MECHANISMS INVOLVED IN MEMORY PROMOTING EFFECT OF JOBELYN IN MICE

EXPOSED TO UNPREDICTABLE CHRONIC MILD STRESS

Solomon UMUKORO

DEPARTMENT OF PHARMACOLOGY AND THERAPEUTICSUNIVERSITY OF IBADAN

IBADAN, NIGERIA

4th International Conference on Translational MedicineOctober 26-28, 2015

Baltimore, USA

Phases of Stress Responses

Stress is an integral component of human life and is considered as any condition that leads to perturbation of the body’s homeostasis (Zhu et al., 2014).

During extreme stress situations, the homeostatic mechanisms of the organism become deficient and the survival of the organism is thereby threatened (Henckens et al., 2009).

Response to stress includes 3 phases: Alarm phase, Phase of Adaptation and Phase of Exhaustion

When stress is sustained over a long period, breakdown in adaptation occurs and this leads to ill health and various pathological conditions (Panossian and Wikman, 2010; Ron de Kloe et al., 2005).

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The Effect of Stress on The Body

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Effects of Chronic Stress on the Brain Produces free radicals that kill brain cells: Cortisol creates a surplus of free radicals — reacting

oxygen species that punch holes in the brain cell walls, making them to rupture and die. Induces a vicious cycle of fear and anxiety: Activate the brain’s “fear center”- amygdala causing a

vicious cycle of even more fear and stress.  It halts the production of new brain cells: Cortisol halts the production of Brain-derived neurotrophic

factor (BDNF)  resulting in  formation of fewer new brain cells. BDNF has been described as the fertilizer” for the brain. Its low levels are associated with depression and Alzheimer’s disease

Depletes critical brain chemicals such as monoamines : Causing depression and propensity to addictions.

Makes one stupid causing the brain to seize up at the worst possible times — exams, job interviews, and public speaking . It impairs memory and making decisions.

Shrinks brain and increases the risk of dementia and Alzheimer’s: Cortisol kills, shrinks and stop the generation of new neurons in the hippocampus, part of the brain that stores memories.

Allows toxins into the brain: by disrupting blood-brain barrier ; brain’s gatekeeper and entering of harmful substances- brain cancer, brain infections etc

Causes brain cells to commit suicide: leading to premature aging on a cellular level, causing cells in both body and brain cells to die prematurely.

Destroys our happiness and peace of mind: It saps the victim of the joy of life leaving the individual with excessive worry , fear, anger , frustration, insomnia, addiction, suicidal thoughts etc

Stress-induced Memory Impairment

Chronic stress

Over secretion of cortisol by

adrenal gland

Excess influx of calcium into brain

cells

Over production of free radicals that

initiates the cascade for neuronal degeneration

Memory dysfunctions

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Phytomedicines: New Hope For Stress Management?

Although stress has been known to have deleterious effects on the body for over a century, drugs which could ameliorate its pathological impacts are yet to be discovered.

The conventional use of stimulants and anabolic steroids for stress management are ineffective in combating the multiple effects of stressors and are also associated with serious adverse effects (Samuelsson and Bohlin, 2009).

However, in contrast to these conventional agents, adaptogens, which are compounds of plant origin, are devoid of such adverse effects (Samuelsson and Bohlin, 2009).

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Desirable Qualities of Adaptogens

Possess multipronged mechanisms of action and act nonspecifically to provide resistance against stressful conditions by regulating the various key elements involved in the stress response system (Panossian and Wikman, 2009).

Boost energy and resilience in the face of stress; promote physical and mental performance Improve defense mechanisms of the body, and enhance longevity (Samuelsson and Bohlin, 2009).

Should have little or no systemic toxicity

A number of medicinal plants such as Panax ginseng, Ginkgo biloba, Ocimum sanctum, Withania somnifera have been shown to possess adaptogenic property and some of them are available commercially for stress management (Panossian and Wikman, 2009).

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Description of Jobelyn (JB)

JB is a food supplement obtained from a potent antioxidant plant Sorghum bicolor - (millet or guinea corn).

S. bicolor has been used for many centuries for treatment of various diseases in Nigerian traditional medicine. It is also given to babies with the belief that it enhances memory functions by the natives.

Clinical indications of JB: JB is formulated as capsules (2-3 capsules daily) and syrups for anemia and joint pains (Erah, 2003) Used by the natives for neurological disorders (Shikoya et al., 2008) Immune booster and energizer Used in a vitamin-like fashion to promote general well beings and to relieve stress (Erah, 2003).

Moreover, no adverse effects have been recorded since the use of JB over the years. Preclinical studies showed that JB was well tolerated by the animals.

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Phytochemicals in Jobelyn

JB is rich in several phytochemicals like polyphenols, apigeninidins, luteolinidins, narigenins, apigenins, luteolins, dimeric 3-deoxyanthocyanidin (Mendes and Carlini 2007; Burdette 2010).

Studies have that these compounds possessed wide range of biological activities including neuroprotection, anti-neuroinflammation, antioxidation and membrane stabilization (Benson et al., 2013; Burdette 2010) suggesting a protective effect against stress-induced memory impairment.

Thus, this study was designed to evaluate the adaptogenic-like property of Jobelyn in mice exposed to unpredictable chronic mild stress (UCMS), an animal model that closely mimic the ways humans encounter stressors on daily basis.

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Materials and Methods

Laboratory animals: Male Swiss mice (20-22 g) obtained from the Central Animal House, University of Ibadan were used

in the study. They were housed in plastic cages at room temperature and fed with rodent pellet diet and water ad

libitum. The animals were handled in accordance with the National institutes of Health (NIH) Guide for the

care and use of Laboratory Animals.

Preparation of JB: Jobelyn-JB (Health Forever Products Ltd, Lagos, Nigeria), donepezil, -DP (Pfizer, USA) and

immipramine, IM (Sigma, Germany). The drugs were dissolved in distilled water and were administered orally per body weight. The control group was treated with distilled water.

The doses of JB used in this study were selected based on results obtained from preliminary investigations.

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Experimental Procedures-I Unpredictable chronic mild stress (UCMS) paradigm: The UCMS protocol as described by Yalcin et al., (2005).

Male Swiss mice (n = 6) were treated orally with JB (5, 10, 25, 50 mg/kg), DP (1 mg/kg) or IM (1 mg/kg) 30 min before exposure to two of the following stressors twice: damp sawdust (3 hours), forced swim test (5 min), hypoxia (15 min), social defeat (30 s), no beddings in cage (overnight), starvation (overnight), tail suspension (6 min), predator (30 s), water deprivation (overnight) and noise (30 min).

This procedure was repeated daily for 14 days before testing for memory.

Non-stress control group received distilled water (10 ml/kg) and not subjected to UCMS, whereas, the stress control group also received distilled water (10 ml/kg) but were subjected to UCMS.

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Experimental Procedures-2 Test for memory: Y-maze paradigm as previously described (Yan et al., 2001). Briefly, mice were placed individually at the centre of the Y-maze and allowed to explore all

the three arms freely for 5 min.

The number and sequence of arm entries were recorded and the apparatus was cleaned after each test.

Estimation of blood glucose and serum corticosterone levels: After behavioral testing, blood was obtained for estimation of blood glucose and serum

corticosterone levels using a commercial glucometer tape (Accu-Chek Actives, Roche Diagnostics India Pvt Ltd) and ELISA kits (Oxford Biomedical Research, USA) according to the manufacturer’s instructions.

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Experimental Procedures-3

Determination of brain concentrations of malondialdehyde (MDA) and glutathione (GSH):

The brain concentrations of malondialdehyde (MDA) and glutathione (GSH), the major biomarkers of oxidative stress were estimated using spectrophotometer as previously described by Okhawa et al., (1979) and Moron et al., (1979) respectively.

Estimation of Acetylcholinesterase (AChE) Activity: The brain concentration of AChE activity, a major marker for cholinergic neurotransmission

was assessed using spectrophotometer (Ellman et al., (1961).

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Experimental Procedures-4

Brain histology and determination of neuronal density: Half of the mice brain tissues were fixed for 7 days in 10% formaldehyde. They were then taken through the processes of dehydration, clearing, infiltration, embedding,

sectioning and staining with H&E stain. Slides of the hippocampus were made and viewed using light microscope for any histological

changes. The population of surviving neuronal cells were then counted for neuronal density in the

hippocampal region.

Statistical analysis: The data were analyzed using Graph Pad Prism software version 5.00. All values were expressed as mean ± S.E.M. Statistical analysis of data was done using One-

way ANOVA, followed by Newman-Keuls post-hoc test.

P-values less than 0.05 (p < 0.05) were considered statistically significant.

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Summary of Results

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Jobelyn Reverses Unpredictable Chronic Mild Stress-Induced Memory Impairment In Mice

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Fig 1: Effect of Jobelyn on unpredictable chronic mild stress (UCMS)-induced memory impairment in mice Values represent the mean ± S.E.M for 6 animals per group. **p < 0.05 compared to non stress control group; *p < 0.05 compared with stressed group (ANOVA followed by Newman Keuls test).

Jobelyn Reduces Oxidative Stress in the Brain of mice Exposed to UCMS

Treatment Dose(mg/kg)

MDA(µmol/g tissue)

GSH(µmol/g tissue)

Non stress control - 20.68 ± 2.86 30.18 ± 1.61Stress control - 32.82 ± 2.76** 22.33 ± 1.35**

JB 5 20.86 ± 2.52* 33.68 ± 1.51*

JB 10 18.35 ± 1.87* 40.73 ± 2.91*

JB 25 16.52 ± 1.95* 46.52 ± 2.01*

JB 50 23.96 ± 2.65* 38.22 ± 2.14*

DP 1 28.75 ± 1.53 26.85 ± 2.27IM 1 22.99 ± 1.87* 35.67 ± 2.32*

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Values represent the mean ± S.E.M for 6 animals per group. **p < 0.05 compared with non-stress control group; *p < 0.05 compared with stressed group (ANOVA followed by Newman Keuls test).

Table 1:

Jobelyn Decreases Brain Acetylcholinesterase (AChE) Activity in UCMS-mice

Treatment Dose(mg/kg)

AChE activityµmol/min/g tissue

Non stress control - 354.000 ± 8.27Stress control - 388.20 ± 8.45**

JB 5 337.80 ± 12.52*

JB 10 328.70 ± 7.45*

JB 25 346.80 ± 9.93*

JB 50 375.20 ± 10.28DP 1 344.50 ± 12.33*

IM 1 340.50 ± 8.24*

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Values represent the mean ± S.E.M for 6 animals per group. **p < 0.05 compared with non-stress control group; *p < 0.05 compared with stress control group (ANOVA followed by Newman Keuls test).

Table 2:

Jobelyn® Reduces corticosterone level in the brain of mice exposed to unpredictable chronic mild stress

Treatment Dose(mg/kg)

Corticosterone conc.(ng/ml)

Non stress control   4.70 ± 0.11Stress control   7.24 ± 0.11**

JB 5 4.27 ± 0.17*JB 10 4.62 ± 0.27*JB 25 4.85 ± 0.17*JB 50 4.87 ± 0.26*DP 1 4.17 ± 0.72*IM 1 3.40 ± 1.44*

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Values represent the mean ± S.E.M for 6 animals per group. **p < 0.05 compared with non-stress control group; *p < 0.05 compared with stress control group (ANOVA followed by Newman Keuls test).

Table 3:

Effect on UCMS-Induced Neuronal degeneration in Mouse Brain

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DP 1 mg/kgIM 1 mg/kg

StressNon -stress

Fig 2: H&E. x 400

Effect of Jobelyn on UCMS-Induced Neuronal degeneration in Mouse Brain

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JB 50 mg/kg

JB 10 mg/kg

JB 25 mg/kg

JB 5 mg/kg

Fig 3: H&E. x 400

Effect of Jobelyn on the population of viable hippocampal neuronal cells in UCMS-mice

Fig. 4:Values represent mean ± S.E.M for 6 animals per group. **P < 0.05 compared with non stress control (ANOVA followed by Newman Keuls test), *P < 0.05 compared with stress group (ANOVA followed by Newman Keuls test). 23

Summary and Conclusions

The results of this study provide evidence that suggest that Jobelyn promotes adaptability to stress and reversed memory impairment induced by chronic stress in mice.

The antioxidant, anti-cholinesterase and neuroprotective activities demonstrated by Jobelyn may be playing a significant role in its positive effect on memory performance in mice exposed to chronic stress.

Moreover, the finding that Jobelyn suppressed the levels of corticosterone; a major indicator of stress response, further supports its use as energizer and in conditions associated with stress related disorders.

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Acknowledgement I thank the organizer of this conference for the golden opportunity to present my work to this

great scientific audience

I also appreciate the efforts of the immediate past HOD in procuring some vital instruments for my Department.

I appreciate the efforts of the Director of Health Forever Product Limited, Lagos, Nigeria (www.health-forever.com) for giving free sample of JB and his support as well.

I also appreciate Dr O. Owoeye of Anatomy Department and Mrs Akindele, Mr Okon and Mr Onome of Department of Physiology for their support and expertise.

My institutions; firstly University of Ibadan for the expected Tefund travel grant support and Osun State University for granting my request to attend this conference

My wife for her support and interest in my research work.

My special thanks to all members of the Neuropharmacology Laboratory (NeuroLAB), UI for their cooperation.

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Members of Neurolab Unit

DR UMUKORO Remi Dr Alabi Tony Adrain Adaeze Dr. Annafi Dr. Olugbemide Gabriel Oritoke Osarume Esther Yomi

DR. ADERIBIGBE Adeoluwa Toyin Ada Nneka Ebi Ben Valliant Sowunmi Niyi Yusuf

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Some Selected References

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Benson KF, Beaman JL, Ou B, Okubena A, Okubena O, and Jensen GS (2013). West African Sorghum bicolor Leaf Sheaths Have Anti-Inflammatory and Immune-Modulating Properties In Vitro. Journal of Medicinal Food 16(3): 230–238.

Eduviere AT, Umukoro S, Aderibigbe AO, Ajayi AM, Adewole FA (2015). Methyl jasmonate enhances memory performance through inhibition of oxidative stress and acetylcholinesterase activity in mice. Life Sciences 132: 20-26.

Heo et al., Naringenin from Citrus junos has an inhibitory effect on acetylcholinesterase and a mitigating effect on amnesia. Dementia and Geratric Cognitive Disorders, 17, 151-157.

Luine V, Villegas M, Martinez C and McEwen BS (1994). Repeated stress causes reversible impairments of spatial memory performance. Brain Res. 639:167–170.

Magarinos AM, Garcia Verdugo JM and McEwen BS (1997). Chronic stress alters synaptic terminal structure in hippocampus. Proc. Natl. Acad. Sci. USA 94:14002–14008.

McEwen BS (2008). Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators. Eur. J. Pharmacol. 583:174–185.

Mineur YS, Belzung C, Crusio WE (2006). Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behaviour and Brain Research 175: 43–50.

Okhawa H, Ohishi N and Yagi K (1979). “Assay for Lipid Peroxides in Animal Tissues by Thiobarbituric Acid Reaction,” Analytical Biochemistry 95: 351- 358.

Panossian A. Wikman G, Kaur P and Asea A (2010). Molecular chaperones as mediators of stress protective effect of plant adaptogens. Heat Shock Proteins and Whole Body Physiol. 5: 351-364.

Rodriguez EL, Gonzalez AS, Cabrera R and Fracchia LN (1988). A further analysis of behavioral and endocrine effects of unpredictable chronic stress. Physiol. Behav. 43:789–795.

Umukoro S, Omogbiya A.I and Eduviere AT (2013). Effect of Jobelyn on intruder and isolation-induced aggressive behavior in mice. Basic & Clinical Physiology & Pharmacology 24 (4): 263-269.

Umukoro S  Eduviere AT, Aladeokin AC, Olugbemide AS (2014). Antidepressant-like property of jobelyn®, an African unique herbal formulation, in mice. Drug Research 64: 146-150.

Watanabe Y, Gould E and McEwen BS, Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res. 588 (1992), pp. 341–345.

Zhu et al., (2014). Alpha-1 and alpha-2adrenoceptors in restraint stress-induced liver injury in mice. PLoS ONE 9: 921..

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