Received on : 11-07-2018
Accpected on : 16-07-2018
Address for correspondence
Dr. Elizabeth Finbarrs
Department of Anatomy
University of Science and Technology,
Parklane, Nigeria
Email: elizabeth.finbarrs-bello@esut.edu.ng
Contact No: +234-8064113179

Original Article

ERA’S JOURNAL OF MEDICAL RESEARCH

VOL.5 NO.1

EFFECT OF ETHANOLIC SEED EXTRACT OF CITRUS LANATUS

(WATERMELON) ON IMMUNOREACTIVITY OF HIPPOCAMPAL

NEUROFILAMENT OF ADULT WISTAR RATS

Elizabeth Finbarrs, Francis Chinedu*, Oluwatomilayo Patience Ojo**

Department of Anatomy

Faculty of Basic Medical Sciences. Enugu State University of Science and Technology, Parklane, Nigeria

Faculty of Basic Medical Sciences. Ebonyi State University, Abakaliki, Nigeria*

Faculty of Basic Medical Sciences, University of Ilorin, Ilorin, Nigeria**

ABSTRACT

Neurofilaments are major protein constituents of the brain, and play important roles in trafficking of molecules between neurons. An experimental study on

thetotal protein content and immunoreactivity of neurofilament protein was undertaken on the hippocampus of adult rats treated with the seed extract of Citrulluslanatus, Omega H3 and soybean oil. Twenty (20) adult wistar rats of average weights 150g were used. The rats were randomly divided into 5 groups of 4 rats per group. Group A served as control, groups B and C received 100mg/kg and 200mg/kg of Citrulluslanatusethanolic seed extracts orally

respectively, while groups D and E received 300 mg/kg of Omega H3 and 5ml/kgof Soybean oil for two weeks respectively. Total serum protein was

estimated biochemically and statistically analyzed using SPSS version 20 group comparison were made by one-way analysis of variance (ANOVA) and P< 0.05 was considered statistically significant. The hippocampus was dissected, fixed, processed and stained for neurofilament by the avidin–biotin immunoperoxidase method. The result revealed total serum protein levels increased significantly in treated groups while immunoreactivity to neurofilamentincreased in all treated groups compared to the control. In conclusion, ethanolic seed extract of Citrulluslanatushas similar effects as Omega H3 and Soya bean oil which justify it use as supplement.

KEYWORDS: CitrullusLanatus, Cognitive, Neurochemistry, Nutrition, Hippocampus

The use of herbs as nutritional supplements dates back to the time of the early man (1). Thepractice has gained popularity amongst the low income earners in the developing countries including Nigeria.In recent times, daily nutritional intakes can scarcely provide the recommended dietary allowance (RDA), that is the average daily level of intake sufficient to meet the nutrient requirements of nearly all (97%-98%) healthy people. Food and drug supplementations become necessary to make up for a balanced diet. Beside the aforementioned plant supplement: the watermelon also known as Citrulluslanatusand a member of thecucumber (Cucubitacea) family is a popular fruit in many parts of the world and notable for its high water content and attractive look (2).

The watermelon fruit contains numerous seeds which are rich in polyunsaturated fats,vitamins, antioxidants, minerals, proteins in addition to the various phytochemicals which are vital for bodily functions(3).Polyunsaturated fatscontainomega -3 fatty acidswhich promote brain health and cognitive functions and use source of oil for cosmetic products (2) and plays significant role in the treatment of diseases such as; urinary

tract infections,diabetes, hypertension and scabies (3-4).

Like the watermelon seeds, vegetable seed oils such as soybean oil is also rich in polyunsaturated fat. Soybean oil is a popular and most widely consumed cooking oil extracted from the soybean seeds. The soybean seeds are prominent for their high protein and oil content such as monounsaturated fatty acid which is believed to be beneficial in the absorption of vital nutrients andenhancement of cognitive functions of the brain(5-8).

On the other hand, Omega H3is a drug supplement that provides well-balanced source of vital nutrients which can easily be lacking in the diet of many. It contains biologically active, health-promoting important nutritional factors accurately balanced and easily absorbed by the body(9). Omega H3 is rich source of fatty acids which are well characterized by high contents of polyunsaturated fatty acids. Hence, it is used as medicine and nutritionalsupplement aimed at increasing brain energy and cognitive functions (10).

Food consumption is an intrinsically motivated behavior with the potential to modulate brain structure and function. It has been an established fact that the brain structure is rich in lipids particularly fatty acids(11). Food

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Absorbance of sample/

EFFECT OF ETHANOLIC SEED EXTRACT OF CITRUS LANATUS (WATERMELON) ON IMMUNOREACTIVITY OF HIPPOCAMPAL....

and dietary supplements containing high content of polyunsaturated fatty acids are known to enhance brain cognitive function and structural integrity. Particularly, areas of the brain involve in cognition such as the cerebrum and hippocampus. These intakes can mobilize raw materials that can be used to strengthen the cytoskeletal integrity in health and diseases. Thus, this study investigated the effects of ethanolic extract of watermelon seed on immunoreactivity of neurofilament on the hippocampus and, compared with that of soybean oil and omega H3. Since, Neurofilaments are intermediate filaments of matured neurons cytoskeleton and among the most abundant proteins in brain (12-13) andmaintains the structural intergrity of the brain by transporting proteins and neurotransmitters throughout the neuron. The neurofilament (NF) immunostaining has been used as a common tool in diagnostic neuropathology and differentiating neurons that are positive for NF from glia which shown negative staining for NF.

2. MATERIALS AND METHODS

2.1 Collection and preparation of plant material

Fresh and richly green matured watermelon fruits which were examined to be free of any form ofpest bites, damage and were obtained from the local market in Abakaliki, Ebonyi State, Nigeria in the month of August, 2017. It was identified by Prof. (Mrs.) M. O. Nwosu of the Department of Plant Science and Biotechnology, University of Nigeria Nsukka. The fresh watermelon fruits were cut open and the seeds were harvested and allowed to air-dry for five days. The watermelon seeds were grounded into fine powder and used for the preparation of the ethanolic extract.The powdered watermelon seed was thoroughly sieved and 250g of the fine powder was submerged in 500mls of 95% ethanol. The mixture was vigorously stirred and allowed to stand for 48hrs. Thereafter, it was filtered by muslin cloth and the filtrate was left to evaporate for 48hrs. The ethanolic extract was then stored in a bottle and preserved in the refrigerator at 4oc for the administration.

2.2 Drugs and Chemicals

Omega H3 capsules (Vitabiotic Ltd, 1 Apsley way. London. NW27HF (England) was purchased from registered pharmacy store in Abakaliki, Ebonyi State. Soybean oil (Grand oil Nig. Ltd. Nigeria) was procured from the opened market in Abakaliki, Ebonyi State. Routine histological reagents of standard and analytical grades were purchased from certified chemical stores. The neurofilamentantibody usedwas manufactured by Novocastra, LEICA. Germany.The doses of drug were selected based on data from literature and drug information leaflet.

2.3 Experimental Animals / Ethical Statement

Twenty-adult wistar rats of both sexes weighting between

120-150g were purchased from the animal house of Department of Anatomy, Faculty Basic Medicine, Ebonyi State University Abakaliki, Nigeria. The rats were randomly assigned to five groups; A, B, C, D and E, the animals were kept for a period of 2 weeks to acclimatize during this period, they were maintained at room temperature in a ventilated cage and were allowed free access to vital growers mesh and water.All protocols used were in accordance with the ethics and research guidelines of the Faculty of Basic Medical Sciences, Ebonyi State University. After two weeks of acclimatization, the control (group A) was kept on animal feed, groups B and C received 100mg/kg and 200mg/kg of theethanolic extract respectively.Watermelon seed has the oral median dose (LD50) is 765 mg/kg (14). Groups D and E were administered with 300mg/kg of Omega H-3 and 5ml/kg of soybean oil daily by orally gavages for 2 weeks.

2.3 Termination of experiment

On the day one post administration the rats in all the groups were bledvia the retro-orbital plexususing capillary tubes and the blood samples were collected into a plain sample tubes. Thereafter, the rats were anesthetized using 50mg/kg/bw of thiopentalsodium(RotexMedica. Trittau. Germany), the transcardiacperfusionwasthen carriedout using 4% paraformaldehyde. The skulls were opened to harvest the brains which were further fixed in 10 % neutral buffered formal saline for 48hours. The cerebellum was removed first then hippocampus was exposedby dividing each cerebral hemisphere into two asymmetrical parts each having an exposed part of the hippocampus and processed for neurofilamentimmunohistochemical study.

2.4 Estimation of total serum proteins

Total serum protein was photometrically measured at alkaline pH 7.0, which allow proteins to form a stable complex with Cu2+. Three test tubes, blank, standard and sample were labeled respectively. To the sample tube, 0.02ml of serum was added, to the standard test tube, 0.02ml of protein standard was added while; 0.02ml of water was added to the blank test tube. One milliliter (1ml) of the protein reagent was added to the test tubes each. This was mixed well and allowed to stand for 25mins at room temperature (20-25oC). The absorbance was taken at 540nm and calculated thus:Total serum

Proteins (in g/dl) =g ofprotein/dl/x 5 = Absorbance of standard

2.5Immunohistochemical study

Fixed specimens were dehydrated in ascending grades (50%, 70%, 90%, 95% and 100%) of alcohol (ethanol), cleared in xylene and embedded in paraffin wax. Serial sections of 10µm thick were obtained using model 0325 rotatory microtome by Shandon Scientific Ltd, United Kingdom. The deparaffinized sections were stained using

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the Avidin - Biotin immunoperoxidase method. TheNeurofilamentantibody (Novocastra, LEICA. Germany)dilution factor used was 1:100 dilutions. The sections were placed on the hot plate at 70 degree for at least 1hour. Sections were brought down to water by passing them onto 2 changes of xylene, then 3 changes of descending grades of alcohol and finally to water. Antigen retrieval was performed on the sections by heating them on a citric acid solution of pH 6.0 using the microwave at power 100 for 15minutes.The sections were equilibrated gradually with cool water to displace the hot citric acid for at least 5min for the section to cool.

Peroxidase blocking was done on the sections by simply covering section with 3% hydrogen peroxide (H2O2) for 15minutes.Sections were washed with phosphate buffered saline (PBS) and protein blocking was performed using avidin for 15minutes.Sections were washed with PBS and endogenous biotin in tissues was blocked using biotin for 15minutes.After washing with PBS, sections were incubated with the respective diluted primary antibody for neurofilament (Novocastra, LEICA Germany) was diluted 1:100 for 60minutes.Excess antibodies were washed off with PBS and a secondary antibody (LINK) was applied on section for 15minutes.Sections were washed and the (LABEL) which is the horseradish peroxidase (HRP) were applied on the sections for 15minutes. A working DAB solution made up by mixing 1 drop (20microns) of the DAB chromogen to 1ml of the DAB substrate. The working solution was applied on sections after washing off the HRP with PBS for at least 5minutes. The brown reactions begin to appear at this moment especially for a positive target.Excess DAB solution and precipitate were washed off with water.Sections were counterstained with Haematoxylin solution for at least 2minutes and blued briefly.Sections were dehydrated in alcohol, cleared in xylene and mounted in DPX.Images were observed, interpreted and captured using Amscope research microscope (Model 3.2. England)

3. RESULTS

TOTAL PROTEIN

Figure 1: Graph showing the total protein content in each group. Each bar represents mean ± S.E.M. P values for group comparisons were obtained by one way ANOVA followed by Student T- test. P =0.05.

Control

100mg/kg Citrulluslanatus

Total Protein (mg/dl)

6

5

4

3

2

1

0

Control

ater

 

W

...

Melon

ater

 

W

...

Melon

Omega

H3

5ml/kg

Soya

 

...

bean

oil

 

Treatment

200mg/kg Citrulluslanatus

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EFFECT OF ETHANOLIC SEED EXTRACT OF CITRUS LANATUS (WATERMELON) ON IMMUNOREACTIVITY OF HIPPOCAMPAL....

300mg/kg Omega H3

2ml/kg Soybeans oil

Figure 2:Photomicrograph of the hippocampal

tissues of the control,Citrulluslanatus, Omega H3and Soybean oil treated rats. Increase immunoreactivity for neurofilament in all treated groups compared to the control.NeurofilamentImmunostain. x200.

4. DISCUSSION AND CONCLUSION

Our findings revealed that the ethanolic seed extract of citrulluslanatus, soybean oil and Omega H3 significantly increased the total serum protein compared to the control. This affirms that the ethanolic seed extract of Citrulluslanatus enhanced the total serum protein. It also comparably increased the total serum protein in a measure similar to the Omega H3and soya beans oil used as standards in the study (figure I).However, no significant difference (p= 0.05) was found to exit between the groups that received 100mg/kg/bw and 200mg/kg/bw of Citrulluslanatusethanolic seed extracts. This implies, the effect of Citrulluslanatus on total serum protein is not dose dependent.

Neurofilaments are axonal and synaptic components of neurons. They are known to modulate synaptic

neurotransmission and behavior by their differential interaction with specific neurotransmitter receptors in the brain (13).Thence, it plays vital role in cognition while its alterations also underlie axonopathyin neurological disorders and neuropsychiatric diseases (15-18).

The immunohistochemical finding revealed increased immunoreactivity to neurofilamentprotein antibody in the groups treated with the Citrulluslanatus seed extract, Omega H3 treated groupand Soybean oil treated groups compared to the control (figure II). These indicate changes in the cytoskeleton of the hippocampal neurons. Nitin andMilind (3) early reported that the consumption of soybean in diet improves memory and reverses memory deficits.It also showed that Citrulluslanatusseed exhibited similar effect as Omega H3and Soybeans in the hippocampus and by implication can enhance cognitive function. Conversely, previous studies confirmed elevatedneurofilament reactivity thus improves cognitive function while, decreasedneurofilament levels resulted into an impaired cognitive function (19-20).Put together, increased total serum protein and neurofilament will provide an enabling environment for reorganization of the cytoskeleton of neurons thereby strengthens the hippocampal area towards achieving functional homeostasis. This finding can be beneficial in the treatment of disease conditions whereby neuron structural integrity is being compromised as in Alzheimer's disease. In conclusion, the present study shows that Citrulluslanatus seed extract has neuroprotective property.

CONFLICTS OF INTEREST

The authors declared that they have no competing interests.

ACKNOWLEDGEMENTS

We thank the staff of the histology and biochemistry laboratories for providing the technical and experimental assistances.

REFERENCES

1.Kafara E. The used of herbs medication. J Trad Med.1994;32: 27-30.

2.Wehner TC. Discription of watermelon (Citrulluslanatus). J. Am food and Med. 1999; 29: 387-397.

3.Nitin B, Milind P. Effect of soybean supplementation on the memory of alprazolam- induced amnesic mice. J Pharm Bioallied Sci. 2010; 2: 144-147.

4.Olamide AA, Olayemi OO, Demetrius OO,

Olatoye OJ, Adefule A. Effects of Methanolic E x t r a c t o f C i t r u l l u s l a n a t u s S e e d o n Experimentally-Induced Prostatic Hyperplasia. EurJMed Plants. 2011; 1:171-179.

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.5 NO.1

Page: 4

Jan - June 2018

ERA’S JOURNAL OF MEDICAL RESEARCH

VOL.5 NO.1

5.List GR, Neff AN,Heakin AJ, Evans CD, Black LT, Mount TL. Introduction to soybean. J Am Oil Chem Soc.1978;55:521-522.

6.Almonor GO, Fenner GP, Wilson RF. Soybean production. J Am Oil Chem Soc. 1998; 75: 591- 596.

7.Kinny AJ. Hexane extraction of soybean oil. J Food and Lipid. 1996; 3: 273-292.

8.Kammal A,Rafur AU, Gimsu MK. The effect of soybean food on hippocampus. J Food Sci and Anal. 2000;12: 38-43.

9.Gray MJ, Smith LM, Brenes R. Effect of Omega

H3 fatty acid on digestive system. Rev Herbal Med 2004; 29-32.

10.Fernando GP. The combined effects of exercise and food in preventing neurological and cognitive disorders. Preventive Med 2011;1: S75-S80.

11.Johnson LA, Wan PJ. Introduction to fats and oil. J Am Oil ChemSoc 2000;2:223-227.

12.Albert B.Neurofilament as part of cytoskeleton. In: Garland science, 4thed. Molecular biology of

the cell,New York Oxford University press; 2002: 34-38.

13.Aidong A,Ralph A.N. Specialized roles of neurofilament proteins in synapses: Relevance to neuropsychiatric disorders.  Brain Res Bulletin 2016;126-128.

14.Damilola AO, Xavier CA, UgochukwuBA, Oti AA.Protective effect of methanol seed extractsof Citrulluslanatus onparacetamol-induced hepatotoxicity in adult wistar rats.Global J Pharma2016; 10 (2): 60-67,

15.Vicker JC, Morrison JH. Alterations in neurofilament protein immunoreactivity in human hippocampal neurons related to normal aging and Alzheimers. Neuroscience.63(1994)1- 13.

16.Kaj B. Cerebrospinal Fluid Protein Biomarkers for Alzheimer's disease. Neuron 2004; 1: 213- 225.

17.Kaj B,Harald H, Michael W, Henrik Z.Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nature Reviews Neurolog 2010;6:131-134.

18.Tortelli R, Ruggierri M, Cortese R, D'Errico E. Elevated cerebrospinal fluid neurofilament light levels in patients with amyotrophic lateral sclerosis: a possible marker of disease severity and progression. Eur J Neurology 2012;19:1561- 1567.

19.Shuyi C, Jie S,Gang Z, Ai G, Yanlin C, Rongxia F, Yanqiu D,Liraglutide Improves Water Maze Learning and Memory Performance While Reduces Hyperphosphorylation of Tau and Neurofilaments in APP/PS1/Tau Triple Transgenic Mice. Neurochem Res 2012; 42: 2326–2335.

20.Ane-Victoria I,Roser SL, Tom B, Leiv OW,Torgeir BW,Anne B, et al., CSF neurofilament light levels predict hippocampal atrophy in cognitively healthy older adults. Neurobiology ofAging 2017; 49:138-144.

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How to cite this article : Finbarrs E., Chinedu F., Ojo O.P., Effect Of Ethanolic Seed Extract Of Citrus Lanatus (watermelon) On

Immunoreactivity Of Hippocampal Neurofilament Of Adult Wistar Rats. EJMR2018;5(1):1-5

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