CHAPTER ONE

1.0 INTRODUCTION

During the last decades, there is increasing interest to unlock the secrets of ancient herbal remedies.

For this purpose, various strategies have been developed e.g., biological screening, isolation as

well as clinical trials for a variety of plants. Based on the screening methodologies, the therapeutic

values of many herbal medicines have already been established. Although, herbal medicines are

obtained from natural sources, and considered as safe for human beings. On the contrary, they

would have some adverse effects due to the presence of other active ingredients. (Bibi et al.2011)

In the worldwide as well as in the developing countries, the most human died due to infectious

bacterial diseases. The bacterial organisms including Gram positive and Gram negative like

different species of Bacillus, Staphylococcus, Salmonella and Pseudomonas are the main source

to cause severe infections in humans. Because these organisms have the ability to survive in harsh

condition due to their multiple environmental habitats. The synthetic antibiotics have the following

limitation: Firstly, these are costly and are out of range from the patient belonging to developing

countries. Secondly, with the passage of time microorganism develop resistance against

antibiotics. Therefore, after some time these antibiotics are not effective against the microbes.

Furthermore, the antibiotics may be associated with adverse effects on the host, including

hypersensitivity, immune suppression, and also allergic reactions. (Chopra et al.1986). On the

other hand, natural products have got incredible success in serving as a guidepost for new

antibacterial drug discovery. Moreover, antibiotics obtained in this way have biological

friendliness nature. As is well known that the bioactive plant extracts is a promising source of

majority of drugs. For example, Quinine (Cinchona) and berberine (Berberis) are the antibiotics

obtained from plants which are highly effective against microbes (Staphylococcus aureus,
Escherichia coli). (Bibi et al. BMC Complementary and Alternative Medicine 2011, 11:52

http://www.biomedcentral.com/1472-6882/11/52)

Antibiotics that show low efficacy in treating human and animal diseases through antibiotic

resistance must be replaced with new drugs to combat the burden of these pathogens. Hence,

medicinal plants are expected to be the best source of obtaining a variety of drugs. In

Ethiopia, different communities have extensively been using medicinal plants to treat different

diseases formany centuries. Such plants, however, need to be investigated for better understanding

of their properties, safety, and efficiency. Medicinal plants including C. aurea (Ait.) Benth., C.

macrostachyus Del., and Withania somnifera (W. somnifera) (L.) Dunal have been tested for their

antimicrobial efficacy in vitro and in vivo in different countries. However, testing the efficacy of

these plants in different agroecological zones can make the evidence more strong since

agroecology (e.g., geographic location and soil type) could affect the accumulation of bioactive

pharmaceutical ingredients that are found in plants.

As reviewed in Mahidol et al. medicinal plants have been used for treating infectious diseases

because of their ease of accessibility as well as their lower side effects and toxicity.

Medicinal plants may provide an exceptional renewable resource for the discovery of potential

novel pharmaceuticals as their constituents have ample structural and biological diversity. In

Ethiopia, many plant species are being used as traditional medicine for the treatment of various

human and animal diseases. Hence, more studies related to the use of medicinal plants as remedial

agents need to be conducted, especially for plants which are useful to control antibiotic resistant

microbes. The aim of this study was to evaluate the efficacy of five plant extracts, which have been
used as traditional medicines by local healers for their in vitro antibacterial activities against three

multidrug resistant bacteria, namely, S. aureus, E. coli, and P. aeruginosa.

Pentadesma butyracea is a multi-purpose tree, which supplies many indigenous people with a

diversity of valuable non-timber forest products (NTFPs) in Benin. The kernel is its main harvested

product and is processed into butter and marketed (Sinadouwirou et. al., 2003). Its leaves, bark

and roots are used to treat various diseases (Houngbédji 1997), and its wood is carved and used

for construction.

1.1 Aim

The broad aim is to pharmacological screen Pentadesma butyracea for it antibacterial activity.

1.2 Objectives

To determine the antibacterial activity of Pentadesma butyracea of Plant Extract against tested

standard organism
 CHAPTER TWO

2.0 LITRATURE REVIEW

Plants have a great potential for producing new drugs and used in traditional medicine to treat

chronic and even infectious diseases (Panda SK et al., 2009). The Phytomedicine are more import

in the treatment of inflammation. In recent years, there is an increasing awareness about the

importance of medicinal plants. Medicinal plants such as Pentadesma butyracea have shown to

exhibit potent anti-inflammatory effect in the treatment of inflammation.

2.1 Pentadesma Butyracea

Pentadesma butyracea is a tree native to the forests of tropical Africa from Sierra Leone to

Cameroon. It has multiple uses, the main one being the manufacture of a kind of butter called

"kpangnan butter" similar to shea butter. The timber is used in cabinetmaking and construction. It

is sometimes called African butter tree.

Pentadesma butyracea (yellow butter tree) belongs to the pantropical family Clusiaceae (37 genera,

1610 species; Gustafson et al. 2002). It is a native African tree species occurring from 200 to 550

m of elevation, in Guineo-Congolian evergreen forests and gallery forests from Guinea-Bissau to

the Democratic Republic of Congo (Bamps 1971). In Benin, where a savanna corridor interrupts

the zonal West African rainforest, it occurs in the Sudanian phytogeographical zone as defined by

White (1983), in highly aggregated stands (Sinsin & Sinadouwirou 2003), mainly in gallery

forests, in savannah woodlands and at foot hills where humidity is elevated. These forest patches

are probably residues of a more continuous forest occurring during the Holocene African Humid

period that fragmented and degraded at the end of the Holocene due to climate changes (Salzmann

& Hoelzmann 2005). More recently, these forest patches, and hence the habitat of P. butyracea,
were additionally reduced in size due to extensive agriculture and logging (Neuenschwander et al.

2011).

In Benin, where P. butyracea is known under the local names Kpangnan (in Nagot), Akoto (in

Anni), Yêkotchépouo (in Otamari) or Sesseido (in Fulani), fully grown trees reach 10–25 m in

height and a diameter at breast height (dbh) of 110 cm. The wood of P. butyracea is used as timber

and firewood; it has very good mechanical properties similar to those of Khaya senegalensis and

Milicia excelsa (Rachman et al. 1987). Flowers are whitish, fleshy, hermaphroditic, producing

many stamens and some nectariferous glands around the superior ovary (Akoegninou et al. 2006).

The fruit of P. butyracea is a berry containing seeds in a yellow pulp (Hawthorne & Jongkind

2006). P. butyracea often reproduces by asexual reproduction via root shoots.

Pentadesma Butyracea scientific classification

Kingdom Plantea

Clade Tracheophytes

Clade Angiosperms

Clade Eudicots

Clade Rosids

Order Malpighiales

Family Clusiaceae

Genus Pentadesma

Species P. Butyracea

Binomial name Pentadesma Butyracea Sabine

2.1.1 Description of Pentadesma butyracea

Pentadesma butyracea is an evergreen, medium-sized to fairly large tree up to 35 m tall; bole

cylindrical, up to 100–150 cm in diameter, sometimes with small buttresses or stilt roots; bark

rough and scaly, inner bark red-brown to brown, finely fissured, exuding bright yellow sap; twigs

angular or ribbed, dark brown to black. Leaves opposite, in dense terminal clusters, simple and

entire; stipules absent; petiole up to 2.5 cm long, stout; blade obovate to oblong-oblanceolate, 9–

25 cm × 3.5–7 cm, base cuneate, apex shortly acuminate, leathery, glabrous, shiny dark green

above, pinnately veined with numerous, parallel lateral veins, ending in marginal vein, with

glandular canals parallel to veins. Inflorescence a terminal thyrse, 1–7-flowered. Flowers bisexual,

regular, 5-merous, yellowish or greenish white; pedicel 1–4 cm long, often curved; sepals free,

ovate, up to 5 cm long, very unequal, leathery; petals free, oblong to ovate, up to 6 cm long, keeled;

stamens numerous, in 5 bundles opposite the petals, 4–6.5 cm long, fused at base; disk glands 5,

alternating with petals, up to 0.5 cm high; ovary superior, ovoid-ellipsoid, 1–2 cm long, 5-celled,

style elongate, ending in 5 linear spreading lobes up to 0.5 cm long. Fruit an ellipsoid to ovoid

berry, 9–15 cm × 6.5–12.5 cm, base with persistent calyx, stamens and disk glands, apex pointed,

wall coarse, brown, leathery, 5–15-seeded. Seeds pyramidal, with flattened sides or irregular, 3–4

cm × 2.5–3 cm, dark brown. Seedling with hypogeal germination; epicotyl reddish, 10–30 cm

long; first leaves opposite, 7–16 cm × 2.5–4.5 cm.

2.1.2 Properties Of Pentadesma butyracea

 The kernel of the seed contains per 100 g dry matter 50 g fat and 1.5–1.8 g unsaponifiable

matter. It also contains an odourless and tasteless resin that is yellowish in colour and toxic.

The fatty acid composition of kanya butter is palmitic acid 3–8%, stearic acid 41–46%,

palmitoleic acid 0.2%, oleic acid 48–51%, linoleic acid 0–2%. Kanya butter is similar to
 shea butter in several characteristics, including slip point, saponification number,

solidification point and fatty acid composition.

 The heartwood of Pentadesma butyracea is yellowish or pinkish brown, distinctly

demarcated from the whitish to pale pink sapwood, which is fairly wide. The grain is

straight to slightly wavy, texture coarse. The wood is heavy, with a density of 850–1000

kg/m³ at 12% moisture content, hard and strong. It air dries slowly with little splitting, but

cupping may occur. The rates of shrinkage are moderately high, from green to oven dry

4.5–4.7% radial and 8.0–8.7% tangential. At 12% moisture content, the modulus of rupture

is 140–270 N/mm2, modulus of elasticity 6900–19,300 N/mm2, compression parallel to

grain 56–101 N/mm2, cleavage 17–19 N/mm, Janka side hardness 8000 N, Chalais-

Meudon side hardness 5.0–12.6.

 The wood saws satisfactorily, but may cause gumming of saw blades and overheating. It

planes, polishes and moulds well and bores satisfactorily, although heating may occur; the

wood holds nails well, but splitting on nailing is rather common. It is not durable, being

susceptible to attack by pinhole borers and marine borers, but fairly resistant to termites.

The heartwood is very resistant to impregnation with preservatives, the sapwood

moderately permeable.

2.1.3 Growth And Development of Pentadesma butyracea

Pentadesma butyracea tree first flower when about 8 m tall. Flowering occurs during a large part

of the year, but mainly during the main rainy season. In Gabon trees flower from March to

September. The flowers produce large amounts of nectar, which is eaten by monkeys; they are

probably important pollinators. In Gabon fruits are produced mainly from October to December,
and in Benin from March to June. They are eaten by elephants and monkeys, which disperse the

seeds.

2.1.4 Ecology of Pentadesma butyracea

Pentadesma butyracea occurs in tropical rainforest on moist or swampy ground, mostly on river

banks. It does not occur where mean annual rainfall is less than 1000 mm. It prefers deep soils. In

Ghana it is strongly associated with leached soils. In Benin it occurs naturally in riparian forest.

2.1.5 Propagation And Planting of Pentadesma butyracea

Pentadesma butyracea is propagated by seed. Freshly harvested, mature and healthy seeds

germinate well, but seeds are very sensitive to desiccation and fermentation. When stored in a dry

place at 25–36°C they lose their viability quickly; at 10–15°C they keep their viability longer, but

it is difficult to break dormancy. The best results are obtained when seeds are stored in jute bags

and are watered regularly. Under natural conditions trees may also regenerate by root suckers.

2.1.6 Harvest of Pentadesma butyracea

In Benin fruits are usually gathered in April–June, mostly by women. After collection, they are

put together under a tree and covered to accelerate fermentation of the fruit pulp and to facilitate

seed extraction. It has been estimated that a woman may collect 15–40 kg of seeds per season.

2.1.7 Handling After Harvest of Pentadesma butyracea

In rural areas, fruits are processed by water extraction, usually the job of women. Gathered fruits

are put together under a tree and covered. After 10 days the fruit pulp has decayed and the seeds

can be extracted easily. Seeds are boiled and then dried in the sun or a kiln to prevent further

rotting. Dry seeds are pounded until they are clean and are turned over daily to prevent mouldiness.
To extract the oil, seeds are crushed and ground into a paste. The paste is boiled in water and the

oil is skimmed off. The oil yield rarely exceeds 35% of the seed dry weight.

Figure 1: Pentadesma butyracea plant.

2.1.8 Medicinal Uses of Pentadesma butyracea

 Kanya butter is a suitable base for topical medicines. Its application relieves chest-pain,

cough in children, strain and abscesses.

 It is used as a substitute for shea butter from Vitellaria paradoxa C.F.Gaertn. when the latter

is rare or cannot be used traditionally, e.g. during treatment of leprosy or epilepsy.

 The leaves serve as a galactagogue vegetable.

 They are believed to make the milk easily digestible and help in teething.
 An infusion of ground roots is used to wash children during weaning, while infusions of

the bark are put in a bath to relieve fever.

 A decoction from the roots is used as vermifuge in Liberia.

 The latex from the bark is applied to the skin against skin parasites.

 In certain regions, the oily presscake is applied externally to animals (e.g. sheep) to treat

galls and is also used to plaster walls of houses (e.g. Tata Somba houses in north-western

Benin).
 CHAPTER THREE

3.0 MATERIAL AND METHOD

3.1 COLLECTION AND EXTRACTION

The plants material was collected from a geographical region Benin City, Edo, Nigeria. The

collected plants were identified by Dr. Dr. P.O Osarumwense, Department of Chemical Sciences,

Olusegun Agagun University of Science and Technology (OAUSTECH) Okitipupa Ondo State

Nigeria after examination of the specimens already presented in herbarium.

The plant material was thoroughly washed under running tap water and dried under shade. Then

the material was grounded to powder form. Powdered material of the plant was soaked in methanol

separately at room temperature. After seven days, the extract was filtered under vacuum through

Whatman filter paper. The residue was again dipped in methanol for additional seven days and

filtered thereafter. The filtrates were combined and methanol was evaporated under vacuum, using

rotary evaporator (Buchi Rotavapor R-200) at 45°C. The quantities of extracts obtained was

200mg.

3.2 BACTERIAL STRAINS

The bacterial strains for this experiment were isolated and Standard reference bacteria were
obtained from the National Referral Bacteriology and Mycology Laboratory, Nigeria Public
Health Institute. Clinical isolates of Escherichia coli, Staphylococcus aureus, Pseudomonas
Aeuriginosa, Bacillus species, Klebsiella pneumonia, and Aspergillus were used.
3.3 FRACTIONATION

Fractionation was carried out by suspending each extract in 250 mL water separately and
partitioning with different organic solvents (hexane, chloroform, ethyl acetate, and methanol) in
order of increasing polarity by using separating funnel. All the fractions of the plant extract were
dried by evaporating respective solvent using rotary evaporator. All extracts were stored at 4°C till
further analysis.

3.4 Antibacterial activity

The agar well diffusion method was performed to exploit antibacterial potential of used extracts.

Each extract (200 mg) was dissolved in 10 mL of 99.9% dimethyl sulfoxide (DMSO) (Sigma-

Aldrich USA) to get 20 mg/mL concentration. Cefotaxime (2 mg/mL) in DMSO was prepared as

positive control. Pure DMSO (99.9%) was used as negative control.

 CHAPTER FOUR

4.0 RESULT AND DISCUSSION

4.1 Antibacterial activity of Plant Extract against tested standard organism

TEST ORGANISM ZONE OF INHIBITION (mm)

Extract 250mg/mL

Escherichia coli 12

Staphylococcus aureus 11

Pseudomonas Aeuriginosa NZ

Bacillus species NZ

Klebsiella pneumonia NZ

Aspergillus NZ

KEY

NZ = No Zone of Inhibition
4.2 DISCUSSION

The plant was tested against ATCC bacterial cultures to determine and investigate their

antibacterial potential. We observed that the crude methanol extract of Pentadesma Butyracea

showed significant antibacterial activities against all the tested bacterial strains. Maximum activity

was conferred against Escherichia coli (12 nm) while minimum was observed against

Staphylococcus aureus (23 mm) with mean inhibition zone diameter 11 nm. Pseudomonas

Aeuriginosa, Bacillus species, Klebsiella pneumonia and Aspergillus shows no zone of inhibition.

The antibacterial activities of the methanol extracts of Pentadesma Butyracea were tested against

microorganisms isolated from the wounds of patients with lymphoedema and standard ATCCs. In

vitro antibacterial activity was tested in the presence or absence of a zone of inhibition in diameter,

the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in

comparison with the reference antibacterial drugs.

Generally, it was observed that bacterial growth inhibition increased as the concentration of the

extracts increased. Pairwise comparison of ANOVA was used to test the variability in

susceptibility of the microorganisms toward the extracts (p < 0.05). No significant difference was

observed in terms of susceptibility between K. pneumoniae isolates and ATCC (p = 0.91), S. algae

isolates and P. aeruginosa ATCC (p = 0.74), E. coli isolates and K. pneumoniae ATCC (p = 0.89),

S. aureus isolates cork borer. Then, a volume of 100 μL methanol extracts of A. aspera, L. inermis

and A. indica leaves (at concentrations of 100 mg/mL, 200 mg/mL, and 400 mg/mL) were

dispensed into the wells. Similarly, 5% Di-methylsulfoxide (DMSO) was dispensed into the

control well, and reference antibiotic discs were placed on the surface of the plate and incubated

for 24 h at 37 °C. For Streptococcus pyogenes, a carbon dioxide incubator was used for incubation.

Each experiment was done three times.

 CHAPTER FIVE

CONCLUSION

In summary, we have described the antibacterial properties of the aqueous fraction of Pentadesma

Butyracea against the tested microbes. Therefore, the extracts of these plants were considered as

suitable candidates for antibacterial drug discovery. The other extracts showed lower activity

which might suggest the lack of bio-active components and/or insufficient quantities in the extract.

Based on our findings, we envision that the discovery of novel antibacterial agent from natural

sources (plants) will help to minimize the adverse effects of synthetic drugs. These findings

support the traditional claim that the medicinal plants have antibacterial activity in wound

infections. Further investigations, however, need to be carried out before recommending their use.

The results of the present study support the traditional uses of these plants practiced by the local

healers. The plants investigated in this study exhibited antibacterial activities against the test

bacterial species. However, it was reported that Pentadesma Butyracea has been used for the

treatment of external and internal parasites. Further researches are needed to be conducted to

evaluate the efficacy of these medicinal plant species on other microbes in different agro ecological

settings and their safety levels as well as their phytochemical compositions.

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