Friday, August 10, 2012

The effect of Ocimum gratissimum on the histology of the lungs of Albino rat

BY
EFIRI EBI CHRISTOPHER

INTRODUCTION
Herbal medicines tend to look primitive and unscientific when compared to synthetic (conventional) drugs, which are thought to be more reliable than those made from plants. Herbal medicine is still the mainstay of about 75-80% of the world population, mainly in the developing countries for primary health care (Kamboj, V. P, 2000). This is primarily because of the general belief that herbal drugs are without any side effects, beside being cheap and locally available. The use of plants for healing purposes predates human history and forms the origin of much modern medicine. Many synthetic drugs originated from plant sources: a century ago, most of the few effective drugs were plant-based. Examples include: Aspirin (which is a chemical copy of the analgesic chemical in the bark of willow trees), digoxin (from fox glove), guanine (from the bark of various cinchona tree species which was used in the treatment of malaria) and morphine (from the opium poppy). (Vicker A. and Zollman C., 1999).
It’s been seen that a lot of plants had played effective roles in the production of drugs hence, we now look at the plant,The perennial plant Ocimum gratissimum (scent leaf) is widely distributed in the tropics of Africa and Asia. It belongs to the family labiatae and it is the most abundant of the genus Ocimum. In the southern part of Nigeria, the plant is called “ effinrin-nia” by the Yoruba speaking tribe, “Nchonwu” in Igbo, while in the northern part of Nigeria, the Hausas call it “Daidoya”. (Effraim K.D et al., 2001).
It is a perennial plant that is woody at the base. It has an average height of 1-3m high. The leaves are broad and narrowly ovate, usually 5-13cm long and 3-9cm wide. It is a scented shrub with lime-green fuzzy leaves. (Wagner et al., 1999)

PLANT CLASSIFICATION
Kingdom Plantae – plant
Subkingdom Tracheobionta – vascular plant
Super division Spermatophyte - seed plant
Division Magnoliophyta – flowering plant
Class Magnoliopsida – Dicotyledon
Subclass Asteridae
Order Lamiales
Family Lamiaceae – mint family
Genus Ocimum L. – basil
Specie Ocimum gratissimum L.-Africa
(Plant classification culled from: USDA 2008)

CONSTITUENTS OF O. GRATISSIMUM
Photochemical evaluation of this plant has shown that it is rich in alkaloid, tannis, phytates, flavonoids and oligosaccharides. It has tolerable cyanogenic content (Ijeh et al., 2004). The volatile aromatic oil from the leaves consists mainly of thymol (32-65%) and eugenol; it also contains xanthones, terpenes and lactones (C.N. Ezekwesili et al., 2004). Characterization of its ethanolic extracts revealed the presence of non – cyclic sesquiterpenes, phenols (Esvanzhuga, 1986).

USES OF OCIMUM GRATISSIMUM
In folk medicine, Ocimum gratissimum is extensively used throughout West Africa as a febrifuge, anti- malarial and anti- convulsant. The crushed leaf juice is used in the treatment of convulsion, stomach pain and catarrh. Oil from the leaves have been found to posses antiseptics, antibacterial and antifungal activities. (C.N. Ezekwesili et al., 2004).
In the coastal area of Nigeria, the plant is used in the treatment of epilepsy, (Osifo, 1992), high fever (Oliver 1980) and diarrhea (Oliver, 1980 and Sofowara, 1993). While in the savannah areas decoctions of the leaves are used to treat mental illness (Abdulrahman, 1992).
Ocimum gratissimum is used by the Ibos of southern Nigeria in the management of the baby’s cord. It is believed to keep the baby’s cord and wound surface sterile. It is used in the treatment of fungal infections, fever, cold and catarrh .(Iwu, 1986).
Clinical trials in creams formulated against dermatological disease have yielded favorable result (Edeoga and Eriata, 2001). Nutritional importance of this plant centers on it’s usefulness as a seasoning because of its aromatic flavor . (C.N. Ezekwesili et al., 2004).

GROSS ANATOMY OF THE LUNGS
The lungs are paired cone-shaped organs which lie in the thoracic cavity. They extend from the diaphragm to just slightly below the clavicle and lie above the rib posteriorly and anteriorly. The lungs are separated from each other by the heart, viscera and the great vessels of the mediastinum. The lungs are attached to the heart and to the trachea by structures that comprise the roots of the lungs.
The roots of the lungs are formed by structures entering and emerging from the lungs at its hila. The hilium of the lung is an area of the medial surface of each lung through which the structures that form the root of the lungs enter and leave the lung. These structures include the main bronchus, pulmonary vessels, bronchial vessels, lymphatic vessels and nerves. The lungs are enclosed by the pleural membrane which consists of the parietal and visceral pleura.
The lungs are divided into lobes by the horizontal and oblique fissures. The right lung has 3 lobes, while the left lung has 2 lobes. The right lung is larger and heavier than the left, but shorter and wider because the right dome of the diaphragm is higher than the heart and the pericardium bulge more to the left. The anterior margin of the right lung is relatively straight, but the anterior margin of the left lung has a deep cardiac notch which overlaps the heart. (Moore and Dailey,1999).
Anatomically each lung consist of the following
1. AN APEX: This is the blunt superior end of the lung. It ascends into the neck for about 1 inch (25cm) above the clavicle into the root of the neck that is covered by the cervical pleura.
2 THREE SURFACES: They include the following
• Costal surface: It is a convex-shaped surface and, it is large and smooth. It is related to the dorsal pleura that separates it from the ribs, coastal cartilages and the innermost intercostals muscles. The posterior part of the surface is related to the bodies of the thoracic vertebra and is sometimes referred to as the vertebral part of the costal surface.
• Mediastinal surface: It is convex-shaped due to its relation to the middle mediastinum which contains the pericardium and the heart. It also contain the hilium and thus receives the root of the lungs around the pleura forms a covering or pleural sleeve.
• Diaphragmatic surface: It is concave-shaped and it forms the base of the lungs that rests on the dome of the diaphragm.

3. THREE BORDERS: They include :
Anterior Border: The costal and mediastinal surfaces meet at this point or border and overlap the heart. The cardiac notch indents the border of the left lungs
• Inferior Border: It circumscribes the diaphragmatic surface of the lungs and separates it from the coastal and mediastinal surfaces.
• Posterior Border: At this border the costal and mediastinal surfaces meet posteriorly. It is broad and rounded and lies in the cavity at the side of the thoracic region of the vertebral column (Moore and Dailey, 1999).
BLOOD SUPPLY TO THE LUNGS
The bronchi, connective tissue and visceral pleura of the lungs receive their blood supply from the bronchial arteries which are branches of the descending aorta. The bronchial veins which communicate with the pulmonary veins drain into the azygos and hemiazygos veins.
The alveoli receive deoxygenated blood from the terminal branches of the pulmonary arteries. The oxygenated blood leaving the alveolar capillaries drains into the tributaries of the pulmonary veins which follow the intersegmental connective tissue septa to the lung root. Two pulmonary veins each leave the lung root to empty into the atrium of the heart. (Snell, 1999).

MICROSCOPIC ANATOMY OF THE LUNGS
Each lung is covered by two pleura, consisting of a layer of mesothelial cells with underlying connective tissue. This connective tissue extends inward between pulmonary lobes and lobules and septa carrying ramification of pleural blood and lymphatics vessels. The respiratory epithelium undergoes progressive transition from a tall pseudo stratified columnar ciliated form in the larynx and trachea to a simple cuboidal non-ciliated form in the smallest airways.
The wall of the respiratory bronchiole is lined with cubiodal epithelium at the proximal portion, but disappears in the distal portion of the respiratory bronchiole. Smooth muscle is located adjacent to the epithelium. A small branch of the pulmonary artery accompanies the respiratory bronchiole and numerous alveoli open into the alveolar duct.
The oval alveoli are lined by squamous epithelium. Adjacent alveoli share a common inter alveolar septum; which contains capillary plexus supported by fine connective tissue fibers, fibroblasts and other cells.

AIM AND OBJECTIVE OF STUDY
In view of its many uses especially in Nigeria and the fact that traditional medicine practitioners prescribe and administer decoctions of the leaves of Ocimum gratissimum to patients without regard to its possible adverse effects, this present investigation is to access its potential toxic effect on the lungs in albino rat if any and to determine the possible histopathological changes that may arise as a result of an intake of the extract.


LITERATURE REVIEW
A lot of research has been carried out on the herb, Ocimum gratissimum. Though, literature search has not revealed any study on the effect of Ocimum gratissimum on the histology of the lung of albino rat, some works closely related to it has been documented.
In 2004, researchers carried out an in vitro study on the ileum of guinea pig. The effect of Ocimum gratissimum extract on intestinal motility, determined by the magnitude of contraction of isolated guinea pig ileum.
Results shows that Ocimum gratissimum extract mimicked the action of adrenalin and nor adrenaline on the isolated guinea pig ileum by abolishing the acetylcholine-induced contraction of the smooth muscle of ileum ( C.N. Ezekwesili et al., 2004).
In 2005, a research work was carried out on the antimicrobial effect of aqueous and ethanolic fraction of two spices, Ocimuim gratissimum and Xylopia aethiopica were analyzed against five pathogenic organisms; Staphylococcus aureus, Esherichia coli, Streptococcus fecalis, Pseudomonas aeruginosa and lactobacilli. The result indicate that ethanolic extracts of Ocimuim gratissimum had a minimum inhibitory concentration (MIC) of 30µg/ml againsT
Staphylococcus aureus, Esherichia coli, Pseudomonas aeruginosa and lactobacilli while for Streptococcus fecalis the MIC was 15µg/ml. Aqueous extract of Ocimuim gratissimum had an MIC of 12.5µg/ml against Staphylococcus aureus, Esherichia coli and Streptococcus fecalis, while for Pseudomonas aeruginosa and lactobacilli the MIC was 6.25µg/ml and 25µg/ml, respectively. (Ijeh I. I. et al., 2005).
The findings suggest that the anti-microbial activity of these spices reside in their aqueous functions and also indicate that very low concentrations are required to achieve anti-microbial effects and justifies the ethno medical use of Ocimuim gratissimum leaves as a plaster to cover wound surface and baby cord. Pseudomonas aeruginosa, Esherichia coli, Staphyloccus aureus are common human commensals and have been incriminated in the infection of wounds (Duguid et al 1985).
The findings also justifies the application of Ocimuim gratissimum in dermatological creams and indicate that effective doses could be achieved at very low concentrations; and also shows that the aqueous fractions of both plants have more potential as an antimicrobial agent than its ethanolic fractions. (Ijeh I. I et al., 2005).
In 2006, a research was carried out on the safety and hypoglyceamic properties of aqueous leaf extract of Ocimum gratissimum in streptozotocin induced diabetic rat. Diabetes mellitus was induced in the rats by single injection of 70mg/kg streptozotocin and graded doses of the aqueous leaf extract of Ocimum gratissimum was administered orally to the experimental diabetic rats for 28 days. Administration of this aqueous leaf extract caused a statistically significant reduction in plasma glucose level in streptozotocin induced diabetic rats. The extract appear non toxic as evidenced by normal serum levels of AST (Aspartate transaminase), ALT (Alanine transaminase), ALP (Alkaline phosphatase), and bilirubin.
This data appear to agree with claimed hypoglycaemic effect of Ocimum gratissimum. Twenty-eight day administration of the extract to non diabetic rats did not produce any significant difference in the plasma glucose concentration of non diabetic extract treated rats compared with control. (U.G. Egesie et al., 2006).
The mechanism of the hypoglycaemic effects of the aqueous extract of this plant is not clear at the moment. However reports are available to show that anti-diabetic plants may affect circulating insulin level. (Lamela et al., 1985).
In studies, in the department of veterinary physiology and pharmacology, university of Ibadan, Nigeria. The effect of aqueous extracts hibiscus sabdariffa calyces and Ocimum gratissimum leaves on the intestinal transit in rats were determined in experimental rats. The leaves of Ocimum gratissimum and hibiscus sabdariffa calyces were oven dried and then pulverized. 10% extracts of both powder were made and administered orally to rats at varying doses. Test rats were given the 10% extract of Ocimum gratissimum and hibiscus sabdariffa at 0.5ml/100g, 1ml/100g, 2ml/100g body weight. Control rats received saline instead of extracts.
After 30minutes, each animal was then given 1.5ml of a dye solution orally. 1hour after administration of the dye each animal was then sacrificed and the intestine carefully dissected out. The result showed that the extract of Ocimum gratissimum and hibiscus sabdariffa caused a reduction in the transit point of the dye, the extract of hibiscus sabdariffa (zobo) was more effective. The reduction in transit point, and hence the increase in transit time by both extracts indicates that the plants could be useful at appropriate doses in the control of diarrhoea. Hibiscus sabdariffa would be more effective in this regard. (Owulade M.O. et al., 2004).
In 2004. Orafidiya et al carried out studies on the acute and sub-chronic toxicity of the essential oil of L. leaf. Oral and intra-peritoneal acute toxicity and the sub-chronic intra-peritoneal toxicity of the essential oil of Ocimum gratissimum Linn, Lamiaceae (Ocimum oil), was investigated. The acute toxicity test involved the oral and intra-peritoneal administration of graded doses of Ocimum oil prepared as a 4% v/v emulsion to 2 groups each of 30 rats and mice. LD50 and LD100 were determined for both routes and species. In the sub-chronic toxicity study, 25 male Sprague-Dawley rats were randomized into 4 test groups (treated with three graded sub-lethal doses of Ocimum oil prepared as a 4% v/v emulsion) and a control. Organs and blood samples were taken for analyses after a 30 day treatment period. A dose-dependent sedative effect of Ocimum oil was observed during the acute toxicity study in mice and rats and in the sub-chronic test in rats. Evidence of treatment, route, and dose-dependent toxicity were detected in both studies. Changes in weight of the testes, hearts, kidneys, intestines and lungs of the rats were statistically insignificant (ANOVA P < 0.05). Data analyses of blood biochemical, haematological and histopathological findings showed significant differences between control and treated groups and revealed that Ocimum oil is capable of invoking an inflammatory response that transits from acute to chronic on persistent administration. While the study revealed that Ocimum oil might be better tolerated when administered orally for systemic delivery, the oil has toxic potentialities that should not be overlooked (Orafidiya et al, 2004).
In a 1998 in vitro study, researcher screened the pharmacological activities of aqueous extract of Ocimum gratissimum in isolated rabbit jejunum, rat stomach and also it analgesic properties in mice. The extract caused a dose dependent inhibition of he rabbit jejunum spontaneous pendular movement. The blocking effect on acetylcholine induced contraction was non-competitive in the rat stomach.
The result of the analgesic study showed that the extract evoked a
prolongation of reaction time of 80% (p< 0.05) over 20minutes observation time with no signs of toxicity.( P.I. Aziba et al., 1998).
In 2005, the antifungal activity of Ocimum gratissimum towards dermatophytes were investigated at Falculdade de farmácia da UFG, Goiánia, Goiós, Brazil. Extracts of Ocimum gratissimum were investigated for in vitro antifungal activity, using agar dilution technique against dermatophytes. The extracts produced antifungal activities against Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes.
These results shows that extracts of Ocimum gratissimum are active in-vitro against human pathogenic dermatophytes. (M.R.R. Silva et al., 2005).
Also in an attempt to explain the scientific basic for the medicinal and nutritional benefit of leafy vegetables, the phytochemical constituent, antioxidant and antimicrobial activity were assessed. In the study, dried leaves of Ocimum gratissimum were extracted with rectified spirit and the extracts were subsequently analyzed for their phytochemical constituent, antioxidant property as typified by its total phenol content, free radical scavenging ability and reducing power, as well as the ability of the extracts to inhibit the growth of some clinically isolated Enterobacteriocea and some fungi.
The result of the study revealed that Ocimum gratissimum extracts contains tannin, saponin, anthraquinone, alkaloids and glycosides. The total phenolic content of the extract was 3.6g/100g, while the reducing power and free radical scavenging ability were 2.4OD and 51.2% respectively. 10mg/ml of the extract inhibited the growth of the following bacteria: Proteus sp, Pseudomonads aeruginosa, Shigella dysentria and Staphylococus; and fungi (Saccharomyces cerevisae and Candida albicans). Furthermore, the inhibition of both the fungi and bacteria were found to be dose-dependent. (Ganiyu Oboh, 2006).
It could therefore be concluded that part of the reasons for the use of Ocimum gratissimum leaves in folk medicine against gastrointestinal disorders and haemorrhoids could be as a result of its antioxidant and antimicrobial property which is hinged on the array of pharmacological active phytochemicals present in the vegetable leaves. (Ganiyu Oboh 2006).
Ocimum gratissimum has shown to have a dose-dependent effect in some organs tested and also have antifungal and antimicrobial properties. In the course of its use in the treatment of different ailment, this work is aim studying if the extract (Ocimum gratissimum) will have any deleterious effect on the lungs.



MATERIALS AND METHOD
MATERIALS
The materials used for the experiment include:
• 20 Albino rats of both sexes
• Plastic cages with iron netting
• Saw dust (litter)
• Animal feed (growers mash) and water
• Laboratory coat and gloves
• Dissecting set and dissecting board
• Fresh leaves of Ocimum gratissimum
• Hand grinder
• Measuring Cylinder and plastic specimen bottle
• Weighing balance
• Water bath
• Dessicator
• Syringes
• Sample bottles
• 10% formal saline
• Cotton wool and anesthesia (Di ethyl ether)
• Graded alcohol (50%, 70%, 95% and absolute alcohol)
• Glass slides and slide rack
• Hot plate
• Xylene
• Paraffin wax
• Embedding mould and pot
• Deepex (DPX) mountant
• Haematoxylin and Eosin stain
• Cover slip
• Microscope (light)
• Slide rack
• Orbit shaker
• Diamond pencil
• Rotatory microtome


METHODS
Collection and identification of plant materials
Fresh leaves of Ocimum gratissimum (scent leaf) were purchased from Mile 3 ultra modern market, Diobu, Port Harcourt. The leaves were identified as Ocimum gratissimum leaves by Dr. B. C Ndukwu, of the Department of Plant Science and Biotechnology, Faculty of Science, University of Port Harcourt, Choba.
3.2.2 Preparation of Ocimum gratissimum (scent leaf) extract
Fresh leaves of Ocimum gratissimium were grinded with hand grinder. The extract were squeezed from the grinded leaves. The concentration of the extract was absolute.
3.2.3 Experimental animals
20 albino rats of both sexes weighing between 125-375g wt were purchased from the animal house, University of Port Harcourt, Choba. They were kept in plastic cages with iron nettings in the experimental laboratory (histology Lab.) of the College of Health Science, University of Port Harcourt. They were allowed to acclimatize for a period of two weeks and fed with growers mash. They were also given tap water at pleasure using water bottles.
Administration of extract
The rats were grouped into 4 of five animals per group according to the weight of the rats. Group 4 serve as control group; group 1, 2 and 3 received the aqueous extract of Ocimum gratissimum at doses of 5, 3 and 2ml/kg body weight respectively.
Administration of extract were carried out orally by the use of syringes in order to determine the actual dose, animals received their doses 3 (three) times weekly for a period of three weeks. At the end of the third week, the animals were sacrificed after ether anesthesia by incision made on the midline of the ventral surface of the rats with the lungs excised. In each sacrifice, the lungs were dissected out, labeled and put into specimen bottles which contain 10% formal saline for further tissue processing.

TISSUE PROCESSING
The organ ( lungs) were fixed in 10% formal saline , which serve as a fixative. This process is known as fixation and it was carried out in order to prevent putrefaction. The method used for tissue processing was the paraffin wax methods. In the course of processing, the tissue was taken through the following processes;
1. DEHYDRATION: This is the removal of water from the tissue. It was done by passing the tissue through an ascending grade of alcohol as follows:
50% alcohol for 2 hours
70% alcohol for 2 hours
95% alcohols for 12 hours (over night)
Absolute alcohol for 2 hours
During the exchange from one grade to another, the tissues were agitated rapidly.
2. CLEARING: The purpose of clearing is to remove all the alcohol content from the tissue sample. This is usually done by the use of xylene. The dehydrated tissues are placed in xylene for 2 hours.
3. IMPREGNATION: The process is also known as infiltration. It helps to remove the clearing agent in the tissue. The lung tissues were transferred from xylene to molten paraffin wax (melting point 54°C-60°C) for 2 hours.
4. EMBEDDING: This process involve immersing the tissue in molten paraffin wax to solidify. This process enables the tissues to be sectioned properly. Metallic embedding moulds were used; after embedding the tissue blocks obtained were now casted onto wooden blocks.
5. SECTIONING: The casted tissue blocks were then trimmed with a microtome at 20-25 microns. The tissue blocks were trimmed so that the tissues would be more visible. They were later placed in ice to ensure hardening of the wax. After trimming the microtome knife was changed and another was set in place on the microtome for tissue sectioning. The ribbons were then floated in a warm water bath (37°C) and then the best ribbons were picked out with forceps and placed on slides. The slides were labeled using diamond pencils and were transferred to a slide rack. The slides were later put in an embedding oven for one hour.
6. STAINING: After sectioning, the staining procedures are as follows:
• The slides were warmed with hot plate and treated with xylene to remove wax for 5-10mins.
• They were dehydrated in descending grade of alcohol, from absolute to 95% alcohol and finally to 70% alcohol (in seconds).
• Slides were rinsed in water.
• They were then stained with haematoxylins (15-25mins)
• They were rinsed in water to remove excess stain (1min).
• The slides were dipped in acid alcohol (in seconds). This is done in order remove excess stain and to help the nucleus absorb the stain.
• The slides were rinsed in water (two changes of water) for 3-5 seconds. This process is known as BLUING and it gives the stain its characteristic background.
• The slides were then stained with Eosin ( 3-5mins) and then washed in water to remove excess stain.
• They were dehydrated in 70%, 90% and then to absolute alcohol.
• The slide were cleared in two changes of xylene; mounted on DPX and then covered with cover-slipped.
• The slide were left to dry before viewing on microscope and photographing.
7. MICROSCOPY: Here the slides were carefully studied under low and high magnification of the light microscope. Comparisons were made with the slides of the control group for detection of histological changes.



RESULTS AND OBSERVATION
The following observations were made in course of this research work and will be described as follow:
During the weekly oral administration of the aqueous extract of Ocimum gratissimum at doses of 5, 3 and 2ml/kg body weight, there was no mortality in all group tested.
5.1 GENERAL OBSERVATION
After administration of the aqueous extract of Ocimum gratissimum the animals in each group look dizzy when compared with the control group (group 4) but regain consciousness after a while; and they usually increase their intake of water immediately after administration.
CHAPTER FIVE
DISCUSSION AND CONCLUSION
DISCUSSION
The study of the toxic or adverse effects of crude drugs of plant origin is essential in order to provide guide to their safe usage and eventually standardization ( Effraim K. D et al 2001).
This is especially pertinent, as traditional medicine practitioners and some individuals often administer such preparations without regard to there possible adverse effect. From the result obtained, it was observed that the animals given higher doses of the extract, showed greater changes in their lungs architecture; when compared to those given lower dose.
Also from the result obtained, the extract (Ocimum gratissimum), when administered showed certain changes in the histology of the rat lung such as congestion of blood vessels, interstitial pneumonitis, which is as a result of the inflammation of the lung tissues. Inflammation result when there is an injury to the tissue which is either caused by bacteria, trauma, chemicals, heat or any other phenomenon (Guyton A.C, 2000).
It was also observed from the results that sections of the lungs showed widening of the interstitium with areas of alveolar damage. This widening occurred as a result of edema. Edema refers to the presence of excess fluid in the body tissues. The edema was more pronounced in group 2 animals.
CONCLUSION
This study has shown the adverse effect of the aqueous extract of the plant, Ocimum gratissimum on the lungs of the albino rat on the histological aspects especially when administered at high doses. It has also shown widening of the interstitium due to edema, congestion of blood vessel, alveolar damage, infiltration by inflammatory cells and interstitial pneumonitis (inflammation of the lung tissues).
All these depend on the level of the doses given. Therefore, extreme cautions should be taken in the administration of the plant extract.
Also researchers should carry out further experiments on the plant Ocimum gratissimum because most of the works had only focused on the antifungal, antimicrobial and phytochemicals of the plant.



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