|Year : 2013 | Volume
| Issue : 1 | Page : 23-27
Methanolic crude leaf extract of Ocimum gratissimum reverses phenylhydrazine-induced anemia in albino wistar rats
Nubila Thomas1, Ukaejiofo Ernest Okem1, Nubila Nkoyo Imelda2, Iyare Eborisiagbon Eghosa3, Chijioke Chioli Pascal2, Ukaejiofo Ayodele Chisolu4, Shuneba Irene Lum5, Agumbah Sochi Jethro1
1 Department of Medical Laboratory Sciences, Faculty of Health Sciences and Technology, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
2 Department of Pharmacology and Therapeutics, Faculty of Medical Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
3 Department of Physiology, Faculty of Medical Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
4 Department of Medical Rehabilitation, Faculty of Health Sciences and Technology, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
5 Department of Medicine and surgery, Faculty of Medical Sciences, College of Medicine, University of Nigeria, Enugu Campus, Enugu, Enugu State, Nigeria
|Date of Web Publication||30-Dec-2013|
Department of Medical Laboratory Sciences, University of Nigeria, Enugu Campus, Enugu State
Source of Support: None, Conflict of Interest: None
Background: The therapeutic and culinary applications of Ocimum gratissimum leaf have been practiced in Central and West Africa for decades. This study was designed and conducted to investigate the haematinic and haemopoietic activities of phenylhydrazine-induced anaemia in Albino wistar rats. Materials and Methods: Twenty-four (24) Albino wistar rats were grouped into four (A, B, C and D) with six rats per cage. Groups B, C and D served as the test groups, while group A was the control. The test groups were gavaged with graded doses (4, 8 and 12mg/kg body weight respectively) of Ocimum gratissimum methanolic crude leaf extract once daily for 9 days. Two (2) mls of venous blood was collected from the ocular plexus from each animal into tripotassium ethylene diamine tetra-acetic acid anticoagulant bottle. This was used to determine complete blood count using automated haematology autoanalyzer (Sysmex KX-21N). Results: Red blood cell (RBC) mean value recorded a dose and duration-related statistically significant decrease (P < 0.05) in group D on day 3. However, there was statistically significant increase (P < 0.05) in the RBC indices. In addition, total white blood cell (WBC) recorded a marginal increase in the mean values throughout the study period though not statistically significant (P > 0.05). Furthermore, lymphocyte recorded both dose and duration-related statistically significant increase (P < 0.01). Conclusion: From the result of this study, it can be concluded that the methanolic crude leaf extract of Ocimum gratissimum possesses both haemopoetic and haematinic potentials which are dose and duration-related.
Keywords: Anaemia, Leaf Extract, methanolic, Ocimum gratissimum, reverses
|How to cite this article:|
Thomas N, Okem UE, Imelda NN, Eghosa IE, Pascal CC, Chisolu UA, Lum SI, Jethro AS. Methanolic crude leaf extract of Ocimum gratissimum reverses phenylhydrazine-induced anemia in albino wistar rats. Niger J Exp Clin Biosci 2013;1:23-7
|How to cite this URL:|
Thomas N, Okem UE, Imelda NN, Eghosa IE, Pascal CC, Chisolu UA, Lum SI, Jethro AS. Methanolic crude leaf extract of Ocimum gratissimum reverses phenylhydrazine-induced anemia in albino wistar rats. Niger J Exp Clin Biosci [serial online] 2013 [cited 2019 Jul 19];1:23-7. Available from: http://www.njecbonline.org/text.asp?2013/1/1/23/123959
| Introduction|| |
Nature has provided a very beautiful environment for us to live and survive. Man through the ages has depended on his immediate environment for solving his numerous problems. 1 An estimated 80% or more of the World's population depends primarily on herbal medicine for the management/treatment of many ailments. Thus, dependence on traditional herbal medicine derived from indigenous plant is especially predominant in developing countries where modern Western medicine often is unavailable or is simply too expensive for the common man.
The plant Ocimum gratissimum also known as the African basil or curry, belongs to the mint family, Lamiaceae. It is a non-woody perennial shrub that is native to Africa and other tropical and subtropical regions of the world. 2 The plant has several uses, ranging from culinary, economic and nutritional to medicinal. ,,
In Nigeria, Ocimum gratissimum is found in the Savannah and Coastal areas, the plant is used in the treatment of epilepsy, high fever and diarrhea, while in Savannah areas decoctions of the leaves are used to treat mental illness. 5 In addition, in the southern part of Nigeria, the Yoruba speaking tribe calls the plant "Efirin", the Igbos call it "Ahuji" or "Nchanwu", the Hausas call it "Daidoya."  In addition, it is popularly used as a condiment in the preparation of local and national soups in Nigeria , or as an infusion prepared in aqueous (boiled in water) or ethanol medium locally referred to as "Ogogoro" in folk medicine preparation.
Furthermore, as a medicinal plant, it has been used in the traditional treatment of various ailments such as diarrhoea, fever, pneumonia and skin diseases.  It also possesses antimicrobial/antibacterial and antifungal. ,,,,, The plant also has anthelmintic activity against Haemonchus contortus and other strongylid nematodes of man and animals. , Anaemia is defined as a reduction in the number of circulatory red blood cells (RBCs), the haemoglobin concentration, or the volume of packed red cell (haematocrit) in the blood. The three major categories of anaemia include: hypoproliferation, maturation defects, and haemolysis/blood loss.It is a hidden epidemic worldwide and can have serious consequences if left untreated. Infants, children, women of childbearing age, pregnant women and the elderly are considered to be particularly vulnerable.
Anaemia is one of the numerous ailments claimed to have been successfully treated with plant materials by traditional medicine practitioners. In China for instance, blood diseases such as malformation of blood circulatory system, anaemia, varicose veins and haemorrhages have been treated with plant materials.  In spite of the long-term nutritional and traditional herbal medicinal uses of Occimum gratissimum, its haematinic potentials are yet to be confirmed in controlled scientific studies.
The present study was designed to investigate the haematinic and haematopoetic properties of methanolic crude leaf extract of Ocimum gratissimum in phenyl-hydrazine-induced anemic Albino wistar rats.
| Materials and Methods|| |
Twenty-four (24) adult male white Albino wistar rats weighing 100-200g purchased from the Animal House of the Department of Physiology University of Nigeria, Enugu Campus, were used for the study. The rats were and grouped into four (4): A, B, C and D according to similar body weight. The control group was A, and received no treatment, while B, C, and D were the test groups. The rats were fed with standard feed and clean tap water ad libitum throughout the duration of the study. They were handled according to the Institutional and National guidelines for the care and use of laboratory animals review committee, University of Nigeria Nsukka, Enugu State, Nigeria.
Induction of Anemia
Anemia was induced using 10mg/kg body weight of phenyl-hydrazine through oral route for 10 days.
Plant Collection, Identification, Extraction and Administration
A sample of the leaf of Ocimum gratissimum was authenticated at the Department of Botany, University of Nigeria by a taxonomist. A voucher specimen was deposited at the herbarium for further reference (UNH No.360 a ). The leaves were then bought fresh from Gariki, a local market in Enugu, Enugu State.
After collection they were allowed to air-dry in the shade for 6 days. This was then ground into fine powder. Two hundred and twenty grams (220g) dry-weight of the powder was dissolved in 1000ml of 98% methanol. This was shaken vigorously at intervals for 2 days. The extract was strained using fine cheese-cloth. The filtrate was then filtered using Whatman filter paper No. 1. This was allowed to stand in an open stainless-steel metallic basin and allowed to evaporate at room temperature (32 ± 2°C). The semi-solid greenish-black residue was reconstituted in graded doses (4, 8, and 12mg/kg body weight) and stored in the refrigerator throughout the study period. This was administered to the test groups (B, C, and D), respectively once daily for 9 days.
Sample Collection and Analysis
Under thiopentone sodium anesthesia, 2ml of venous blood was painlessly collected aseptically from the retro-bulbar plexus of the median canthus of the eye on days 3, 6 and 9. This was immediately delivered into tripotassium ethylene diamine tetra-acetic acid (K3 3 EDTA) anticoagulant bottles. This was well mixed by gentle inversion and used for the determination of hematological parameters (complete blood count) using an automated hematology autoanalyzer (Sysmex KX-21N) following the manufacturer's guidelines. All samples were analyzed within 2 hours of collection.
Statistical analysis was carried out using statistical software Graph-Pad Prism. One way analysis of variance (ANOVA) and Students' "t "- test was adopted for comparison. The data were expressed as mean ± standard error (SE). P < 0.05 was considered significant.
| Results|| |
RBC mean value showed a statistically significant decrease (P < 0.05) in group D (12mg/kg). Mean cell hemoglobin concentration (MCHC) and mean cell volume (MCV) mean values revealed a statistically significant increase in groups C (8mg/kg) and D, while MCV and mean cell hemoglobin (MCH) mean values similarly revealed a statistically significant decrease (P < 0.01) in groups C and D, while lymphocyte mean values revealed a statistically significant increase (P < 0.01) when compared with the control group on day 3 of oral crude extract administration [Table 1].
|Table 1: Comparison of the mean ± S.E of the hematological parameters after 3 days of oral crude extract administration with the control group|
Click here to view
MCV mean values showed a statistically significant increase (P < 0.01) in groups B and D. Similarly, MCH mean value showed a statistically significant increase (P < 0.05) in the same groups B and D, while platelet mean value recorded a statistically significant decrease (P < 0.05) in groups C and D when compared with the control group on day 6 of oral crude extract administration [Table 2].
|Table 2: Comparison of the mean ± S.E of the haematological parameters after 6 days of oral crude extract administration with the control group|
Click here to view
Packed cell volume (PCV) mean value showed a statistically significant increase (P < 0.05) in group B. However, MCHC mean value showed a statistically significant decrease (P < 0.05) in group C. MCV mean value revealed a statistically significant increase in group C and group D (P < 0.05). Similarly, MCH mean value showed a statistically significant increase (P < 0.05) in group C while platelet mean value showed a statistically significant decrease (P < 0.05) in groups C and D when compared with the control group on day 9 of crude extract administration [Table 3].
|Table 3: Comparison of the mean ± S.E haematological parameters after 9 days of oral crude extract administration with the control group|
Click here to view
| Discussion|| |
In Africa, anemia associated with malnutrition and infections are major causes of both infant and adult morbidity and mortality. Ocimum gratissimum has been used in most tropical countries as both vegetable and herb due to its high nutritional content and medicinal value in the alleviation of many ailments due to inaffordability of the cost of modern health care and orthodox medicine. This study thus aimed at investigating the hematopoetic and hematinic potentials of the methanolic crude leaf extract of Ocimum gratissimum on phenylhydrazine-induced anemia in Albino wistar rat. RBC mean value recorded a dose and duration related statistically significant decrease (P < 0.05) in group D on day 3. This indicates that at high dose, the methanolic crude leaf extract of Ocimum gratissimum might have had some suppressive effects on hemopoesis in general and erythropoesis in particular. The most likely mechanism could have been that the synthesis of erythropoietin- which is one of the major hormones that stimulates the production of RBCs in the bone marrow, might have been adversely influenced. It could also be that the synthesis of the progenitor stem cells in the bone marrow could have been affected. However, these two suggestions are subject to further investigation and confirmation. This result was in consonant with some previous reports. ,
Interestingly, hemoglobin (Hb) and PCV demonstrated though not statistically significant (P > 0.05) marginal increase in their mean values throughout the study period. This indicates that at this stage of study, (day 3), the test groups that had been anemic due to phenylhydrazine administration have started recovering maybe actually due to the administration of the extract or by the natural physiological compensation by the bone marrow. This was further elucidated by statistically significant increase in the RBC indices. This was in disagreement with the result of an earlier report by Olusegun et al.  and Okon et al. Hence one might be tempted to suggest that Ocimum gratissimum methanolic crude leaf extract is most likely to have some haematinic potentials only as was indicated by a statistically significant reduction (P < 0.05) in the platelet count mean value throughout the study. This might also indicate that the methanolic crude leaf extract of Ocimum gratissimum could also possess some anti-thrombotic activity and hence, most likely to predispose long term consumers to bleeding or coagulation disorders as earlier suggested by Ephraim et al.,  Olusegun et al. and Okon et al. 
In addition, total white blood cell (WBC) recorded a marginal increase in the mean values throughout the study period though not statistically significant (P > 0.05). This indicates that the Ocimum gratissimum methanolic crude leaf extract could have mild leucopoetic activity, which could be both dose and duration dependent. It could also be suggested that the phenylhydrazine might not have destroyed or adversely affected not only the RBCs as supposed, but also might have probably destroyed maybe in a smaller amount of the leucocytes as was further elucidated by a statistically significant decrease (P < 0.01) in the neutrophil count on day 3 and in the ANOVA in group C. Hence, it could be assumed that, with increase in duration of exposure and maybe concentration of the methanolic crude leaf extract of Ocimum gratissimum coupled with the bone marrow recovery both the total WBC and neutrophil counts are likely to increase indicating it's antibacterial and antifungal properties as earlier reported by Okon et al. 
Furthermore, lymphocyte count recorded both dose- and duration-related statistically significant increase (P < 0.01) in this study. This indicates that the methanolic crude leaf extract of Ocimum gratissimum could have some lymphopoietic stimulatory activity. This result was in contrast to the other earlier reports by Ephraim et al.,  Olusegun et al.,  and Okon et al.  who recorded a statistically significant decrease even though they worked for a longer period as compared with this present study. Hence, it could be suggested that Ocimum gratissimum methanolic crude leaf extract possesses antibacterial, antifungal, and antiviral effects, hence could be used to remedy most infectious diseases although this is subject to further investigation.
| Conclusion|| |
From the result of this study, it can be concluded that the methanolic crude leaf extract of Ocimum gratissimum possesses both hematinic and leucopoietic potentials, which are dose- and duration-related.
| References|| |
|1.||Olusegun RJ, Josiah O, Lugman AO, Ayokunle O, Sikiru AB. Effects of aqueous extract of Ocimum gratissimum on haematological parameters of wistar rats. Biokemistri 2008;20:33-7. |
|2.||Tucker AO, DE Baggio T. The Big Book of Herbs, a comprehensive Illustrated Reference to Herbs of Flavour and Fragrance. Loveland COY: Inter-Weave Press; 2000. p. 30-4. |
|3.||Memphill I. The Spice and Herb Bible. Toronto: Robert Rose, 2000. p. 120. |
|4.||Brown D.: The Herb Society of America New Encyclopedia of Herbs and their uses. New York: D.K; 2001. p. 31. |
|5.||Onajobi FD. Smooth muscle contracting lipidic-soluble principles in chromatographic fractions of Ocimum gratissimum. J Ethnopharmacol 1986;18:3-11. |
|6.||Oboh G, Akindahunsi AA. Change in the ascorbic acid, total phenol and antioxidant activity of some sun-dried green leafy vegetables in Nigeria. Nutr Health 2004;18:29-36. |
|7.||Oboh G. Effect of blanching on the antioxidant property of some tropical green leafy vegetables. Food Sci Technol. (LWT) 2005;38: 513-7. |
|8.||Holetz FK, Veda-Nakamaru T, Filho BP, Cortez DA, Morgado-Diaz JA, Nakamaru CV. Effects of essential oil of Ocimum gratissimum on the trypanomastid Herpetomas samuelpessoai. Acta Protozool 2005;42:269-76. |
|9.||Sofowora A. Medicinal Plants and Traditional Medicine in Africa. Ibadan: John Wiley and Sons Limited; 1993. p. 142-6. |
|10.||Nwosu CO, Eke NC, Adamu M. Antihelminthic efficacy of aqueous extract of Ocimum gratissimum Linnaeus (Labiata) against nematode parasites of small ruminants. Niger J Exp Appl Biol 2005;6:145-8. |
|11.||Offiah VN, Chikwendu UA. Anti-diarrhoeal effects of Ocimum gratissimum leaf extract in experimental animals. J Ethnopharmacol 1999;68:327-30. |
|12.||Holm, Y. Bioactivity of basil.In: Ratmo Hiltumen R, Holm Y, editors. Basil, The Genus Ocimum. Australia: Harwood Academic Publishers; 1999. p. 20-4. |
|13.||Ngossoum MB, Ngang JJ, Tatsadjieu LN, Jirovetz L, Buchbauer G, Adjoudji O. Antimicrobial study of essential oils of Ocimum gratissimum leaves and Zanthoxylum Xanthoxylides fruits from Cameroun. Fitoterapia 2003;74:284-7. |
|14.||Iwalokun BA, Gbenle GO, Adewole TA, Smith SL, Akinsinde KA, Omonigbehin EO. Effects of Ocimum gratissimum essential oil at subinhibitory concentrations on virulent and multidrug resistant Shigella strains from Lagos Nigeria. APMIS 2003;4: 477-82. |
|15.||Pessoa LM, Morias SK, Bevilaqua CM, Luciano JH. Anthelmintic activity of essential oil of Ocimum gratissimum Linn and eugenol against Haemonchus contortus. Vet Parasitol 2002;109:59-63. |
|16.||Richard H. Chinese Herbal Medicine: Ancient art and modern Science. New York: Shocker Brook; 1978. p. 49-52. |
|17.||Ephraim LD, Salami HA, Osewa TS. Effects of aqueous leaf extract of Ocimum gratissimum on haematological and biochemical parameters in Rabbits. Afr J Biomed Res 2000;3:175-9. |
|18.||Okon UA, Ita SO, Ekpenyong CE, Davies KG, Inyang OI. Reduction of platelet and lymphocyte counts and elevation of neutrophil counts in rats treated with aqueous leaf extract of Ocimum gratissimum. Afr J Biochem Res 2011;5:303-6. |
[Table 1], [Table 2], [Table 3]