ISOLATION AND ETHNO-PHARMACOLOGICAL INVESTIGATION OF THE ACTIVE PRINCIPLE OF GUIERA SENEGALENSIS USED IN THE MANAGEMENT OF PSYCHOSIS

PHARM (MRS.) LYDIA DOOSUUR IOR

DEPARTMENT OF PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES.

UNIVERSITY OF JOS

1. ABSTRACT

Herbal medicines play an important role in health care program worldwide. The search for novel pharmacotherapy from medicinal plants for psychosis has progressed significantly in the past decade and their therapeutic potential has been assessed in a variety of animal models. The aim of this study is, to isolate potential bioactive compounds from Guiera senegalensis and to test the isolates for their efficacy in treating psychotic disorders, in the faculty of Pharmaceutical Sciences University of Jos. The extracts will be subjected to bioassay-guided fractionation using standard chromatographic procedures. Active compounds which are expected to be isolated  will be subjected to biological assays using different  animal models of psychotic disorder which include; Apormorphine climbing test in mice, Forced swim test in mice, Phenorbarbitone sleeping time and Elevated plus maze. The study is therefore expected to  provide potential drug compounds  which could be useful in the treatment of psychosis and also to contribute  to the knowledge of the pharmacology of the plants being investigated.

BIOGRAPHICAL SKETCH
NAME   Ior Lydia  Doosuur	POSITION TITLE Lecturer 1
EDUCATION/TRAINING
INSTITUTION AND LOCATION	DEGREE	MM/YY	FIELD OF STUDY
University of Jos   University of Jos	B. Pharm   MSc Pharmacology	Feb/2004   April/2012	Pharmacy   Pharmacology
West African Post graduate College of Pharmacists	FPCPharm	March/2014	Clinical Pharmacy

 

1. PERSONAL STATEMENT

The goal of this research is to isolate potential bioactive principles from Gueira senegalensis leaves and to test the isolates for their antipsychotic property in the faculty of Pharmaceutical Sciences University of Jos. During my master of science studies in Pharmacology, I received training in laboratory research on phytomedicine involving whole animals such as mice, rats and guinea pigs, in ethno pharmacology as well as neuropharmacology, and in the last seven years as a junior Faculty in Pharmaceutical sciences I have been exposed to different areas of research on both central and peripheral diseases, as well as bioassay guided fractionation and isolation of natural products. While undergoing my fellowship program in clinical pharmacy with the West African postgraduate college of Pharmacists, I was privileged to do my residency at the Psychiatric Department of JUTH and I witnessed the devastating effect of mental illness such as Psychosis and depression on patients. I also noticed the high cost as well as the serious adverse effects associated with the drugs used in the treatment of mental disorders which often leads to medication non-compliance and eventual rebound psychosis. These factors motivated me to carry out an ethnobotanical survey of plants used traditionally in the treatment of mental illness in plateau state, Nigeria, in the bid to discover more affordable, safer, accessible and more efficacious drugs used in the treatment of mental illness. Preliminary screening revealed that some of the plants found in the survey are very promising and there is need to carry out further studies on them.

ROLE: PRINCIPAL INVESTIGATOR

2.  POSITIONS

Assistant Lecturer, Department of Pharmacology, University of Jos            2009 – 2012

M.Sc. Pharmacology, University of Jos                                                          2012

Lecturer II, Department of Pharmacology, University of Jos                                    2012 – 2015

Fellow West African College of Pharmacists                                                 2014

Lecturer I, Department of Pharmacology, University of Jos.                         2015 to date   

 

OTHER EXPERIENCE AND PROFESSIONAL MEMBERSHIPS

2006-   Member, Pharmaceutical Society of Nigeria (PSN)

2006-   Member, Association of Lady Pharmacists (ALPs)

2009-   Member, Nigerian Association of Pharmacists in Academia (NAPA)

2010-   Institution Based SIWES Supervisor

2011-   Member, West African Society of Pharmacologists (WASP)

 

PUBLICATIONS

1. Ior, L. D,  Uguru, M. O,  Olotu, P. N, Ohemu T. L.   and Ukpe A.   (2011): “Evaluation of analgesic and anti-inflammatory activities and phytochemical screening of the leaves extract of Paullinia pinnata (Sapindaceae)”Journal of Chemical and Pharmaceutical Research. 3(4): 351-35
2. Ior, L. D,  , Otimenyin, S. O. Umar, M  (2012): “Anti-Inflammatory and Analgesic Activities of the Ethanolic Extract of the Leaf of Syzygium Guineense in Rats and Mice, IOSR Journal of Pharmacy.2(4):.33-36
3. Amagon, K. I, Wannang, N. N, Iliya, H. A, Ior .  L. D. and Chris-Otubor G. O,  (2012): Flavonoids Extracted from Fruit Pulp of Cucumis metuliferus Have Antiviral Properties, British Journal of Pharmaceutical Research 2(4): 249-258
4. Olotu, P. N.  Chindo, I. Y.  Gushit, J. S Ajima, .U. Ohemu, T. L, Ior, L. D, Wannang, N. N. Mmuo M. I. and Onche E. U.  (2013): Pesticidal potential of the leaves of Ocimum basilicum linn. and Hyptis spicigera lam. on Callosobruchus maculatus F. (cowpea weevils) family lamiaceae. J.ournal of  Natural  Products and  Plant Resource.,3 (5):31-35
5. Ior, L. D.  Wannang, N. N, Ior, C. A.  and Amagon, K. I. (2015): In vivo assessment of the antimalarial activity of Cassia Singueana and Cymbopogon Citrutus. Der Pharma Chemica, 7(3):272-278.
6. Ior, C.A. Ayanbimpe, G. Ior, L.D. Okechalu J. Chris- otubor, G.O.(2015):  Enviromental isolation of Cryptococcus neoformans in Jos. Scientific research Journal. 3(6): 15-17
7. Otubor, G. O, Dangiwa, D.A, Ior L. D. and Anukam, N. C. (2015)  Assessment of Knowledge, attitudes and practices of mothers in Jos North regarding immunization. IOSR journal of Pharmacy. 5(6) PP. 34-45

BUDGET

 

	DESCRIPTION	QUANTITY	UNIT PRICE  (N)	TOTAL PRICE (N)
A	Chemicals for extraction
1	Methanol	10 Litres	15,000/2.5L	60,000
2	Dichloromethane	10 Litres	15,000 /2.5L	60,000
	Chemicals for fractionation
3	Hexane	10 Litres	15,000/2.5L	60,000
4	Ethyl acetate	10 Litres	15,000/2.5L	60,000
5	Buthanol	10 Litres	12,000./2.5L	48,000
	Solvents for fractionation via Chromatography
6	Ethyl acetate	10 Litres	15,000 /2.5L	60,000
7	Chloroform	10 Litres	18,000 /2.5L	72,000
8	Methanol	10 Litres	15,000 /2.5L	60,000
9	Acetone	5 Litres	11,000 /2.5L	22,000
10	Butanone	5 Litres	11,000 /2.5L	22,000
11	Toluene	5 Litres	12,000 /2.5L	24,000
12	Sephadex LH 20100	100 gram	100,000	200,000
13	Silica gel 60A,230-400 mesh	1	50,000	50,000
14	Pre-coated TLC plates	1	70,000	70,000
	Bioassay
15	Swiss albino mice	100	250	25,000
16	Apomorphine Hcl	1 g	207,000	207,000
17	Drugs/ Chemicals		300,000	300,000
18	Research assistant	1	360,000	360,000
19	Laboratory consumables		100,000	100,000
20	Statistical Analysis		50, 000	50,000
	TOTAL			1,910,000

           

4. BUDGET JUSTIFICATION

• Solvents and Chemicals are required for plant extraction, fractionation and isolation of compounds.
• Chromatography: Adsorbents and support of various types are needed for the chromatographic separation to be carried out. TLC plates are needed for preparative purposes.
• Biological assay: animals are required for biological assays and evaluation of the plant’s efficacy using various models.
• Research assistant  to assist in collection of plant and authentication, animal care and feeding, etc.
• Drugs and Chemicals: These will be purchased from sigma aldrich through their representatives and include pure chemicals such as ketamine, clozapine, pentobarbitone sodium etc which will be used as standard drugs in the bioassay for comparism with isolates.
• Apomorphine hydrochloride: Use to induce psychosis will also be purchased from sigma aldrich.
• Laboratory consumabls include methylated spirit, cotton wool, glass wares, formalin,syringes,cannulas, normal saline, distilled water etc
• Statistical analysis: The services of a biostatistician will be required to help analyse the data from the study.

 

5. PROJECT NARRATIVE

This project will involve various neurobehavioral models of psychosis bearing in mind the complex nature of psychotic disorder characterized by positive, negative and cognitive symptoms. Animal models that depict these symptoms will be challenged with the isolates that will be obtained from the fractionation and isolation of crude extract of the plant. The efficacy of the isolates will be compared with that of both negative control (normal saline) and positive control (standard drugs), the results and broad conclusions will be directly relevant to the better understanding of the efficacy of G. senegalensis.

6. RESEARCH PLAN

1. AIM

Is to isolate potential bioactive principless from Guiera senegalensis leaves and to test the isolates for their antipsychotic properties.

 SPECIFIC AIMS

• To isolate bioactive principles from the plant
• To test for the antipsychotic activity of the isolates

2. BACKGROUND AND SIGNIFICANCE

Psychotic disorders are chronic recurrent neuropsychiatric disorders that alters the quality of life of the sufferers and have been a major public health concern. ¹ Although these disorders are not very frequent, they are among the most severe and impairing medical diseases.² Active psychosis was ranked the most disabling condition after quadriplegia and dementia in a WHO multi-country study.³They cause enormous suffering for patients and their family members. As the average age of onset for many psychotic disorders is at the most critical period of educational, occupational and social development, their consequences often lead to lifelong disability. Economic costs to society consist of the expense of treatment and loss of productivity. The costs of psychotic disorders were estimated to be the third largest of brain diseases in Europe in 2010, after mood disorders and dementia. ⁴ Globally, 14 per cent of the global burden of disease is attributed to mental illness – with 75 per cent of those affected being found in low-income countries – which includes a broad spectrum of diagnoses, from common mental illnesses such as anxiety and substance abuse, to severe illnesses like psychosis. Mental disorders accounted for 5 per cent of the total burden of disease and 19 per cent of all disability in Africa. Thus, mental illness is a major cause of morbidity and a burden to the patients, their families and society. ⁵ Furthermore, the high prevalence of communicable diseases, including malaria, tuberculosis and HIV/AIDS, are closely associated with mental illness. ⁶ Drugs prescribed for the treatment of psychosis can be categorized as typical and atypical. The typical class of antipsychotics are effective against the positive symptoms, i.e. hallucinations, delusions, etc. And also possess extra-pyramidal side effects. Whereas the atypical class are effective in ameliorating the positive, negative as well as the cognitive symptoms and possess lesser extra-pyramidal side effects but in turn hold greater risk of cardiovascular diseases, diabetes and agranulocytosis etc. Regular intake of these agents may also increase oxidative load and thereby further enhance the progression of the disease.^{7, 8, 9} Since current drug treatments are limited by poor efficacy and tolerability, there is therefore need for alternative remedies and herbal medications for the treatment of psychotic disorders.

3. PRELIMINARY DATA

An ethnobotanical survey of plants used traditionally in the treatment of mental illness was carried out in some local government areas of Plateau state and a total of 45 medicinal plants species were found. Preliminary screening of these plants revealed that G. senegalensis has very promising antipsychotic properties that can be harnessed for future use.

4. EXPERIMENTAL DESIGN/ METHODS

PLANT COLLECTION AND IDENTIFICATION

The fresh leaves of G. senegalensis will be collected from Bassa LGC of Plateau  State, Nigeria.  The plants  will first be  identified  on field using keys and description given in the Flora of west Tropical Africa. ¹⁰ Its botanical identity will further be confirmed and authenticated at the herbarium of the College of Forestry Jos.

 EXTRACTION

The stem-bark of G. senegalensis will be sorted  to eliminate unwanted particles and dead matter, after which they will be air dried under shade, then ground to powder and preserved. ¹¹  The dried powdered leaves of the plant will be extracted successively by maceration using petroleum ether, ethyl acetate, methanol, N hexane and distilled water in that order. The extracts will be concentrated to dryness in vacuo on a rotary evaporator. This would yield petroleum ether, ethyl acetate, methanol and hexane extracts of  the plant.

FRACTIONATION AND ISOLATION

Bioassay of  all the extracts  mentioned above will be carried out. The extract with the best neuropharmalogical activity will be selected for further analysis.

Thereafter chromatographic separation (TLC and column) will be carried out to further fractionate and isolate compounds from active extract. Column chromatography will entail the use of Baeckstrom Separo equipment for Accelerated Gradient Chromatography (AGC) which is a form of Medium Pressure Liquid Chromatography (MPLC) on silica adsorbent/ support (normal / RP-18 bonded). Judicious use of this in combination with thin-layer chromatography – (both analytical and preparative) is expected to yield pure compounds.

Since this is a bioassay guided research,  fractionation process will be bioactivity-driven, the extract, fractions and isolated compounds will be tested for biological activity.

BIOLOGICAL ASSAYS

The fractions and isolated compounds will be tested for antipsychotic activity in vivo using  mice. For each of the experiments, the mice would be grouped in to five groups of five mice each;

Group 1 would serve as negative control, normal saline would be administered to the mice.

Group 2, 3, and 4 would serve as test groups and would receive graded doses of the extracts.

Group 5 would serve as positive control group and would be given a standard antipsychotic drug (clozapine).

Duration of treatment – The duration of treatment for each test is 21 days, the mice will be pretreated with ketamine/ apomorphine for 14 days, after which the mice will receive either the test drug or control on the 15^th – 21s^t day one hour after pretreatment with psychotic agent.

The following test will be carried out:

a. Inhibition of Apomorphine Climbing in Mice

This will determine the neuroleptic potentials of the extracts. Mice will be divided into 5 groups of 5 mice each. The groups would be treated with normal saline (10 ml/kg), different doses of isolate, or clozapine (2 mg/kg), orally one hour post treatment, each mouse would be treated with apomorphine (4 mg/kg). Each mouse will then be observed for climbing behaviors. The average climbing scores would be determined for each group. ¹²

b. Elevated plus maze test

Ketamine-induced hyperlocomotion would be used to screen for the antipsychotic effect of extract (positive symptom) as previously described by Yamamoto et al., would be adopted with brief modification. After pre-treatment for 14 days, the mice from the different groups would be treated with either test/control drug, and placed in the centre of the maze and allowed to explore it. The duration of immobility(s) and number of line crossed would be recorded for 5 minutes using a stopwatch. After each mice session, the observation chamber would be cleaned with 70% ethanol to remove residual odour.¹³

3. Forced Swimming Test:

The antipsychotic effect of the extracts would be screened using ketamine-enhanced immobility in forced swim test paradigm, that is predictive of the negative symptoms of schizophrenia, which is reflected as a state of despair in mice as described by Chindo et al.¹⁴ The reduction in the immobility time serves as a specific and selective index of antidepressant activity that can be used to alleviate the negative symptoms of schizophrenia. Each mouse from the various groups would be pre-treated with a sub-anaesthetic dose of KET (30 mg/kg, i.p.) once daily for 14 days. After which, each mouse would be placed in a standardized transparent glass cylinder (height 46 cm, diameter 20 cm) containing water at 25 °C to a depth of 30 cm and forced to swim for 6 min (pre-test session) 1 h after the last treatment with ketamine for habituation. Twenty-four hours (24 h) later, the mice would be treated for 7 days with test/control drug and KET respectively, and forced to swim individually, while carrying out observations.

d. Ketamine-induced cognitive dysfunction (Y- maze)

The effect of the extract on spontaneous alternation performance would be assessed using a Y-maze test, which allows the evaluation of cognitive searching behaviour, as an index for the cognitive dysfunction of psychosis as described by Monte et al.¹⁵ In this protocol, mice would be divided into 5 groups as stated above. The groups would be treated with sub anaesthetic doses of ketamine once daily for 14 days, followed by either the test or control drugs on day 15 -21. After the last treatment each mouse would gently be placed individually in the Y- maze apparatus, at the end of arm A, and allowed to explore all the three arms (labelled A, B, C) freely for 5 minutes, taking the following parameters: the number of arm visits and sequence (alternation) of arm visits visually. After each mice session, the observation chamber would be cleaned with 70% ethanol to remove residual odour.

Animal disposal

On completion of the experiment in accordance with the animal experiment protocol, the animals will be disposed using euthanasia procedures, the mice will be injected with an anesthetic agent (pentobarbital sodium) and killed by exsanguination under anesthesia. When the mice are confirmed dead, the dead mice would be returned to the animal house for proper disposal in the incinerator and the cages would be properly cleaned and disinfected.¹⁷

 Statistical Analysis

The data collected would be analyze using STATA statistical package. The data will be expressed as mean ± standard error of mean (SEM), and analyzed using one-way analysis of variance (ANOVA), followed by post hoc test for multiple comparisons where appropriate. P < 0.05 will be considered statistically significant.

Result/Expected Outcome of the Study

The study is expected to  provide potential drug compounds  which could be useful in the treatment of psychotic disorder and also to contribute  to the knowledge of the pharmacology of the plant being investigated.

5. FUTURE DIRECTIONS

Characterization And Structure Elucidation

 The compounds isolated will be characterised and their chemical structures elucidated by joint application of spectroscopic techniques – UltraViolet (UV), Infra Red (IR), Nuclear Mass Resonace (NMR) (proton & C-13; 1-D & 2-D) and MS. Toxicological studies would be conducted on whole animals, clinical trials would be conducted to test the efficacy and safety of the substance on human subjects.

7. ETHICAL CLEARANCE (VERTEBRATE ANIMALS)

The research will be carried out in accordance with the US guidelines (NIH publication #85-23, revised in 1985) for laboratory animal use and care. The animals will be cared under standard laboratory conditions of a 12 h light/dark cycle in room with controlled temperature and humidity. Food and water will be available ad libitum.

The ethical committee of the Animal house, University of Jos has approved the proposal for the study to be carried out using animals. The ethical Clearance is attached to this document.

8. REFERENCES
9. Ehmann T, Yager J, Hanson L: (2004) Early psychosis: a review of the treatment literature. Child Mental Health Policy Res Program 7:1–52.
10. Insel T.R. Rethinking schizophrenia. Nature (2010) 468:187-193.  Isohanni M, Jones PB, Moilanen K, Rantakallio P, Veijola J, Oja H, Koiranen M, Jokelainen J, Croudace T, Järvelin M. Early developmental milestones in adult schizophrenia and other psychoses. A 31-year follow-up of the Northern Finland 1966 Birth Cohort. Schizophrenia Research 52:1- 19. 
11. Ustün TB. (1999) The global burden of mental disorders. The American Journal of Public Health;89:1315-1318.
12. Gustavsson A, Svensson M, Jacobi F, Allgulander C, Alonso J, Beghi E, Dodel R, Ekman M, Faravelli C, Fratiglioni L, Gannon B, Jones DH, Jennum P : (2011) Cost of disorders of the brain in Europe. European Neuropsychopharmacology ;21:718-79.
13. Amuyunzu-Nyamongo M. (2013).  The social and cultural aspects of mental health in African societies, Commonwealth Health Partnerships pg 59- 63.
14. Freeman, M., Patel, V., Collins, P. Y. and Bertolote, J. (2005). 'Integrating mental health in global initiatives for HIV/AIDS'. The British Journal of Psychiatry 187 (1):1-3.
15. Meltzer H (2010) Antipsychotic agents & Lithium. In: Basic & Clinical Pharmacology, ed. ^eds. Katzung B G, Masters S B & Trevor A J, McGraw-Hill Companies.
16. Meyer JM (2011) Pharmacotherapy of Psychosis and Mania. In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, eds. Brunton L, Chabner B & Knollman B, McGraw-Hill.
17. Reus VI (2008) Mental Disorders. In: Harrison's Principles of Internal Medicine, ed.^eds. Fauci A S, Braunwald E, Kasper D.L, Hauser S.L, Longo D.L, Jameson J.L & Loscalzo J, McGraw-Hill
18. Hutchinson , J and Dalziel, J.M. (1954). Flora of West Tropical Africa Vol 1(1). Crown agents for Oversea Governments and Adminstrations Millbank, London. Pp 51.
19. Evans, W. C. (2009). ‘Trease and Evans’ Pharmacognosy. Fourteenth Edition.  W. B. Saunders Company Ltd. London. Pp119-120
20. Pandy V, Narasingam M, Mohamed. Z (2012) Antipsychotic-like activity of Noni (Morinda citrifolia Linn.) in mice. BMC Complementary and Alternative Medicine 12:186. http://www.biomedcentral.com/1472-6882/12/186
21. Yamamoto M, Mizuki Y, Suetsugi M, Ozawa Y, Ooyama M, Suzuki M.( 1997)  Effects of dopamine antagonists on changes in spontaneous EEG and locomotor activity in ketamine treated rats. Pharmacology Biochemistry and Behavior. 57:361- 365.
22. Chindo AB, Adzu B, Yahaya TA, Karniyus SG. (1999) Ketamine-enhanced immobility in forced swim test: A possible animal model for the negative symptoms of schizophrenia. Progress in Neuro- Psychopharmacology & Biological Psychiatry.;38:310-316.
23. Monte AS, Greicy CS, Roger SM, Joanna KS, Júnia VS, Rafaela CC, Bruna MM, Ribeiro DF, Silvânia MM. (2013) Prevention and reversal of ketamine-induced schizophrenia related behavior by minocycline in mice: Possible involvement of antioxidant and nitrergic pathway. Journal Psychopharmacology. 2013;11:1032-1043.
24. Chatterjee M, Verma P, Maurya R & Palit G. (2011) Evaluation of ethanol leaf extract of Ocimum sanctum in experimental models of anxiety and depression. Pharm Biol. 49: 477-483.
25. AVMA Guidelines for the Euthanasia of Animals, 2013 Edition
26. MENTOR

My proposed mentor for this research is Prof Otimenyin Sunday Oritsemenyin, Professor of Pharmacology, Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Jos.