ANTI-INFLAMMATORY AND SPASMOLYTIC EFFECTS OF AQUEOUS-METHANOL EXTRACT OF OCIMUM GRATISSIMUM LEAVES

ABSTRACT

The leaves of Ocimum gratissimum were investigated as a possible drug for the management of forced labour, hepatopathy and gastro intestinal upset. Phytochemical analysis of the aqueous- methanol extract of the plant revealed the presence of alkaloids, flavonoids, phenols, saponins, tannins, glycosides, terpenoids, reducing sugar in varying quantities. Anti-inflammatory activity of the extract at doses of 100, 150 and 200 mg/kg body weight showed a significant decrease in egg albumin-induced paw oedema in earlier and later stages of the inflammation (p < 0.05) when compared  with  that  of  the  control.  Hepatoprotective  effect  of  the  extract  showed  a  dose dependent shield of the liver from the potent trichloromethyl free radical, a metabolite of carbon tetrachloride as seen on the histopathology of the liver. The liver marker enzyme (AST, ALT and ALP) activities were significantly reduced in the treated groups (p < 0.05) when compared with that of the untreated group. Glutathione concentration and superoxide dismutase activity also increased in all the treated groups (p < 0.05) when compared with that of the untreated group 2. There was non significant difference in GSH concentration and SOD activity in all the treated groups (p > 0.05) when compared with that of the normal (negative control). Catalase activity also significantly increased in a dose dependent manner. The effect of the crude extract and its fractions on rat uterus showed two active components that exhibited a biphasic function. At lower doses (below 1 mg/ml) the extract relaxed smooth muscle preparations while doses above 1 mg/ml contracted them. Higher doses of the crude extract potentiated oxytocin contractile effect and lower doses inhibited it. The crude extract and all the fractions inhibited acetylcholine- induced contractions in a dose dependent manner. The effect of the fractions on rat uterus showed  that  n-hexane and  ethyl  acetate fractions  contained  the relaxant  components  which inhibited oxytocin and acetylcholine-mediated contraction in dose dependent manner while the methanol fraction contained the contracting component. The extract also showed the same effect on   adrenoceptors   of   rabbit   jejunum.   At   lower   doses,   it   mimicked   the   effect   of sympathomimetics: adrenaline and noradrenaline, abolishing the spontaneous contractions, tonic strength and relaxing rabbit jejunum in a dose dependent manner. However at higher doses above 1 mg/ml, it contracted the tissue. The extract can also be useful in the treatment of adverse effects of cholinergic agonists such as salivation, lacrimation / sweating, gastro intestinal hyperactivity, diarrhoea and emesis. Gas chromatography and mass spectrometry analysis of the extract revealed the presence of thymol and carvacrol with both having 78.79% abundance. Other components included alpha D glucopyranose (alpha D-glucose), carbamic acid, aromatic compounds, organic acids and esters. This study has provided evidence for the continued use of this plant in the treatment of preterm delivery owing to it spasmolytic activity.

CHAPTER ONE

INTRODUCTION

Plant foods, especially vegetables, contribute substantially to both local diets and ethnomedicine in developing countries (Okafor, 1980; Gbile and Adesina, 1987). The use of plant materials as spices, condiments and for medicinal purposes dates back to the history of mankind (Ogunyemi,

1979). Recently, the exploitation of wild plants for medicinal purposes has gained wider acceptance in many countries of the world. This is primarily because of the belief that herbal drugs, besides being cheap and locally available, are without side effect. A century ago, most of the effective drugs were plant based. Examples include: digoxin (from foxglove) and morphine (from opium poppy). This led to the recognition of the value of traditional medical systems particularly of Asian origin and the identification of medicinal plants from indigenous pharmacopoeia.

Traditional medicine involves the use of herbs, animal and minerals in prevention and treatment of diseases. However, herbal medicine is the most widely used of the three. Right from the cradle, parts or all plant species have been used in medicine. Although many of the claims ascribed  to  herbal  medicine  have  not  been  proved  scientifically,  some  herbs  have  been extensively studied, thereby warranting their use as alternative to or complements of orthodox medicines. A major criticism associated with the use of herbal medicine is the absence of scientific evaluation of their safety profiles, since many of them have turned out to be toxic (Ernst, 2005; Yeung et al., 2008). About 80% of the world population is dependent wholly or partially on plant-based drugs (WHO, 1996). Nigeria and most developing countries of the world, rural and urban dwellers, literate or illiterate rely heavily on herbal preparations for treatment of various diseases despite availability of orthodox medicine (Nwabuisi, 2002).

1.1       Ocimum gratissimum

Ocimum gratissimum Linn locally referred to as ‘scent leaf’ is a vegetable commonly consumed as flavouring agent in soups. It belongs to the family of Lamiaceae and is widely distributed in the tropical regions of the world (Okigbo and Ogbonna, 2006). It is an erect, multi-branched perennial shrub that grows up to a height of two metres with a tap root and many adventitiousroots. It is mainly used as spice to flavour foods and meats (Okigbo, 1977). Its use as spice is known to reduce microbial load and extend the shelf life of foods. Scent leaf-enriched pepper soup is widely consumed in the tropics as an appetizer, stimulant and as refreshment or as a comfort food in homes, restaurants, hotels and hospitals. It is especially eaten in periods of recuperation from illness. The medicinal value of the plant lies in its component phytochemicals which produce definite physiological actions on human body (Proestos et al., 2006). Alkaloids, flavonoids, tannins, which contain phenolic compounds, are quite outstanding in this respect. Phenolic compounds are indeed well known as free radical scavengers, metal chelators, reducing agents, hydrogen donors, singlet oxygen quenchers (Proestos et al., 2006). Among all families of the plant kingdom, members of the Lamiaceae have been used for centuries in folk medicine.

1.1.1    Morphology of Ocimum gratissimum

Ocimum gratissimum is an aromatic, perennial herb, 1-3 m tall with erect stem, round- quadrangular, much branched, glabrous or pubescent, woody at the base, often with epidermis peeling in strips. The leaves measure up to 10 x 5 cm, and are ovate to ovate-lanceolate, sub- acuminate to acuminate at apex, cuneate and decurrent at base with a coarsely crenate, serrate margin, pubescent and dotted on both sides. The leaves show the presence of covering and glandular trichomes. Stomata are rare or absent on the upper surface while they are present on the lower surface. Ordinary trichomes are few, while the long ones up to 6-celled are present on the margins mostly; the short ones which are 2 celled, are mostly found on the lamina. Petioles are up to 6 cm long and racemes up to 18 cm long. The peduncles are densely pubescent. Calyx is up to 5mm long, campanulate and 5-7 mm long, greenish- white to greenish-yellow in colour. Nutlets are mucilaginous when they are wet. Fruit consisting of four, dry, one-seeded nutlets enclosed in the persistent calyx (Kritikar and Basu, 1993).

1.1.2    Geographical Distribution of Ocimum gratissimum

Ocimum gratissimum L is widely distributed throughout India, often cultivated in Ceylon, Java, tropical Africa, South America, Nigeria and Asia (Aruna and Sivaramakrishina, 1990). It is also found in some states of North India like Jammu, Punjab, Haryana and also cultivated in Kerala

and commonly known as Ramtulsi (Hindi); Shrubby basil, Fever plant, Fever leaf (English);

22

Banjere (Punjabi); Bantulsi, Ramtulsi (Bengali); Avachibavachi, Ramtulsi (Gujrati); Rama tulsi

(Malyalam); Nimma tulsi, Ramtulsi (Kannad) and Elumicham tulasi (Tamil).

1.1.2.1 Taxonomy of Ocimum gratissimum

Kingdom                    Plantae plant

Super division            Spermatophyta seed plant Division                       Tracheophyta, vascular plants Class                           Magnoliopsida  Dicotyledon Subclass                      Asteridae

Order                           Lamiales

Family                        Lamiaceaemint family

Genus                         Ocimum L. Basil

Specie                         Ocimum gratissimum  L

1.1.2.2 Common names

This herb is commonly known by names such as African basil, wild basil, shrubby basil, tree basil, East Indian basil, clove basil in English; menthe gabonaise in French; ruku-ruku rimba) in Indonesia, ruku-ruku in Malay, Clove basil and wild basil in Hawaii. Some of its other common names are Menthe gabonaise, Basilic sauvage and Basilic in French.  In Nigeria, it is variously called “Nchuanwu”, “Ahunji”, in Igbo, “Efirin” in Yoruba, “Ihiri eziza” in Bini, “Dai doya tagida” in Hausa or “Ntion” in Efik and is found in the wild or cultivated throughout the tropics and subtropics. In West Africa, O. gratissimum is commonly found around village huts and garden (Iwu, 1993) and cultivated for medicinal and culinary purposes.

1.2       Pharmacological Activities / Medicinal Uses of O. gratissimum

1.2.1    Antioxidant Activity of O. gratissimum

Antioxidants that are chemicals protect the body from damage caused by harmful molecules called free radicals. They prevent the oxidation of cells from toxins and reactive oxygen species. Living cells possess an excellent scavenging mechanism to avoid excess ROS induced cellular injury. However, with ageing and under influence of external stresses, these mechanisms become inefficient and dietary supplementation of synthetic antioxidants are required. In this context, aromatic plants, particularly their essential oils, are being evaluated for antioxidant activity. Ocimum  plants  contain  large  amounts  of  antioxidants  other  than  vitamin  C,  vitamin  E, flavonoids and carotenoids  (Baratta et al., 1998). The presence of many pharmacologically active compounds in Ocimum species provides them protection against free radical induced oxidative damage of cellular components. The antioxidant capacity of basil essential oils has been reported (Baratta et al., 1998).

The comparative antioxidant activity of essential oils of four Ocimum species (O. basilicum, O. canum, O. gratissimum and O. sanctum) by DPPH bioassay has been reported (Bunarathep et al., 2005) and it was found that O. gratissimum had the highest activity followed by O. sanctum, O. canum and O. basilicum with EC50 values 30.20, 767.82, 8 343.19 and 47 057.45 μg/ml respectively (Bunarathep et al., 2005). The antioxidant capacity of free volatile aglycons from basil (O. basilicum) by two different methods which were 2,2-diphenyl-1-picrylhydrazyl radical

scavenging method (DPPH) and ferric reducing/antioxidant power assay (FRAP). DPPH method shows that free volatile aglycones possess good antioxidant properties comparable with that of the  essential  oil  and  well-known  antioxidant  butylated  hydroxytoluene,  but  less  than  pure eugenol (Politeo et al., 2007). The results obtained by FRAP method showed that these compounds are somewhat less effective antioxidants than essential oil and butylated hydroxytoluene.

1.2.2    Anti-inflammatory Potential of O. gratissimum

The  anti-inflammatory  action  of  O.  gratissimum  has  been  established  and  it  arises  from inhibition of prostaglandin synthesis and neutrophil/macrophage chemotaxis. In vitro studies also revealed that these bioactive compounds inhibited nuclear factor-ĸB (NF-ĸB) activation induced by tumour necrosis factor (TNFα) and blocked cyclooxygenase activity (COX-2) in lipopolysaccharide-stimulated macrophages. COX-2 expression was triggered by growth factors, cytokines and LPS (Kim et al., 2003). Eugenol and carvacrol showed reduced inflammation by decreasing TNF-α and infiltration of neutrophils during pulmonary infection in animals. These compounds  when  administered  at  dosages  of 160  mg/kg  body weight  showed  reduction  in alveolar collapse and PMN infiltration in the lungs (Raghavenra et al., 2006). Eugenol also protected chemical-induced cellular dysfunction of macrophages and balanced the pro/anti- inflammatory mediators in mouse peritoneal macrophages.

1.2.3    Antimicrobial Properties of O. gratissimum

The indiscriminate use of antimicrobial agents has resulted in the emergence of a number of drug-resistant bacteria and fungi. To overcome the increasing resistance of pathogenic microbes, more effective antimicrobial agents with novel mode of action must be developed. Essential oils derived from several ocimum species have been reported to be active against several gram- positive and gram-negative bacteria as well as against yeasts and fungi due to their terpenic constituents. Recently, essential oils and extracts of certain plants have been shown to have antimicrobial effects (Lang and Buchbauer, 2012) as well as imparting flavour to foods (Burt, 2004). Janssen et al. (1998) investigated the antimicrobial potential of four Ocimum species viz.,

O. canum, O. gratissimum, Ocimum trichodon and O. urticifolium grown in Rwanda against

Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Trichophyton mentagrophytes var. interdigitale. They found that O. canum was the most effective amongst the studied oils.

1.2.4    As Skin Permeation Enhancers

The delivery of drugs via skin routes has been extensively studied. Dermal and transdermal drug delivery are often limited by poor permeability of the stratum corneum (SC) to drugs, which precludes their crossing the skin at therapeutic rates. The barrier properties of the SC can be reduced by the use of natural products. Ocimum gratissimum is receiving considerable interest as enhancers to improve drug permeation. Essential oil of sweet basil (O. basilicum) was evaluated as skin permeation enhancer to promote the percutaneous absorption of drugs and reported it as noble enhancers (Fang et al., 2002).

1.2.4.1 Cardiovascular and Anti-lipidaemic Actions of Ocimum gratissimum

Both Ocimum gratissimum oil and eugenol decrease the blood pressure in conscious deoxycorticosterone acetate (DOCA)-salt hypertensive rats exhibiting its cardiovascular effects. In an earlier research, Lahlou et al. (2004) found that intravenous treatment of normotensive rats with O. gratissimum oil and its main constituent (eugenol) dose dependently decreased mean aortic pressure. Eugenol was also reported to induce vasorelaxant effects on rat and rabbit thoracic aorta as well as on rat mesenteric vascular bed (Nishijima et al., 1999). Essential oil from O. sanctum leaves has been shown to have the potential for lipid-lowering action. The anti- hyperlipidaemic ability of the essential oil extracted from O. sanctum leaves in rats fed with high cholesterol diet was investigated (Suanarunsawat et al., 2009). The researchers found no significant difference in body weight gain, food intake, and heart weight in all groups of rats. They also found that phenylpropanoid compounds, the main composition of O. sanctum essential oil was responsible for the lipid lowering effect in high cholesterol rats (Suanarunsawat et al.,

1999). In another finding by treating with O. sanctum oil to rats fed with a high cholesterol diet, it was found that essential oil suppressed the high serum lipid profile and atherogenic index. They concluded that essential oil of O. sanctum has lipid-lowering and antioxidative effects that protect the heart against hypercholesterolemia.

1.2.4.2 Immunomodulatory and CNS Activity of Ocimum gratissimum

In current competitive life, stress is a very common problem for human beings. Accumulation of more free-radicals due to stress leads to adverse effects on various vital organs and tissues of the human body. Ocimum essential oil has gained special attention due to its biological properties. However, little is known about its immunomodulatory effects. The immunomodulatory effect of ocimum oil occurs via modulating GABAergic activity which influences both humoral and cell- mediated immunological parameters in native non-stressed as well as stressed animals (Mediratta et al., 2002). It has been reported that terpenes have a protective effect against pentylenetetrazole and picrotoxin-induced convulsions (Librowski et al., 2000). Modulation of glutamergic and GABAergic transmission are mechanisms indicated for anticonvulsant action on monoterpenes. Linalool,  the most  abundant  constituent  of  Ocimum  oil,  has  anticonvulsant  activity  against pentylenetetrazole-induced convulsion and also through inhibition of glutamergic transmission as well as through suppression of voltage-gated current (Elisabetsky et al., 1995).

1.2.4.3 Medicinal Uses of O. gratissimum

O.  gratissimum  has  been  used  extensively  in  the  traditional  system  of  medicine  in  many countries. In the North east of Brazil, it is used for medicinal, condiment and culinary purposes. The flowers and the leaves of this plant are rich in essential oils so it is used in preparation of teas and infusion (Rabelo et al., 2003). In the coastal areas of Nigeria, the plant is used in the treatment of epilepsy, high fever and diarrhoea (Effraim et al., 2003). In the Savannah areas, decoctions of the leaves are used to treat mental illness. O. gratissimum is used by the Igbos of South Eastern Nigeria in the management of the baby’s cord, to keep the wound surfaces sterile. It is also used in the treatment of fungal infections, fever, cold and catarrh (Ijeh et al., 2005). Brazilian tropical forest inhabitants use a decoction of O. gratissimum roots as a sedative for children.  People  of  Kenya  and  sub  Sahara  African  communities  use  this  plant  for  various purposes which include; the leaves are rubbed between the palms and sniffed as a treatment for blocked nostrils, they are also used for abdominal pains, sore eyes, ear infections, coughs, barrenness, fever, convulsions, and tooth gargle, regulation of menstruation and as a cure for prolapse of the rectum (Matasyoh et al., 2007).

O. gratissimum has biological activity such as antidiabetic, antiseptic, antidiarrhoea (Gbolade, 2009; Akinyemi et1 al., 2004; Ezekwesili et al., 2004). The leaves of O. gratissimum are used as a laxative and its infusion serves as a relief for respiratory disorders, headaches, fever, diarrhoea, dysentery, pile and convulsion (Idu et al., 2005; Danziel, 1980). The whole plant is used as a remedy for gonorrhoea, catarrh conditions, cough, constipation, ringworm, flatulence, hypertension and anaemia. Its leaves are used as sponge to remove skin blemishes and have been formulated into skin creams used for treating dermatological disorders as well as toothpastes used in maintaining oral hygiene (Odebiyi and Sofowora, 1978).

1.3       Phytochemistry

Phytochemistry is the study of natural bioactive substances found in plants that work with nutrients and dietary fibre to protect against diseases (Doughari et al., 2009). These non-nutrient plant chemical compounds or bioactive components are often referred to as phytochemicals (‘phyto- from Greek – phyto meaning ‘plant’) or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pests (Abo et al., 1991; Liu, 2004; Doughari et al., 2009). Studies indicate that phytochemicals, working together with nutrients found in fruits, vegetables and nuts, may help slow the ageing process and reduce the risk of many diseases including cancer, stroke, high blood pressure, cataracts, osteoporosis and urinary tract infections (Gao et al., 2001).

They can have complementary and overlapping mechanisms of action in the body including antioxidant effects, modulation of detoxification enzymes, stimulation of the immune system, modulation of hormone mechanisms as well as antibacterial and antiviral effects (Gao et al.,

2001). Medicinal plants are of great importance to the health of individuals and communities. The medicinal value of these plants lies in some chemical substances that produce definite physiological actions on the human body. Many medicinal plants are used as spices and food plants. They are also sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes (Okwu, 2001). Medicinal herbs are significant sources of synthetic and herbal drugs. Medicinal plants have active ingredients which are responsible for most of the biological activities they exhibit (Fukumoto and Mazza, 2000).

1.3.1    Phytochemical Constituents of Plants

Phytochemicals are a heterogeneous group of chemical compounds with numerous biologically active substances that have potential disease inhibiting-capabilities (Akinmoladun et al., 2007).

Phytochemicals   (plant   chemicals)   are   bioactive   substances   of   plants   that   have   been associated with the protection of human health against chronic degenerative diseases (Fukumoto and Mazza, 2000). These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1998; Okwu, 2005). More than 900 different phytochemicals have been found in plant foods and more probably will be discovered (Polk, 1996).

1.3.2     Alkaloids

Alkaloids are natural products that contain heterocyclic nitrogen atoms; they are basic in character.   The   name   was   derived   from   “alkaline”   and   it   was   used   to   describe   any nitrogen-containing base. These are the largest group of secondary chemical constituents. They are made largely of ammonia compounds consisting mainly of nitrogenous bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring. The compounds have basic properties and are alkaline in reaction, turning wet red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid, typically as 1°, 2° or 3° amines, contribute to the basicity of alkaloid (Firn, 2010). Alkaloids generally exert pharmacological activity particularly in mammals such as humans.

Many of our most commonly used drugs are alkaloids from natural sources and new alkaloidal drugs are still being developed for clinical purpose (Roberts and Winks, 1998). Most alkaloids with biological activity in humans affect the nervous system, particularly the action of neurotransmitters, example, acetylcholine, adrenaline, noradrenaline, gamma-aminobutyric acid (GABA), dopamine and serotonin (Schmeller and Wink, 1998). They react with acids to form crystalline salts without the production of water (Firn, 2010). The majority of alkaloids exist in the solid state such as atropine, some as liquids containing carbon, hydrogen, and nitrogen. Most alkaloids are readily soluble in alcohol. Though they are sparingly soluble in water, their salts are usually soluble. The solutions of alkaloids are intensely bitter. These nitrogenous compounds function in the defence of plants against herbivores and pathogens, and are widely exploited as pharmaceuticals,  stimulants,  narcotics,  and  poisons  due  to  their  potent  biological  activities

1.3.3     Flavonoids

Flavonoids are an important group of phytochemicals widely distributed among the plant flora. It is made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. Flavonoids are also referred to as bioflavonoids. They are organic compounds that have no direct involvement in the growth or development of plants. They are plant nutrients that when consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the human body. Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than 4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960). Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids, flavanones, flavonols and anthocyanidines.

Flavones are characterized by a planar structure because of a double bond in the central aromatic ring. Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli and berries (Bernatora et al., 2002). Flavonones are mainly found in citrus fruit, an example is narigin. Flavonoid is involved in scavenging of oxygen- derived free radicals (Nijveldt et al., 2001). It has been identified as a potent hypolipidaemic agent in a number of studies. Experimental studies showed that flavonoids enhance vaso-relaxantprocess (Bernatora et al., 2002) and prevent platelet activity-related thrombosis.

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