INTRODUCTION:

Mitochondria not only involved in the production of energy by oxidative phosphorylation it also plays an important role in the regulation of reactive oxygen species (ROS) induced oxidative stress in the heart.^[1] Superoxide (O^2-) and hydrogen peroxide (H₂O₂) as part of ROS are involved in regulating the cell growth and death of cardiac myocytes. Prolonged oxidative stress and impairment of energy production in the mitochondria leads to MI.^[2] Thus, mitochondria serve both as an energy source and target of ROS mediated injury in failing heart.

ISO induced oxidative stress is a known standard method and it is a synthetic catecholamine and β-adrenergic receptor. ISO causes Ca²⁺ overload in myocardium,^[3]mitochondrial disruption,^[4] alterations in mitochondrial respiration,^[5] depletion of adenosine tri phosphate (ATP) and inhibiting the activities of tricarboxylic acid (TCA) cycle enzymes.^[6]The involvements of free radicals in the pathophysiology of diseases are managed by supplementation with antioxidants.^[7]

Antioxidants may offer resistance by scavenging free radicals, inhibiting lipid peroxidation (LPO) and by other mechanisms to prevent diseases.^[8] The antioxidant activities are higher in medicinal plants and are relatively low cost with minimum side effects and hence these herbal drugs are prescribed widely.^[9] The world health organization (WHO) has expected that 80% of people in developing countries relies on traditional medicine, often plant drugs, for their most vital health care needs. Medicinal plants play a vital role in the discovery of novel drugs used in modern medicine.^[10]

Medicinal plants may contain a spacious range of free radical scavenging phytoconstituents such as alkaloids, phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, tannins, terpenoids (including carotenoids), etc. These phytochemicals and some other endogenous metabolites are rich in antioxidants. Researches in plants represent an invaluable source for discovering new substances as drugs which contain a large number of secondary metabolites.^[11] Z. armatum DC is a medicinal plant fit into the family of Rutaceace which is commonly known as Indian prickly ash, Nepal pepper (or) Toothache tree. It has many chemical constituents which were reported to have several biological activities.^[12,13] The genus Zanthoxylum is also reported to harbor several metabolites that show anti-proliferative activity.^[14] Z. armatum fruits are used as age-old herbal remedies. The present study was targeted on the ameliorative effect of Z. armatum fruit, on mitochondrial antioxidants against MI.

MATERIALS AND METHODS:

Collection of plant material and preparation of extract:

The plant Z. armatum fruit was collected from Kolli hills, India. The taxonomic identity of the plant was confirmed from the ABS Botanical Conservation, Research and Training centre, Salem, Tamilnadu, India. (Voucher Specimen No: AUT/ECP/101). Extract was prepared by the maceration procedure from dried fruits using 50% ethanol for 5 days. Extract was concentrated using rotary evaporator. Isoproterenol hydrochloride was purchased from Sigma Chemical Co., St. Louis, MO, USA. All the other chemicals and reagents used were of analytical grade.

Animals:

Male Wistar albino rats (Rattus norvegicus) weighing 100–120g were obtained from animal house of PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India. They were housed in polypropylene cages under a 12:12 hr light and dark cycle at around 37°C. The rats had free access to tap water and food. They were fed on a standard pellet diet (AVM Cattle and Poultry Feeds, Coimbatore) and water ad libitum. The clearance of the ethical committee for experimentation on animals was obtained before the start of the experiment (Proposal No: 158/PO/bc/99/CPCSEA). The experiment was carried out according to the guidelines of the Committee approved by the Animal Ethical Committee of PSG Institute of Medical Sciences and Research, Coimbatore.

Induction of MI:

ISO hydrochloride was used to induce MI in rats. Animals were injected subcutaneously with freshly prepared ISO hydrochloride in sterile normal saline at a dose of 20mg/100g body weight.

Experimental design:

Animals were divided into six groups of six rats in each group and the grouping of animal is shown in table 1.

Table 1: Treatment schedule

Groups	Diet/Treatment
GI- Normal control rats	Standard rat pellet for 30 days
GII - Z. armatum fruit treated rats	Hydroethanolic extract of Z. armatum fruit 400mg/kg body weight for 30 days (oral intragastric tube).
GIII – ISO treated rats	ISO (20mg/100g body weight) injected subcutaneously twice at an interval of 24hr on 28^th and 29^th day.
GIV- Rats pretreated with Z. armatum fruit (low concentration) + ISO	Hydroethanolic extract of Z. armatum fruit (200mg/kg body weight for 30 days) + ISO (20mg/100g body weight) subcutaneously twice at an interval of 24hr on 28^th and 29^th day.
GV- Rats pretreated with Z. armatum fruit (high concentration ) + ISO	Hydroethanolic extract of Z. armatum fruit (400mg/kg body weight for 30 days) + ISO (20mg/100g body weight) subcutaneously twice at an interval of 24hr on 28^th and 29^th day.
GVI - Rats pretreated with standard drug + ISO	Standard drug verapamil (1mg/ kg body weight for 30 days) + ISO subcutaneously twice at an interval of 24hr on 28^th and 29^th day.

At the end of the experimental period i.e., 12 hr after the second dose of ISO injection, all the rats were scarified by cervical dislocation under mild chloroform anesthesia. The heart tissue was excised immediately and thoroughly washed with ice-cold physiological saline and it was used for various biochemical estimations.

Separation of heart mitochondrial fraction:

Heart mitochondria were isolated by the method of Takasawa et al., (1984)^[15] with slight modifications. The heart tissue was put into ice cold medium containing 250 mM sucrose, 0.5 mM EDTA, 50mM Tris HCl (pH 7.4) and homogenized. The minced blood-free tissue was then resuspended in 20 mL of isolation medium containing 0.1% (w/v) defatted bovine serum albumin and transferred to a 50-mL glass homogenizer. The suspension was incubated for 1 min (4°C) and then rehomogenized. The homogenate was subjected to differential centrifugation at 4°C to isolate mitochondria. Mitochondrial fraction was finally resuspended in the same buffer (final concentration 0.2% v/v) in ice for 15 min. To determine lipid peroxidation (LPO), the mitochondrial pellet was dissolved in a buffer consisting of 175 mM KCl, 10 mM Tris (pH 7.4) so as to remove sucrose.

Estimation of thiobarbituric acid reactive substances (TBARS) in mitochondrial fraction:

The concentration of TBARS in the heart mitochondrial fraction was estimated by the method of Fraga et al., (1988).^[16] 0.1 ml of tissue homogenate was treated with 2 ml of TBA-TCA-HCl reagent (0.37% TBA, 0.25 M HCl and 15% TCA, 1:1:1 ratio) and placed for 15 min in a water bath and then cooled and centrifuged at 3500 ×g for 10 min at room temperature. The absorbance of clear supernatant was measured at 535 nm against a reference blank. The values were expressed as nmol of TBARS produced/ mg protein.

Estimation of lipid hydroperoxides in mitochondrial fraction:

The concentration of lipid hydroperoxides in the heart mitochondrial fraction was estimated by the method of Jiang et al., (1992).^[17] 1.8 ml of the Fox reagent was mixed with 0.2 ml of tissue homogenate and incubated for 30 min at room temperature, and the absorbance was measured at 560 nm in a UV-Visible spectrophotometer. Lipid hydroperoxides were expressed as nmol/100 mg of protein.

Assay of antioxidant enzymes in heart mitochondrial fraction Assay of SOD:

The activity of SOD in the mitochondrial fraction was assayed by the method of Kakkar et al., (1984).^[18] 0.5 ml of tissue homogenate was diluted to 1 ml with double distilled water. Then, 2.5 ml of ethanol and 1.5 ml of chloroform, both chilled, were added. This mixture was shaken at 4^oC and then centrifuged. To an appropriately diluted 0.2 ml of supernatant, 1.2 ml of sodium pyrophosphate buffer, 0.1 ml of phenazine methosulphate, 0.3 ml of nirtobluetetrazolium, 0.2 ml of nicotinamide adenine dinucleotide- reduced (NADH) and double distilled water in a total volume of 3 ml. The reaction was started by the addition of NADH. After incubation at 30^oC for 90 s, the reaction was stopped by the addition of 1ml of glacial acetic acid. The reaction mixture was stirred vigorously and shaken with 4 ml of n-butanol. The intensity of the chromogen in the butanol layer was measured at 560 nm against butanol blank in a UV- spectrophotometer. A system devoid of enzyme served as control. One unit is defined as the enzyme concentration required inhibiting the chromogen production by 50% in one minute at 560nm. The activity was expressed as Units/100 mg protein.

Assay of CAT:

The activity of catalase in the mitochondrial fraction was assayed by the method of Sinha, (1972).^[19] To 0.9 ml of phosphate buffer, 0.1 ml of tissue homogenate and 0.4 ml of hydrogen peroxide was added. After 60 s, 2 ml of dichromate acetic acid mixture was added. The tubes were kept in a boiling water bath for 10 min and the colour developed was read at 620 nm in a UV-Visible spectrophotometer. Standards in the range of 2-10 μmoles of H₂O₂ were taken and proceded as test with blank containing the reagent only. The activity was expressed as nmol of H₂O₂ consumed/min/mg protein.

Assay of GSH-Px:

The activity of GSH-Px in the mitochondrial fraction was assayed according to the method of Rotruck et al., (1973).^[20] To 0.2 ml of trisbuffer, 0.2 ml of EDTA, 0.1 ml of sodium azide and 0.5 ml of tissue homogenate were added. To this mixture, 0.2 ml of GSH followed by 0.1ml of hydrogen peroxide was added. The contents were mixed well and incubated at 37⁰C for 10 min along with a tube containing all the reagents except the sample. After 10 min, the reaction was arrested by the addition of 0.5 ml 10% TCA, centrifuged and the supernatant was estimated for GSH by the method of Ellman, (1959). The activity was expressed as μmol of GSH consumed/min/100 mg protein.

Assay of GST:

The activity of GST in the mitochondrial fraction was assayed by the method of Habig et al., (1974).^[21] The reaction mixture containing 1 ml of phosphate buffer, 0.1 ml of 1-Chloro-2,4-dinitrobenzene (CDNB) and 0.1 ml of the tissue homogenate was made up to 3 ml with double distilled water. The reaction mixture was pre-incubated at 37⁰C for 15 min. To this, 0.1 ml of reduced GSH was added, and the change in absorbance was measured at 340 nm for 3 min at 30’s interval in a UV Visible spectrophotometer. The activity was expressed as nmoles of CDNB conjugated/min/100 mg of protein.

Estimation of GSH:

The concentration of GSH in the mitochondrial fraction was estimated by the method of Ellman, (1959).^[22] A known weight of heart tissue was homogenized in phosphate buffer. From this, 0.5 ml was pipetted out and precipitated with 2 ml of 5% TCA. To 1 ml of the supernatant, 0.5 ml of Ellman’s reagent and 3 ml of phosphate buffer were added. The yellow colour developed was measured at 412 nm in a UV-Visible spectrophotometer. A series of standards was treated in a similar manner along with a blank containing 3.5 ml of phosphate buffer. The levels were expressed as nmol/100 mg of protein.

RESULTS:

Effect of hydroethanolic extract of Z. armatum fruit on Lipid peroxidation in heart mitochondria:

Free radical mediated lipid peroxidation and lipid hydroperoxides are related to the membrane integrity and permeability of mitochondria. The degree of mitochondrial membrane TBARS and lipid hydroperoxides of control, ISO induced MI rats (GIII) and hydroethanolic extract of Z. armatum fruit treated rats (GIV and GV) were shown in table-2. Mitochondria from MI heart rats (GIII) showed a significant increase in the levels of TBARS and lipid hyroperoxides compared to normal control rats (GI). Pretreatment with hydroethanolic extract of Z. armatum fruit (200 and 400 mg/kg) had a protective effect by reducing the level of lipid peroxidation in the mitochondrial fraction. There is no significant difference between standard drug treated rats (GVI) when compared to the normal rats (GI).

Table 2: Effect of hydroethanolic extract of Z. armatum fruit on Lipid peroxidation in heart mitochondria

Groups	TBARS (nmoles /mg protein)	Lipid hydroperoxides (nmol/100mg of protein)
I	4.74+ 0.21	6.94+ 0.31
II	4.01+ 0.19	6.29+ 0.17
III	8.74+0.42^a	12.2+0.26^a
IV	7.52+0.41^a,b,c	10.84+0.51^a,b,c
V	5.21+ 0.75^a,b	8.42+ 0.46^a,b
VI	5.79+ 0.61^a,b	8.12+ 0.13^a,b

Values are mean + SD of six samples in each group. a,b,c- significant at 5% level (p<0.05). Group comparison: a- GI vs GII, GIII, GIV, GV, GVI: b- GIII vs GIV, GV, GVI: c- GVI vs GIV, GV.

Effect of hydroethanolic extract of Z. armatum fruit on enzymic antioxidants in heart mitochondria:

Table – 3 shows rats treated with isoproterenol significantly decreased the activities of SOD, CAT and other endogenous antioxidant enzymes (GPx, GR, GST) in the heart mitochondria, when compared to normal control rats (GI). The level of GSH also reduced in the heart mitochondria. Oral pretreatment with hydroethanolic extract of Z. armatum fruit (200 and 400 mg/kg) to isoproterenol induced rats daily for a period of 30 days significantly increased the activities of antioxidant enzymes in the heart mitochondria, when compared with isoproterenol-induced untreated rats (GIII). There is no significant difference between control (GI) and plant alone treated rats (GII).

Values are mean + SD of six samples in each group. a,b,c- significant at 5% level (p<0.05). Group comparison: a- GI vs GII, GIII, GIV, GV, GVI: b- GIII vs GIV, GV, GVI: c- GVI vs GIV, GV.

1 unit (U) of SOD = amount of enzyme causing 50% inhibition of NBT reduction /min/mg/protein.

DISCUSSION:

Mitochondria are involved in the production and regulation of cellular bioenergetics supply in the form of ATP and electron transport. During the past two decades, it has become clear that other than energy generation, mitochondria produces reactive oxygen species (ROS) as side products of respiration. Excessive production of ROS in mitochondria as a result of damage to electron transport complexes that leads to ischemic myocardium in the heart.^[23] ISO induced oxidative stress is a well known standard method and it is a synthetic catecholamine and β-adrenergic receptor. Acute β-adrenergic receptor stimulation rapidly generates ROS.

Lipid peroxidation has been proposed to be a major mechanism of ROS attack. Lipid peroxidation or reaction of oxygen with unsaturated lipids produces a wide variety of oxidation products. The main primary product of lipid peroxidation is lipid hydroperoxides (LOOH). Generation of lipid peroxides during ischaemia could damage the mitochondrial membrane and affect its function. Increased levels of TBARS and lipid hydroperoxides indicated the increased lipid peroxidation in the mitochondria of the heart in the myocardial infarcted rats. Thus, accelerated lipid peroxidation damages both the structure and the function of the heart mitochondria in ISO treated rats.

Pretreatment with hydroethanolic extract of Z. armatum fruit significantly decreased the levels of TBARS and lipid hydroperoxides in the mitochondria of isoproterenol-induced rat heart. Hydroethanolic extract of Z. armatum fruit contain secondary metabolites which protect lipids against oxidative damage. Previous results also confirmed the anti-lipid peroxidative effect of hydroethanolic extract of plants against isoproterenol-induced mitochondrial damage in myocardium.^[23,24] Furthermore, in the present study ISO depresses total cellular antioxidant capacity by down regulating activities of SOD, CAT, GPx, GST and reduced glutathione, leading to the loss of membrane integrity, inducing heart contractile dysfunction myocyte toxicity and finally producing myocardial necrosis. This observation is concerned with the earlier findings that decreased activities of these enzymes by ROS may effect the heart mitochondria substrate oxidation resulting in reduced oxidation of substrate, reduced rate of reducing equivalents to molecular oxygen and depletion of cellular energy.^[25] The extent of recovery of the myocardium can be improved if mitochondrial damage is prevented or reduced by the supplement with medicinal plants.

Mitochondrial localization of catalase has been considered to be a peroxisomal marker enzyme and has been used as an indicator of peroxisomal proliferation.^[26] GSH is an important antioxidant that protects the mitochondrial membrane in response to the oxidative stress from lipid peroxidation.^[27] It can act as the electron donor for GPx in animal cells, and also directly reacts with ROS. In addition, oxidized product of GSH can be reduced by the NADPH-dependent reaction catalyzed by glutathione reductase.^[28] GPx is the most important peroxidase for H₂O₂ removal in mammals. In mitochondria, the decreased GSH level and increased oxidized GSH are the reasons for inactivated GSH-dependent enzymes, such as GPx and GST in isoproterenol-induced myocardial infarcted rats. Thus, maintaining intracellular GSH content in mitochondria can protect cardiomyocytes from cellular damage.

Further, it is present in relatively large amounts within the cytosolic and mitochondrial compartments of heart. GST is a phase II detoxification enzyme that catalyzes the conjugation of reduced GSH with an array of xenobiotic and endogenous electrophiles.^[29,30] Pretreatment with Z. armatum fruit significantly increased the activities of SOD, CAT, GPx, GST and reduced glutathione in the mitochondrial fraction of ISO induced rats. Z. armatum may act as an antioxidant by scavenging ROS and also improving the endogenous antioxidant system in ISO induced rats.

The present study results suggest that the increased generation of ROS due to ISO was associated with mitochondrial damage and dysfunction in the heart, which were characterized by an increased lipid peroxidation in the mitochondria, decreased oxidative capacity attributable to low enzyme activities. Z. armatum fruit protected rat heart mitochondria against ISO induced oxidative stress may be due to its endogenous antioxidant and scavenging of free radical properties.

ACKNOWLEDGMENTS:

The authors gratefully thank the Department of Biochemistry, PSG College of Arts & Science, Coimbatore for their support.

CONFLICT OF INTEREST:

There is no conflict of interests.

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Received on 18.08.2017 Modified on 11.09.2017

Research J. Pharm. and Tech 2018; 11(2):681-686.

DOI: 10.5958/0974-360X.2018.00128.2

Groups	SOD (U/100mg of protein)	CAT(nmoles Of H₂O₂consumed/min/mg protein)	GPx(µmoles Of GSH consumed/min/ 100mg protein)	GST (nmoles of CDNB conjugated/ min/100mg protein)	Glutathione (nmole/100mg protein)
GI	13.24+ 0.11	14.71+0.21	2.96+ 0.3	49.94+ 4.31	5.71+0.12
GII	12.96+ 0.12	13.92+0.31	2.61+ 0.28	37.71+ 3.03	5.95+0.23
GIII	5.17+0.30^a	6.72+0.07^a	1.32+0.17^a	11.21+1.06^a	2.79+0.17^a
GIV	9.72+0.03^a,b,c	9.08+0.71^a,b,c	1.95+0.91^a,b,c	24.48+4.02^a,b,c	4.11+0.72^a,b,c
GV	11.9+0.31^a,b	11.91+0.92^a,b	2.74+ 0.05^a,b	30.26+2.09^a,b,c	5.17+0.02^a,b
GVI	11.1+ 0.01^a,b	12.12+ 0.48^a,b	2.41+0.02^a,b	36.21+1.73^a,b	4.91+ 0.18^a,b