Influence of Momordica charantia on oxidative stress-induced perturbations in brain monoamines and plasma corticosterone in albino rats.

OBJECTIVES: The objective of this study was to evaluate the antistress activity of Momordica charantia (MC) fruit extract on stress-induced changes in albino rats and also to explore attenuating effects of MC on in vitro lipid peroxidation in rat brain.
MATERIALS AND METHODS: In this study, Wistar albino rats (180-200 g) were used. Plasma corticosterone and monoamines-5-hydroxy tryptamine (5-HT), norepinephrine (NE), epinephrine (E) and dopamine (DA) in cortex, hypothalamus and hippocampus regions of brain were determined in animals under different stressful conditions. Ethanolic fruit extract of MC, at doses of 200 and 400 mg/kg, was used. The oxidative stress paradigms used in in vivo models were acute stress (AS) and chronic unpredictable stress (CUS). Panax quinquefolium (PQ) was used as a standard in in vivo models and ascorbic acid was used as a reference standard in the in vitro method.
RESULTS: Subjecting the animals to AS (immobilization for 150 min once only) resulted in significant elevation of plasma corticosterone levels and brain monoamine levels. Pretreatment with MC at doses of 200 and 400 mg/kg p.o. significantly countered AS-induced changes and a similar effect was exhibited by PQ at 100 mg/kg p.o. In the CUS regimen (different stressors for 7 days), plasma corticosterone levels were significantly elevated whereas the levels of 5-HT, NE, E, and DA were depleted significantly. Pretreatment with MC (200 and 400 mg/kg) attenuated the CUS-induced changes in the levels of above monoamines in cortex, hypothalamus, and hippocampus regions of brain and plasma corticosterone in a dose-dependent manner. Furthermore, MC extract (1000-5000 μg/mL) exhibited a significant quenching effect on in vitro lipid peroxidation indicating its strong antioxidant activity which was compared with ascorbic acid.
CONCLUSIONS: This study reveals the antistress activity of MC as it significantly reverted the stress-induced changes, and the activity might be attributed to its antioxidant activity since stress is known to involve several oxidative mechanisms.

Introduction

Several stressful conditions are responsible for the etiopathogensis of various psychotic disorders in which the functional identity of neurotransmitters is challenged. Out of various neurotransmitters—noradrenaline (NA), dopamine (DA), and 5-hydroxy tryptamine (5-HT) are the important monoamines which are widely distributed in brain, and their functional role is well established during stressful conditions.[1] The hypothalamic–pituitary–adrenal (HPA) axis is activated during stressful conditions. Chronic stressful conditions lead to consistent hyperactivity of the HPA axis which in turn lead to increased secretion of adrenocorticotrophic hormone (ACTH) from adenohypophysis[2] and are known to adversely affect the normal psychosomatic homeostasis. Various biological amines such as catecholamines, histamine, and 5-HT have been implicated in the regulation of the HPA-axis.[3] Their effects on ACTH are predominantly indirect, exerted via activation of hypothalamic corticotrophin releasing hormone (CRH) neurons.[4]

Many herbs reported in the ancient literature have potent antistress activity and their utilities in the current scenario need to be unveiled. The drugs of plant origin are gaining increasing popularity and are being investigated for remedies of a number of disorders including adaptogenic (antistress) activity.[5]

Momordica charantia Linn. (MC), a climber belonging to family Cucurbitaceae, is commonly known as bitter gourd or bitter melon in English and karela in Hindi. It is cultivated throughout the world for use as vegetable as well as medicine. Its popular medicinal uses focused research so ever and in the last few decades several hundred studies that have been carried with MC, using modern tools, credit MC with anti-diabetic, antiviral, anti-tumor, antibacterial, antioxidant, antiulcer, anti-inflammatory, hypocholesterolemic, hypotriglyceridemic, hypotensive, immunostimulant, and insecticidal properties.[6] However, antistress-activity of MC has not been explored earlier apart from its use as traditional remedy for so many physiological and pathological disorders. Hence, this study has been undertaken to investigate the effect of ethanolic extract of MC on altered HPA axis activity and levels of monoamines such as 5-HT, NE, E, and DA in cortex, hypothalamus and hippocampus regions of brain and plasma corticosterone during stressful conditions. A standard adaptogenic plant, Panax quinquefolium (PQ), commonly known as American ginseng has been used as a reference standard. Furthermore, the effect of MC on in vitro lipid peroxidation was also studied to correlate this effect with its in vivo benefits. In this method ascorbic acid (AA), a standard antioxidant was used as a reference standard.

Materials and Methods

Chemicals

5-HT, NE, E, DA and corticosterone and crude root powder of PQ (M/S Sigma Chemicals, USA), butylated hydroxy toluene (BHT) (Ranbaxy Fine chemicals, Mumbai), ascorbic acid (Loba Chemicals, Mumbai), and other chemicals and solvents of HPLC grade/analytical grade were used.

Preparation of Momordica charantia Fruit Extract

Fresh unripe fruits of MC (1 kg) were shade dried, seeds were discarded and the dried pulp was made into a coarse powder, then it was extracted with 95% ethanol by Soxhlet extraction for 8 h. The extract so obtained was evaporated in a Rota flash evaporator under reduced pressure. The final yield was 7.4%.

Animals

Adult Wistar albino rats of either sex (180–200 g), fed with standard pellet diet and water ad libitum were used. They were kept under a controlled light/dark cycle and temperature (25 ± 3° C). This experiment was complied with the guidelines for animal experimentation of our laboratory and was approved by Institutional Animal Ethics Committee (IAEC Reg. No. 926/ab/06 CPCSEA-2006, approval No. 09/2009-10).

Stress Procedures

Rats were divided into the non-stress group (NS), acute stress (AS) and chronic unpredictable stress (CUS) groups, and drug-treated groups for both AS and CUS. Each group consists of 6 rats. The NS group received vehicle (distilled water) only. In AS, different groups of animals were administered with extracts of MC (200 mg and 400 mg/kg, p.o.) or PQ (100 mg/kg, p.o.) daily for 3 days. On the third day, except the NS group, rats were subjected to AS (immobilization for 150 min. once only) and killed immediately. In CUS, vehicle/drugs were fed daily 1 h prior to exposure to stress regimen up to seven consecutive days. The CUS regimen involves subjecting the animals to two different stressors of variable intensity on every day in an unpredictable manner for 7 days as described earlier.[7] Various stressors include immobilization stress (150 min), forced swimming (20 min) overnight soiled cage bedding, foot shock (2 mA for 20 min) day–night reversal and fasting (12 h).

Estimation of Plasma Corticosterone

Immediately after the last stress regimen, blood was collected in EDTA-coated tubes kept in ice and centrifuged at 1000 × g for 20 min. at 4 °C. Plasma was separated and aliquots were stored at –70 °C for corticosterone estimation. An High Performance Liquid Chromatography (HPLC)/Ultraviolet (UV) system was used for quantification of plasma corticosterone according to Woodward and Emery[8] using dexamethasone as an internal standard. Briefly, 500 μL of plasma containing known quantity of dexamethasone was extracted with 5 mL of dichloromethane (DCM). The DCM extract was evaporated to dryness and dissolved in 100 μL of the mobile phase. Twenty microliters of the extract was injected into a HPLC system for quantification. The mobile phase consisted of methanol:water (70:30) at a flow rate of 1.2 mL/min and corticosterone was detected at 250 nm using an UV detector. A gradient HPLC (Shimadzu) with a LC-10AT pump, UV detector (Shimadzu), and RP-C18 column (250 mm × 4.6 mm ID; Gemini 5 μm 110A) was used.

Estimation of Monoamines

The various biogenic amines in rat brain were estimated by the method of Wagner et al.[9] The levels of 5-HT, NE, E, and DA were measured by using HPLC coupled with an electrochemical detector (ECD). The rats in all the groups were killed by perfusion of heart with ice-cold normal saline under light anesthesia. After killing, the brain was rapidly removed, and the cerebral cortex, hypothalamus, and hippocampus were dissected on an ice-cold plate.

The isolated brain regions were homogenized with 0.17 M perchloric acid by a Teflon homogenizer. Dihydroxy benzylamine (DHBA) was the internal standard. Twenty microliters of the sample were injected into the HPLC system (Shimadzu) which is connected to an isocratic pump (model: LC-10AT, Shimadzu) and reverse phase column (Lichrospher RP C-18, Shimadzu) for separation of biological amines. The reaction products were detected with an electrochemical detector (ICS-3000, DIONEX) which was coupled to the HPLC system and set at a potential of +0.60 V. The mobile phase contains citric acid, disodium hydrogen orthophosphate, EDTA, octane-1-sulfonic acid, sodium salt, and 14% methanol (pH –4.0) at a flow rate of 0.8 mL/min. The concentration of neurotransmitters was expressed as nanograms/gram wet weight of brain tissue.

Statistical Analysis

The values of above parameters were expressed as mean ± SEM. The statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnette's multiple comparison tests. P < 0.05 was considered to be statistically significant.

In Vitro Lipid Peroxidation

Randomly selected rats were fasted overnight. After killing, the whole brain except cerebellum was removed quickly. It was further processed to get 10% homogenate in 0.15 M KCl,[10] using a Teflon homogenizer. Reaction mixture (0.5 ml) was prepared by adding 0.1 ml of MC extract (1000–5000 μg/ml) or ascorbic acid (1000–5000 μg/ml), 0.1 ml of brain homogenate, 0.1 ml of 0.15 M KCl, 0.1 ml of 0.06 mM ascorbic acid, and 0.1 ml of 15 mM FeSO4. This reaction mixture was incubated at 37 °C for 30 min. An equal volume of TBA:TCA [1:1, 1 ml] was added to the above reaction mixture followed by addition of 1 mL of BHT. This final mixture was heated on a water bath for 20 min at 80 °C and cooled, centrifuged and absorbance read at 532 nm[11] using a spectrophotometer [Shimadzu 160 IPC]. The extent of lipid peroxidation in rat brain homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS). The percentage inhibition of lipid peroxidation was calculated as per the formula:

The concentration of MC or AA for 50% inhibition of lipid peroxidation (IC50 value) was found by linear regression analysis.

Results

Effect of Momordica charantia on Acute Stress-Induced Changes in Brain Monoamines

In acute stress, the levels of 5-HT, NE, E and DA in cortex, hypothalamus and hippocampus were significantly elevated in comparison to the NS group [Figure 1]. Pretreatment with MC at doses 200 mg/kg and 400 mg/kg significantly decreased (P < 0.01) the AS-induced elevation in levels of 5-HT, NE, E, and DA in a dose-dependent manner. Similarly pretreatment with PQ (100 mg/kg) also significantly decreased the elevated levels of 5-HT, NE, E, and DA induced by AS.

Figure 1

Bar diagram representing the changes in the levels of brain monoamines in AS. Results were expressed as mean ± SEM, n=6. P < 0.01 when AS group compared with NS control group, P < 0.01 when the treated groups compared with AS group

Effect of Momordica charantia on Chronic Unpredictable Stress Induced Changes in Brain Monoamines

In contrast to AS, continuous stress exposure for 7 days led to significant depletion in the levels of 5-HT, NE, E, and DA in all the regions, i.e. cortex, hippocampus and hypothalamus, when compared to the NS group. Pretreatment with MC and PQ significantly countered the CUS-induced changes resulting in significant elevation in levels of above monoamines in a dose-dependent manner (P < 0.01) [Figure 2].

Figure 2

Bar diagram representing the changes in the levels of brain monoamines in CUS. Results were expressed as mean ± SEM, n=6. P < 0.01 when CUS group compared with NS control group, P < 0.01 when the treated groups compared with CUS group

Effect of Momordica charantia on Plasma Corticosterone Levels

In the acute stress and chronic unpredictable stress regimen, there was significant elevation in plasma corticosterone levels. Pretreatment with the ethanolic extract of M. charantia (200 mg and 400 mg/kg body wt.) significantly reduced (P < 0.01) both AS- and CUS-induced elevation of plasma corticosterone levels in a dose-dependent manner. Similarly, PQ (100 mg/kg body wt.) which was used as a reference standard also significantly countered the elevation of plasma corticosterone levels induced by AS and CUS [Figure 3].

Figure 3

Bar diagram representing the changes in the levels of plasma corticosterone in AS & CUS. Results were expressed as mean ± SEM, n=6. P < 0.01 when AS & CUS groups compared with NS control group, P < 0.01 when the treated groups compared with AS & CUS groups

Effect of Momordica charantia on Lipid Peroxidation

Different concentrations of the MC fruit extract ranging from 1000 to 5000 μg/mL showed better activity in quenching lipid peroxides with an IC50 value of 3444 μg/mL. Similarly ascorbic acid, which was used as a reference standard at concentrations of 1000–5000 μg/mL, also showed a significant inhibition of lipid peroxides with an IC50 value of 4502 μg/mL [Figure 4].

Figure 4

Graph representing the effect of MC and AA on lipid peroxidation in rat brain (mean ± SEM). (IC50 value of MC – 3,444 μg/ mL, IC50 value of AA – 4,502 μg/mL)

Discussion

Stress is an unavoidable phenomenon that affects the body system at various levels. Acute stressful conditions activate the monoaminergic system leading to an increase in the extracellular levels of NE, E, DA, and 5-HT in cortex and hippocampus regions of brain.[1213] Serotonin is a potent stimulator of ACTH secretion and has been found to activate the release of CRH from hypothalamus.[414] In some earlier reports, serotonergic pathways were not involved in swim-stress induced, hypoglycemia-induced and foot shock-induced corticosterone secretion.[1516] However, serotonergic neurons in the hypothalamus are activated in response to restraint stress (immobilization stress) and endotoxin exposure and 5HT1A, 5HT2A and 5HT2C receptors are involved in the mediation of the restraint and ether stress-induced release of ACTH.[17] Above literature reports support the hypothesis that the effect of different forms of stress on the HPA axis is mediated through different neurotransmitters system.[2] ACTH from anterior pituitary acts on adrenal cortex and makes it to release corticosteroids (corticosterone in rodents and cortisol in humans).

In our study, the brain levels of 5-HT, DA, NE, and E were elevated along with plasma corticosterone levels in AS indicating the activation of the HPA axis. From our findings, it can be confirmed that not only 5-HT, elevated levels of NE, and E, DA might also be responsible for the activation of the HPA axis and their possible regulatory role in the release of ACTH which was consistent with the earlier reports. Both the doses of MC were effective in normalizing the AS elevated levels of 5-HT, DA, NE, and E in the cortex, hypothalamus and hippocampus regions. We also observed that pretreatment with PQ significantly reverted back the AS-induced changes in the levels of 5HT, DA, NE, and E in different regions of brain.

Stress effects depend on the type and duration of stressor. In contrast to AS, overload by stressors of unpredictable schedule leads to disruption of the homeostatic conditions prominently. CUS is considered to be more clinically relevant model to observe stress manifestations. Severe stressful conditions decrease monoamine levels which are mainly due to increased stress sensitization and their preferential and higher utilization during severe stressful conditions.[18] In our study, CUS significantly decreased monoamines in all the brain regions. MC at doses of 200 mg and 400 mg/kg was effective in normalizing 5-HT, DA, NE, and E in the cortex, hypothalamus and hippocampus regions. Similar effects were observed by pretreatment with PQ in the CUS model.

Plasma corticosterone level, being an immediate stress effector, is considered to be an important marker to evaluate stress response. The elevated plasma corticosterone under stressful conditions is necessary to maintain the energy balance which includes increased plasma glucose, triglyceride, and creatine kinase levels.[7] In this study, AS and CUS exposure resulted in elevated plasma corticosterone and the elevation was more in AS than in CUS. The increase during AS exposure is due to the novelty of the stressor, whereas the decrease in its response during CUS is due to the excessive usage of stored glucocorticoids for the demand and slow operation of compensatory mechanisms such as increased ACTH secretion and adrenal hypertrophy.[7] Pretreatment with both the doses of MC, 200 mg and 400 mg/kg, and PQ 100 mg/kg, effectively reduced the AS- and CUS-induced elevation in the levels of plasma corticosterone.

Oxidative stress has been implicated in the pathology of many diseases and antioxidants may offer resistance against oxidative stress by scavenging free radicals, inhibiting the lipid peroxidation, and by many other mechanisms and thus prevent disease.[19] In this study, MC extract has shown strong antioxidant activity by attenuating the lipid peroxidation in rat brain suggesting a decrease in oxidative damage (IC50 value =–3444 μg). Preincubation of rat brain homogenate with the extract showed significant inhibition of lipid peroxidation at all the concentrations in a dose-dependent manner indicating that the extract protects the brain from Fe2+/ascorbate system-induced lipid peroxidation. The standard antioxidant, ascorbic acid (AA), also exhibited the similar effect.

Previous phytochemical investigations revealed the presence of glycosides, saponins, triterpines, steroids, vitamin C and A, phytochemicals such as momorchins, momordinol, momordicins, charantin, cucurbitacins, diosgenin, goyaglycosides, goyasaponins, etc.,[20] in Momordica charantia, and the observed antioxidant activity of MC might be due to the presence of above antioxidant constituents.

In earlier reports, it was mentioned that oxidative lipid damage, termed lipid peroxidation, produces a progressive loss of cell membrane fluidity and increases membrane permeability of ions like Ca2+.[21] The principal mechanism of neurotransmitter release in both peripheral and central nervous system and also in many hormone-secreting cells is exocytosis where by the neurons release neurotransmitters in response to an increase in the intracellular calcium concentration.[22] Stress causes sympathetic stimulation (direct arteriolar vasoconstriction) leading to local ‘hypoxia’ or ‘ischemia’ in vital organs.[23] It is known that ischemia also causes depolarization of neurons and the release of large amount of glutamate and in ischemic brain damage, the loss of neurons following interruption of blood supply to the brain is not simply due to the cells dying for lack of oxygen, a complex cascade of events takes place including production of free radicals.[24] Earlier there was no evidence regarding the reason for the excessive release of brain monoamines during stressful conditions. In this study, basing on the earlier reports cited above, it was hypothesized that the excessive release of 5-HT, DA, NE, and E might be due to partial depolarization or increased Ca2+ permeability of 5-HTergic, DAergic, NEergic neurons in brain resulting from local ischemia during stressful conditions. Therefore, excessive generation of free radicals and lipid peroxidation might be the underlying mechanism for the stress-related changes in brain monoamines which in turn increase the levels of corticosteroids through HPA axis activation.

Pretreatment with MC extract significantly attenuated all the stress-induced changes, i.e. change in brain monoamines and plasma corticosterone confirming its antistress activity and this activity might be attributed to its strong antioxidant activity. Literature reports in which Sen et al.[25] suggested that the presence of a variety of compounds in the plants and their antioxidant activity might be responsible for their antistress activity, lending support to our hypothesis.

This study provides evidence for the effectiveness of MC against different models oxidative stress with diverse etiologies and its regular use as vegetable by humans is beneficial and scientific. Our investigation will help in rationalizing the Ayurvedic use of the MC, particularly in conditions where oxidative stress is likely to be involved as part of the disease etiopathogenesis.

Conclusions

This present study reveals the antistress activity of MC as it significantly reverted back the stress-induced changes and the extract also exhibited attenuating effect on lipid peroxidation in brain indicating its protective effect against oxidative stress. The antistress activity of MC might be attributed to its antioxidant activity since stress is known to involve several oxidative mechanisms.