35 (25-38%) and is used in manufacture of perfumes, colored soaps and synthesis of Vitamin A (Shah, et al., 2011).
Anti-microbial activity: The ethanolic extracts of the leaves of Lemon grass showed potential antibacterial property against Staphylococcus aureus. Flavonoids and Tannins found in the extract are responsible for the activity (Danlami, 2011).
Anti-fungal activity: Candida albicans is an important pathogen of human infections;
moreover, other species can be associated with some infections. The anti-fungal activity of lemongrass and citral against Candida species was studied and the study showed that lemongrass oil and citral have a potent in vitro activity against Candida spp. (Silva et al., 2008).
Anti-protozoan activity: The family Trypanosomatidae harbours protozoans that are agents of important illnesses in humans, animals and in plants. This family also includes some lower trypanosomatids such as Crithidia, Blastocrithidia, and Herpetomonas, monoxenous protozoans usually found in insect hosts. The essential oil extracted from Cymbopogon citrates showed anti-protozoan activity against Crithidia deanei (Pedrose et al., 2006).
36 digestible carbohydrates and lipids. It is easy to cultivate and provides an inexpensivemeans of combating vitamin and mineral deficiency in less developed regions of the world. Ocimum gratissimum is found throughout the tropics and sub-tropics, both wild and cultivated. Its greatest variability occurs in tropical Africa, where it probably has its origin in India (Osuji et al., 1995).
It has interesting medicinal properties (Gill, 1992). It has been described to have other species in the flora of tropical West Africa (Nwinyi et al., 2009). These include: O. viride Linn, O. suave Linn, O. bacilicum Linn and O. canum Sims.
37 Plate 6: Leaves of Occimum gratissimum
38 2.17.1. Morphology of Ocimum gratissimum
Occimum gratissimum is a shrub up to 1.9m in height with stems that are branched. 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, pubscent and dotted on both the 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 upto 5mm long, campanulate and 5-7 mm long, greenishwhite to greenish-yellow in colour. Nutlets are mucilaginous when they are wet (Bhat, 2003).
2.17.2. Traditional Uses of Ocimum gratissimum
Occimum 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 purpose. 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 (Akinmoldun et al., 2007). O. gratissimum is used by the Ibos of Southeastern 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 (Cristiana et al., 2006). People of Kenyan and sub Saharan African communities‟ use this plant for various purposes like viz., the leaves are rubbed between the palms and sniffed as a treatment for blocked nostrils, they are also
39 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). In India, the whole plant has been used for the treatment of sunstroke, headache, influenza, as a diaphoretic, antipyretic and for its anti-inflammatory activity (Tania, et al., 2006;
Prajapati et al.,2003). The tribes of Nigeria use the leaf extract in treatment of diarrhoea, while the cold leaf infusions are used for the relief of stomach upset and haemorrhoids. The plant is commonly used in folk medicine to treat different diseases such as upper respiratory tract infections, diarrhoea, headache, diseases of the eye, skin diseases, pneumonia, cough, fever and conjunctivitis (Adebolu and Salau, 2005).
2.17.3. Medicinal Uses of Ocimum gratissimum
The essential oil of Ocimum gratissimum contains eugenol and shows some evidence of antibacterial activity (Celso et al., 2002). A study on goats found that the essential oil has anthelmintic activity (Pessoa et al., 2002). A test on guinea pigs found evidence that the essential oil relaxes the muscles of the small intestinal disorders (Socorro et al., 2002). The ocimum oil is active against several species of bacteria and fungi (Trichophyton rubrum and Trichophyton mentagrophytes) (Oboh et al., 2009). A previous screening of crude extracts of plants used in traditional medicine showed that the essential of Ocimum gratissimum inhibited growth of Herpetomonas samuelpessoai (Holetz et al., 2002). African basil is used for a variety of reasons.
In culinary, it is used in salads soups, pastas, vinegars and jellies in many parts of the world. The anti-diarrhea agent and for the treatment of conjunctivitis by instilling directly into the eyes; the leaf oil when mixed with alcohol is applied as a lotion for skin infections, and taken internally for bronchitis. The dried leaves are shuffled to alleviate headaches and fever among other uses (Iwu, 1990). Although, convectional antibiotics have been very useful in orthodox medicine it has been argued by many that its concomitant use with herbal extracts is not desirable as one
40 normally antagonizes the activity of the other. Considering the fact that Ocimum gratissimum is used in most local dishes or foods to achieve a variety of purposes, there is need to ascertain if its characteristics, antagonizes or acts as a synergy when used together with conventional antibiotics. In addition, despite the fact that the various extracts of Ocimum gratissimum have been tested in vitro and shown to be active against some bacterial and fungal isolates (Nakamura et al., 2004; Silva et al., 2005), specific strain differences supposes that a lot more status of bacteria and fungi across other regions be tested to ascertain their in vitro activity against this spice.
2.17.4. Antifungal Activity of O. gratissimum.
Occimum gratissimum is reported to have wound healing properties. Study was carried out to investigate the effect of honey as well as those of surfactants on the antibacterial activity of the essential oil of O. gratissimum. The antibacterial activity of dispersions of ocimum oil (2%) in methanol, honey, a macrogol blend, nonionic and ionic emulsifiers were assessed by cup–plate method using type bacteria and wound isolates. Honey enhanced the antibacterial activity of ocimum oil to a greater extent than the macrogol blend. The activity of ocimum oil emulsion in cetrimide (cationic) was lower than obtained for cetrimide solution. Emulsion of the oil in sodium lauryl sulphate (anionic) exhibited a slightly higher activity than the solution of the surfactant alone. Although Tween 20 (nonionic) and aqueous methanol had no activity, the emulsion of the oil in Tween 20 showed lesser activity than the oil solution in methanol. Honey‟s inherent antibacterial activity, surfactant charge interaction and the effect of emulsification were adduced to the observed differences in antibacterial activity of the ocimum oil formulations.
Findings indicated that honey was a suitable base for ocimum oil especially in the treatment of infected wounds (Orafidiya et al., 2006). An investigation of antifungal activity of the essential oil obtained by steam-distillation (1.1% w/w) of the aerial parts of O. gratissimum and of an
41 ethanolic extract from the steam-distillation residue was carried out using the agar diffusion method. The results revealed that the essential oil inhibited the growth of all fungi tested, including the phytopathogens, Botryosphaeria rhodina, Rhizoctonia sp. and two strains of Alternaria sp., while the extract from the residue was inactive. The antifungal activity of eugenol was evaluated against a species of Alternaria isolated from tomato and Penicillium chrysogenum. The minimal inhibitory concentrations of eugenol were 0.16 and 0.31 mg/disc for Alternaria sp. and P.chrysogenum, respectively (Terezinha et al., 2006). Cryptococcal infection had an increased incidence in last few years‟ due to the explosion of acquired immunedeficiency syndrome. O. gratissimum has been reported earlier with in vitro activity against some bacteria and dermatophytes. In vitro activity of the ethanolic crude extract, ethyl acetate, hexane, chloroform fractions, essential oil, and eugenol of O .gratissimum was studied using an agar rdilution susceptibility method towards 25 isolates of Cryptococcus mneoformans. All the extracts of O. gratissimum studied showed activity in vitro towards C. neoformans. Based on the minimal inhibitory concentration values the most significant results were obtained with chloroform fraction and eugenol. It was observed that the chloroform fraction inhibited 23 isolates (92%) of C. neoformans at a concentration of 62.5 μg/ml and eugenol inhibited 4 isolates (16%) at a concentration of 0.9 μg/ml (Janine et al., 2005). The antibacterial activity of different extracts from the leaves of O. gratissimum was tested against Staphylococcus aureus, Escherichia coli, Salmonella typhi and Salmonella typhimurium, pathogenic bacteria that cause diarrhoea. Extracts evaluated included cold water extract, hot water extract and steam distillation extract. Only the steam distillation extract had inhibitory effects on the selected bacteria and the minimum inhibitory concentration ranged from 0.1% for S. aureus to 0.01% for E. coli and S.
typhimurium, and 0.001% for S. typhi (Adebolu and Salau, 2005).
42 2.18. Phytochemical Constituents of Plant Materials
Several authors have reported that plants contain a wide variety of bioactive constituents (Edeoga et al., 2005). These bioactive constituents of plants have been demonstrated to possess antimicrobial properties. Banso and Adeyemo (2007) reported the antimicrobial activity of alkaloids, glycosides, flavonoids and tannins from plant stem bark. The research of plants‟
bioactive substances has contributed immensely for the betterment of mankind through the provision of value added economic returns, manufacturing of natural plant production industry, provision of better health care and increase in export earning in both rural and urban areas (Tona, 2002).
2.18.1. Phytochemical constituents of Occimum gratissimum
The result of Ameh (2010) confirmed that the leaf extract of Ocimum gratissimum is rich in phytochemicals, the following phytochemicals were detected: glycosides, saponins, tannins, flavonoids, proteins, steroids and terpenoids. However, the extract of the plant material was devoid of alkaloids, resins, oils and acidic compounds. Terpenoids occurred in high concentrations. Glycosides and tannins were present in medium concentrations while saponins, flavonoids and steroids were found in low concentrations. On the same hand, the report of Udochukwu et al. (2015) showed that V. amygdalina and O. gratissimum contains the following phytochemicals: oxalate, phytate, tannins, saponins, flavonoids, cyanogenic glycosides, alkaloids, anthraquinone, steroid and phenol. In terms of concentration in (mg/100g), V.
amygdalina contained higher levels of bioactive compounds than O. gratissimum save for phytate and cyanogenic glycosides. The result of Ladipo et al. (2010) on the phytochemcial analysis of O. gratissimum revealed the presence of alkaloids, tannins, saponin, steroids, cardiacglycoside, flavonoid, terpenoids and phenol. While the result of Nweze et al. (2004)
43 revealed the presence of alkaloids, tannins, glycoside, saponin, resins, cardiac glycoside, steroidal terpens and flavonoids, this is similar to the results of Ladipo et al. (2010). Oladosu-Ajayi et al., (2017) documented that O. gratissiumu contains the same phytochemical compounds as the V. amygdalina but it was revealed from the results Erinle (2012) that more of these compounds can be found in the latter.
2.18.2. Phytochemical constituents of Cymbopogon citratus
The results of Umar et al. (2016) showed that phytochemicals abounds in the leaf of C.
citratus. The following phytochemicals were detected: flavonoids, carbohydrates, steroids, tannins, alkaloids, steroids, and phytosteriods) tested were all detected, except glycosides and phenols that were absent in the acetone and chloroform leaf extracts This agrees with the findings of Sofowora (1984) and AOAC (1990), who reported that many results of phytochemical composition of the ethanol leaf extract of Cymbopogon citratus shows that it contains alkaloids, saponins, tannins, anthraquinones, steroids, phenols, and flavonoids. Each of these phytochemicals is known for various protective and therapeutic effects. For instance, phenol is known to be an erythrocyte membrane modifier. Conversely the documentation of Mohammed and Bassem (2014) on the phytochemical constituent of C. citratuss revealed that the constituents of lemon grass extracts mostly belong to monoterpene, sesquiterpene, and phenolic acids. This phytochemical composition analysis was found to be in accord with previously reported studies (Barbosa et al., 2008; Negrelle and Gomes, 2007)
2.18.3. Phytochemical constituents of Carica papaya
Nath and Dutta (2016) documented that the phytochemical analysis of the leaves Carica papaya showed that the leaves contained tannin and saponins. The result showed that the levels of tannin and saponin in the plant extracts tested were 2.65% and 3.57mg/ml respectively.
Tannins bind to produce rich protein and interfere with protein synthesis. They are known to
44 exert anti-microbial activities by iron deprivation, hydrogen bounding or specific interactions with vital proteins such as enzymes in microbial cells (Scalbert, 1991). They are also observed to have remarkably activity in cancer prevention (Li et al., 2003) showed tannins to be useful in treatment of inflamed or ulcerated tissues. The presence of saponins supports the fact that pawpaw leaf has cytotoxic effects such as permealization of the intestine as saponins are cytotoxic (Okwu and Okwu, 2004), since it contains tannin and saponin, (Gangwar et al., 2015).
Workers have found antimethanogenic activity in the papaya leaves. Tannin and saponin are responsible for reducing methanogenesis in rumen liquor.This is in contrast with the result of Ayoola and Adeyeye (2010) who reported that the phytochemical analysis of the leaves of C.
papaya showed that the leaves contained saponins, cardiac glycosides, and alkaloids. Tannin was absent in the leaves. According to Ikeyi et al. (2013) Carica papaya leaves contain Alkaloid, Saponin, Tannin, Glycoside and Flavonoids and this is in agreement with the work of (Willson et al., 2007). Also, from the result of Ikeyi et al. (2013), it was revealed that phytochemicals can be extracted more when the leaves are dried and pulverized to powered form.
2.18.4. Phytochemical constituents of Citrus sinensis (Orange peel)
Osarumwense et al. (2013) reported that the phytochemical analysis of C. sinensis peel showed the presence of reducing sugar, saponins, cardiac glycosides, tannins and flavonoids. The presence of these constitute gives an indication of the medicinal value of the Citrus sinensis peels, for example, flavonoids have been found to have antioxidant properties, antibacterial and antimicrobial properties (Qian and Nihorimbere, 2004).
2.18.5. Phytochemical constituents of Vernonia amygdalina (Bitter leaf)
The reports of some researchers have shown that V.amygdalina contains varying quantities of the different phytochemicals. Offor (2014) reported that V. amygdalina contains high levels of flavonoids, saponins and β-Caroteinoids. On the same hand, the research of
45 Orjiakor et al. (2016) revealed that Vernonia amygdalina leaves contain anthracene, glycosides, steroids, flavonoids, proteins, carbohydrates, reducing sugars, saponins and tannins were present.
While the documentations of Udochukwu et al. (2015) revealed that V. amygdalina contains bioactive compounds which include: oxalate, phylate, tannins, saponins, flavonoid, cyanogenic glycosides, alkaloids, anthraquinone, steroid and phenol, hence it can serve as good sources of useful elements and bioactive compounds. An empirical research conducted by Usunobun and Okolie (2016) on the biological active ingredient of V. amygdalina showed presence of flavonoids, alkaloids, saponins, tannins, triterpenoids, steroids, reducing sugars and cardiac glycosides and absence of anthraquinones, the result also indicated that Vernonia amygdalina leaves, besides serving as good source of pharmacologically active phytochemi-cals may also be useful as supplements in human and animal nutrition. Other researchers proved that the leaf of V.
amygdalina is rich in saponins, sesquiterpenes and flavonoids (Igile, 1994; Heftman, 1997, Sofowora, 2004).
46 CHAPTER THREE
MATERIALS AND METHODS 3.1. Sources of Materials
The yam tubers with signs of rot and healthy yam (Dioscorea rotundata Poir) tubers were obtained Umuariaga market in Umudike Abia State Nigeria. The neem leaves (Azadirachta indica L.), bitter leaves (Vernonia amygdalina L.) and scent leaves (Ocimum gratissimum L.) were collected from a home garden in Abia state, while Paw-paw (Carica papaya), Lemon grass (Cymbopogon citratus L.) and orange bark (Citrus sinensis L.) were obtained from the staff quarters of The National Root Crops Research Institute, Umudike. The plants were identified by Prof. R. N. Okigbo and authenticated by Mr Tochukwu Egboka of the Department of Botany, Nnamdi Azikiwe University, Awka.