as their effect on the activity of ACE, arginase, cholinesterase in cyclosporine-induced hypertensive rats.

2.2. Experimental rats The use of experimental animals was approved by the departmental ethical committee and the National and Institutional guidelines for animal protection and welfare were followed strictly. The rats were maintained at room temperature (25 °C) with free access to food and water. They were allowed to adapt to their new environment for 2 weeks prior to the commencement of induction and treatment. 2.3. Experimental groups and protocols After acclimatization, the experimental rats were distributed into 7 groups of male rats (n = 6) as follows. Group 1: Control (n = 6), rats were given 0.2 ml of distilled water daily for 7 days; Group 2: Hypertensive group (CSA induced hypertensive rats; negative control group), rats (n = 6) were given CSA (25 mg/kg/day) [31] i.p for a period of seven without treatment; Group 3: Captopril group (Positive control) (n = 6), CSA-induced hypertensive rats treated captopril (ACE inhibitor, 10 mg/kg/day, [32]) administered orally once daily; Group 4: rats (n = 6), CSA-induced hypertensive rats treated with caffeic acid (10 mg/kg/day) via oral administration; Group 5: rats (n = 6), CSA-hypertensive rats treated with caffeic acid (15 mg/kg/day) via oral administration; Group 6: rats (n = 6), CSA-hypertensive rats treated with chlorogenic acid (10 mg/kg/day) via oral administration; Group 7: rats (n = 6), CSA-hypertensive rats treated chlorogenic acid (10 mg/kg/day) via oral administration. It should be on note that administration of CSA and treatments regimes were done concurrently. 2.4. Hemodynamic parameter determination In all rats, systolic blood pressure (SBP) and heart rates (HR) were measured in awake animals, by non-invasive tail-cuff plethysmography (Kent Scientific; RTBP1001 Rat Tail Blood Pressure System for rats and mice, Litchfield, USA). Rats were conditioned with the apparatus before measurements are taken. The measurement of SBP and HR were done at the end of the experiment. The experiment lasted for 7 days, thereafter, the animals were anesthetized with Pentobarbital, the animals were dissected, and blood from the inferior venacava of the heart was collected into ethylene diamine tetra acetic acid-containing tubes without any protease inhibitors and centrifuged at 3000g for 15 min in an MSC bench centrifuge. The clear supernatant obtained (plasma) was used in estimation of enzyme activities and other biochemical indices. The tissues (lung, heart and kidney) were removed and rinsed in ice-cold normal saline after which they were blotted and weighed. The tissues were minced with scissors in three volumes of ice-cold 50 mM Tris−HCl buffer (pH 7.4) and homogenized in a Teflon-glass homogenizer. The homogenates were centrifuged for 10 min at 5000 × g to yield a pellet that was discarded. The clear supernatants obtained were used for various biochemical assays [33]. 2.5. Determination of ACE activity The plasma ACE activity was determined as described by Cushman and Cheung [34]. The substrate [hippuryl-histidylleucine (Bz-Gly-HisLeu)] was purchased from SigmaAldrich. The amount of cleaved hippuric acid from hippuryl-histidyl-leucine was measured by the enzymatic method. 50 μL of sample and 150 μL of 8.33 mM of hippurylhistidylleucine (Bz-Gly-His-Leu) in 125 mM Tris−HCl buffer (pH 8.3) were incubated at 37 °C for 30 min. After incubation, the reaction was arrested by adding 250 μL of 1 M HCl. The Gly–His bond was then cleaved, and the hippuric acid produced by the reaction was extracted with 1.5 mL ethyl acetate. Next, the mixture was centrifuged to separate

2. Material and methods 2.1. Samples and chemicals Chemicals: caffeic acid, chlorogenic acid, HHL, L-arginine and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, 451

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Nelson and Kiesow [39]. This assay involves the change in absorbance at 240 nm due to CAT-dependent decomposition of hydrogen peroxide. For CAT assay in the plasma and tissue homogenates, the tissue was homogenized in 0.1 M potassium phosphate buffer, pH 7.4, at a proportion of 1:5 (w/v). The homogenate was centrifuged at 2000 × g for 10 min to yield a supernatant that was used for the enzyme assay. The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.4), 10 mM H2O2 and 20 μl of the supernatant. The rate of H2O2 reaction was monitored at 240 nm for 2 min at room temperature. The enzymatic activity was expressed in units/mg protein (one unit of the enzyme is considered as the amount of CAT that decomposes 1 mmol of H2O2 per min at pH 7 at 25 °C)

the ethyl acetate layer; then, 1 mL of the ethyl acetate layer was transferred to a clean test tube and evaporated. The residue was redissolved in distilled water, and its absorbance was measured at 228 nm. The plasma ACE activity was expressed as μmol HHL cleaved/ min. 2.6. Determination of arginase activity Arginase activity in the plasma and heart tissue was determined by measuring the rate of urea production using α-isonitrosopropriophenone (9% in absolute ethanol) as previously described by Kaysen and Strecker [35]. Briefly, 50 μl of samples were added into 75 μl of Tris−HCl (50 mmol/l, pH 7.5) containing 10 mmol/ l MnCl2 and was pre-incubated at 37 °C for 10 min to activate the enzyme. The hydrolysis reaction of L-arginine by arginase was performed by incubating the mixture containing activated arginase with 50 μl of Larginine (0.5 mol/l, pH 9.7) at 37 °C for 1 h and was stopped by adding 400 μl of the acid solution mixture [H2SO4/H3PO4/H2O = 1:3:7 (v/v/ v)]. For calorimetric determination of urea, α-isonitrosopropiophenone (25 μl, 9% in absolute ethanol) was then added and the mixture was heated at 100 °C for 45 min. After placing the sample in the dark for 10 min at room temperature, the urea concentration was determined spectrophotometrically by the absorbance at 550 nm. The amount of urea produced was used as an index for arginase activity. The arginase activity was expressed as μmol urea produced/min/mg protein.

2.11. Reduced glutathione Reduced glutathione (GSH) was determined by the method of Ellman [40]. 1 ml of supernatant was treated with 500 μl of Ellman’s reagent (19.8 mg of 5,5′dithiobisnitrobenzoic acid in 100 ml of 0.1% sodium citrate) and 3.0 ml of 0.2 M phosphate buffer (pH 8.0). The absorbance was read at 412 nm in spectrophotometer. 2.12. Total protein determination Protein was measured by the Coomassie blue method according to Bradford [41] using serum albumin as standard. 2.13. Data analysis

2.7. Determination of cholinesterase (acetylcholinesterase and butrylcholinetrase) activity

The values were expressed as mean ± standard deviation (SD). The mean differences in each group were analysis by one‐way ANOVA using Graph pad prism 5.0, followed by Duncan’s multiple range tests at the level p < 0.05.

The AChE enzymatic assay was determined according to the method of Ellman et al. [36]. The reaction mixture (2 ml final volume) contained 100 mM K+-phosphate buffer, pH 7.5 and 1 mM 5,5′-dithiobisnitrobenzoic acid (DTNB). The method is based on the formation of the yellow anion, 5, 5′- dithio-bis-acid-nitrobenzoic, measured by absorbance at 412 nm during 2 min incubation at 25 °C. The enzyme (40–50 mg of protein) was pre- incubated for 2 min. The reaction was initiated by adding 0.8 mM acetylthiocholine iodide (AcSCh) for acetylcholineterase assay while 0.8 mM butrylthiocholine (BcSCh) was used for butrylcholineterase. All samples were in triplicate readings and the enzyme activities were expressed in Units/mg of protein.

3. Results 3.1. Evaluation of the effect of CAA and CHA on SBP and heart rate in CSA-induced hypertensive rats Intraperitoneal (i.p) administration of CSA (25 mg/kg body weight) caused significant (P < 0.05) increases in the final SBP and heart rates measured through the use of tail-cuff when compared to the normotensive (control group) rats, confirming the hypertensive model. The normotensive group (hypertensive group) was placed on an equal volume of distilled water. Also, we noticed that administration of captopril (anti-hypertensive drug) 10 mg/kg body weight, CAA and CHA (10 and 15 mg/kg body weight) noticeably exhibited blood pressure lowering adjudge by significant reduction in SBP and heart rate in the cyclosporine-induced hypertensive rats as shown in Figs. 1 and 2. Our results show clearly that CAA and CHA could reduce SBP & heart rate.

2.8. Nitric oxide level (NOx) determination NOx content was estimated in a medium containing 70 μl of the sample, 70 μl of 2% vanadium chloride (VCl3) in 5% HCl, 70 μl of 0.1% N-(l-naphthyl) ethylenediamine dihydrochloride and 2% sulphanilamide (in 5% HCl) in 1:1 ratio. After incubating at 37 °C for 60 min, nitrite levels, which correspond to an estimative level of NOx, were determined spectrophotometrically at 540 nm, based on the reduction of nitrate to nitrite by VCl3 Miranda et al. [37]. The nitrite and nitrate levels were expressed as nanomole of NOx/mg protein.

3.2. Effect of CAA and CHA on ACE activity in cyclosporine induced hypertensive rats

2.9. Determination of tissue lipid peroxidation The results of the study also revealed that there was significant alteration (P < 0.05) in the activity of ACE in the plasma of CSA-induced hypertensive rats when compared to the normotensive rats. The result revealed that ACE activity in the plasma of hypertensive rats increased significantly when (P < 0.05) when compared with to the rats in the control group (the normotensive group). However, CAA, CHA (10 and 15 mg/kg body weight) as well as captopril (20 mg/kg body weight) treated groups had significantly lower levels of ACE activity in the plasma in a dose dependent manner (10 and 15 mg/kg body weight) when compared with the CSA-induced hypertensive group (untreated group) as presented in Fig. 3. Also, when the effect of CAA and CHA on plasma ACE activity in the treated groups were compared with the hypertensive group (untreated hypertensive rats). Chlorogenic acid

The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. [38]. Briefly 300 μl of tissue homogenate, 300 μl of 8.1% SDS (Sodium dodecyl sulphate), 500 μl of Acetic acid/ HCl (PH = 3.4) and TBA (Thiobarbituric acid) were added, and the mixture was incubated at 100 °C for 1 h. Thereafter, the thiobarbituric acid reactive species (TBARS) produced was measured at 532 nm and calculated as Malondialdehyde (MDA) equivalent. 2.10. Catalase (CAT) activity The determination of CAT activity in the plasma and tissue homogenates were carried out in accordance with a modified method of 452

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Fig. 1. Effect of caffeic and chlorogenic acid on systolic blood pressure in cyclosporine induced hypertensive rats. Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid Fig. 3. Effect of caffeic and chlorogenic acid on plasma ACE activity in cyclosporine induced hypertensive rats. Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

Fig. 2. Effect of caffeic and chlorogenic acid on heart rate in cyclosporine induced hypertensive rats. Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). & P < 0.05 non-significant difference when compared with either normotensive or CSA-treated group with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

Fig. 4. Effect of caffeic and chlorogenic acid on heart acetylcholinesterase activity in cyclosporine induced hypertensive rats. Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). & P < 0.05 non-significant difference when compare either normotensive or CSA-treated group with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

significantly reduced ACE level in the plasma than caffeic acid. and heart of CSA-induced hypertensive rats was significantly (P < 0.05) increased when compared to the normotensive rats as shown in Fig. 6(A & B). The captopril, caffeic acid and chlorogenic acid treated groups had significantly (P < 0.05) lower plasma and heart level of arginase activity when compared to the hypertensive rats. Nevertheless, both phenolic acids exhibited a non-significant difference when their effect on arginase activity in hypertensive rats were compared with each other.

3.3. Effect of CAA and CHA on acetylcholinesterase (AChE) and butrylcholinesterase (BChE) activity in CSA- induced hypertensive rats Figs. 4 and 5 revealed a significant (p < 0.05) elevation in the activity of acetylcholinesterase (AChE) and butyrlcholinesterase (BChE) in the heart of CSA-induced hypertensive rats when compared when compared to the rats in the normotensive group. Administration of CAA and CHA (10 and 15 mg/kg body weight respectively) produced a significant (P < 0.05) decrease in the activity of AChE and BChE in the heart of treated hypertensive rats when compared to the hypertensive rats (untreated rats).

3.5. Effect of CAA and CHA on nitric oxide (NOx) level in CSA-induced hypertensive rats Nitric oxide level in the plasma and heart of CSA-induce hypertensive rats (rats placed on cyclosporine 25 mg/kg body weight only without treated) was significantly reduced when compared with control (normotensive) group as presented in Fig. 7(A & B). However,

3.4. Effect of CAA and CHA on arginase activity in CSA- induced hypertensive rats The activity of arginase using L-arginine as substrate in the plasma 453

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Fig. 5. Effect of caffeic and chlorogenic acid on heart butyrylcholinesterase activity in cyclosporine induced hypertensive rats. Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). & P < 0.05 non-significant difference when compare either normotensive or CSA-treated group with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

administration of captopril (10 mg/kg/day), caffeic acid and chlorogenic acid (10 and 15 mg/kg body weight) caused a dose-dependent significant (P < 0.05) increase in the plasma and heart NOx level of the treated groups when compared to the induced (untreated hypertensive rats) group. NOx level in caffeic acid and chlorogenic acid treated group are not significantly difference. 3.6. Effect of CAA and CHA on malondialdehyde (MDA) level in CSAinduced hypertensive rats Furthermore, the level of MDA in plasma and heart increased significantly (P < 0.05) in the CSA-induced hypertensive rats when compared with rats in the normotensive group as shown in Fig. 8(A & B). The level of MDA in the plasma and heart of captopril, CAA and CHA treated hypertensive rats was significantly (P < 0.05) reduced when compared to the hypertensive (untreated) rats. Nevertheless, caffeic acid significantly reduced MDA level in the plasma of hypertensive rats than chlorogenic acid while a non-significant difference was observed in the heart MDA level when the effect of CAA and CHA were compared.

Fig. 6. (a) Effect of caffeic and chlorogenic acid on plasma arginase activity in cyclosporine induced hypertensive rats. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSAtreated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid. (b) Effect of caffeic and chlorogenic acid on heart arginase activity in cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group). CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

3.7. Effect of on catalase activity in CAA and CHA CSA-induced hypertensive rats

and 15 mg/kg/day) significantly (P < 0.05) improved glutathione concentration in all the treated groups when compared with hypertensive group.

Fig. 9(A & B) revealed the effect of CAA and CHA on the activity of catalase in the kidney and lung of hypertensive rats. The catalase activity in the hypertensive rats was significantly reduced when compared to the control (normotensive) rats. Treatment with captopril, CAA and CHA significantly (P < 0.05) increased catalase activity in the plasma and heart of the treated rats when compared to the hypertensive rats (untreated rats).

4. Discussion The cardiovascular protective effect of CAA and CHA were assessed in CSA-induced hypertensive rats. The CSA involvement in the cardiovascular disorders has been linked to alteration of renin-angiotensinaldosterone system [42], inhibition of nitric oxide synthase [43], impaired renal function [44] as well as increased the generation of ROS [45,44]. The intraperitoneal administration of cyclosporine 25 mg/kg body weight for 7 days caused hypertension in a rat model. This was evidenced by a significant increase in the SBP and HR. The observed increase in the SBP and HR in the induced-hypertensive rats accomplished with significant increase in the activity of ACE in the plasma coupled with significant decreased in NO level in plasma and heart

3.8. Effect of CAA and CHA on glutathione level in CSA- induced hypertensive rats The result as shown in Fig. 10(A & B) revealed that the concentration of reduced glutathione in the heart and kidney of CSA-induced hypertensive rats was significantly (P < 0.05) reduced when compared to the normotensive rats. Administration of captopril, CAA and CHA (10 454

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Fig. 8. (a) Effect of caffeic and chlorogenic acid on plasma malondiadehyde (MDA) level in cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSAtreated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid. (b) Effect of caffeic and chlorogenic acid on heart Malondiadehyde (MDA) level in cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

Fig. 7. (a) Effect of caffeic and chlorogenic acid on nitric oxide level in plasma of cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid. (b) Effect of caffeic and chlorogenic acid on nitric oxide level in heart of cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

Zn2+ and the disulfde bridge residues at the active site of the ACE determine the degree of observed inhibition in the ACE actiivty [50,51]. These interactions between phenolic acid and residue moieties at the active site of ACE (Zn2+ and the disulfde bridge) might be among the possible through which CAA and CHA exhibit blood pressure lowering ability [51]. The level of inhibition of ACE activity by phenolic acids hinge on the kind of interactions between the Zn2+ and the disulfide bridge residue at the active site of the protein (ACE) and the hydroxyl and carboxylic group of the phenolic acid [50,51]. These interactions may have resulted in the observed blood pressure lowering ability of CAA and CHA on ACE activity [51] The extent of inhibition of ACE activity by phenolic acids depends upon the kind of interactions between the Zn2+ and the disulfde bridge residue at the active site of the protein (ACE) and the hydroxyl and carboxylic group of the phenolic acid [50,51]. These interactions between phenolic acids and residue moieties (Zn2+ and the disulfide bridge) at the active site of ACE might have accounted for the observed

(Figs. 3 and 7), these events also affirm the hypertensive effect of cyclosporine [46]. This study revealed that CAA and CHA exhibited blood pressure lowering properties justified by SBP and HR lowering ability in the hypertensive rats as shown in Figs. 1 and 2. The blood pressure lowering ability could be linked to reduction in plasma ACE as presented in Fig. 3. ACE activity involved in the conversion of a non-vasoconstrictive angiotensin I to angiotensin II (ANG II), which is a potent vasoconstrictor and enhancer of bradykinin degradation [47]. The vasopressor influence of ANG II responsible for blood pressure regulation and the elevated plasma level of ANG II has been linked to the pathogenesis and development of hypertension [48]. The observed reduction in the plasma ACE activity in the phenolic acids treated groups could be as a result of decrease in the vascular ACE synthesis and secretion [49]. Reduction in the ACE activity brings about a reduction in the plasma level of angiotensin II and effective therapeutic management of hypertension could be achieved. Research findings have revealed that interaction of phenolic acids hydroxyl and carboxylic group(s) with the

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Fig. 9. (a) Effect of caffeic and chlorogenic acid on kidney catalase activity in cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid. (b) Effect of caffeic and chlorogenic acid on lung catalase activity in cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

Fig. 10. (a) Reduced glutathione level in the heart of cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid. (b) Reduced glutathione level in the kidney of cyclosporine induced hypertensive rats; Values were expressed as mean ± S.D. for 6 rats in each group. *P < 0.05 when compare normotensive with CSA alone (hypertensive group); **P < 0.05 when compare CSA-treated with CSA alone (hypertensive group); CSA = Cyclosporine; CAA = caffeic acid; CHA = Chlorogenic acid.

acetylcholinesterase and butyrylcholinesterase in cyclosporine-hypertensive rats as shown in 4 & 5 agreed with our previous studies on the inhibitory effect of CAA and CHA on the activities of cholinesterase in vitro and in vivo studies [20,6]. Also, in ensuring holistic management of hypertension, the activity of AChE and BChE need to downregulated both in the healthy and hypertensive individual. Nitric oxide (NOx) known as a potent vasodilator plays a prominent role in smooth muscle dilatory processes. The nitric oxide (NOx) limiting factors plays an important role in several diseases associated with vasoconstriction. The up-regulation of arginase activity in the smooth muscle of hypertensive subjects causes ample reduction in the availability NO releasing substrate (L-arginine) to eNOS. Thus, limiting efficiency of nitric oxide-mediated vasodilatory processes. Our present studies showed a significant increase in the arginase activity in the plasma and heart of CSA-induced hypertensive rats (Fig. 6). CAA and CHA treated groups showed a significant reduction in the activity of arginase. Also, the phenolic acids offer a protective effect against the impaired endothelium-dependent relaxation which could occur as

blood pressure lowering attributes of CAA and CHA on ACE activity [51]. In addition, it has been reported that acetylcholine (ACh) play a key role in the relaxation and vasodilation of smooth muscle [52,21]. Research finding has shown that administration of ACh infusion into the renal artery of an experimental rats increased cortical and papillary blood flow [53]. Also, ACh released from the cholinergic nerves contributes to the smooth muscle vasodilatory processes through nitric oxide synthase (NOS) mechanism [13]. Nevertheless, the vasodilatory effect of ACh in the artery and smooth muscle is halted by the activity of acetylcholinesterase and butyrylcholinesterase which hydrolyze ACh to acetate and choline [20]. Hence, reduced ACh bioavailability to muscarinic receptor bring about reduction in nitric oxide (NO) production, thereby promotes smooth muscle constriction. In the present study, the activity of acetylcholinesterase and butrylcholinesterase were significantly increased in the heart of CSA-induced hypertensive rats when matched with control (rats without cyclosporine administration) (Figs. 4 and 5). The reducing effect of CAA and CHA on the activity of 456

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acid than caffeic acid.

result of altered NO production. As earlier discussed, reduction in the activity of acetylcholineterase and butrylcholineterase activities ensure ample amount of acetylcholine for muscarinic receptors. While, inhibition of arginase activity by phenolic acids ensure abundant amount of L-arginine for eNOS for NO production, prevent inactivation of NO by super oxide anion radicals [54] and reduce the precursor polyamine and polyproline which responsible for vascular thickening and stiff ;ness [10,11,55] as well as exhibiting antihypertrophic effect by reducing availability of polyamines which are implicated in the cell growth/ proliferation [56,57]. Interestingly, there is considerable increase in the concentration of NO in the treated-hypertensive rats compared with untreated-cyclosporine induced hypertensive rats (Fig. 7). These observations could as a result of the accumulative inhibitory effects of caffeic acid and chlorogenic acid on cholinesterase as well as arginase activity in the treated-hypertensive rats. It should be on note that cyclosporine administration significantly impaired NO generation via inhibiting eNOS [58]. Cyclosporine has been linked to redox state imbalance, generation of free radicals which play prominent roles in the onset oxidative stress as well as cyclosporine induced toxicity [59,45,60]. Also, finding has shown that cyclosporine administration caused a significant elevation of MDA level alongside with over expression of HO-1 gene expression after treatment [44]. Interestingly, over expression HO-1 is a characteristic feature in deducing altered cellular redox state [61]. Finding from our present study revealed that administration of cyclosporine significantly increased the generation of oxidative biomarkers in the serum and heart of hypertensive rats. These effects were evidenced by an increased in the plasma and heart MDA as well as reduced in the activity of catalase and depreciated level of reduced glutathione in the hypertensive rats as shown in Figs. 8–10 respectively. These findings strongly agreed with previous studies on redox state imbalance observed during cyclosporine administration [62,63], suggesting that cyclosporine–induced hypertension is also associated with increased myocardial oxidative stress. As shown in Fig. 8(A & B), CAA and CHA administration caused a dose-dependent (10 and 15 mg/kg body) reduction in MDA level in the cyclosporine-induced hypertensive rats. This observation also confirms previous reports on the antioxidant properties of CAA and CHA via-a-vis prevention of ROS induced oxidative damage which could be monitored through MDA equivalent compounds as well as the activity of antioxidant enzymes and nonantioxidant enzymes [20,21]. Interestingly, the activity of catalase (kidney and lung) and the level of reduced glutathione (GSH) (kidney and heart) in the hypertensive rats as shown in Figs. 9 and 10 respectively. The released of toxic metabolites from cyclosporine metabolism might account for the depleting level of antioxidant indicators. Catalase and GSH which both acts as defense against oxidative stress in the biological system [64], helps in the management of oxidative stress. The observed lower heart and plasma level of catalase and GSH reveals that these antioxidant indicators have been used-up so as to minimize the level of circulating free radical generated. The antioxidant capacity of caffeic acid and chlorogenic acid play a protective role in stabilizing antioxidant and non-antioxidant enzymes in the treated group as revealed by a dose-dependent increase in the activity and the level of catalase and reduced glutathione (Figs. 9 and 10).

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