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 Aqueous (cold and hot water) and ethanol extracts of Piper guineense seeds were screened for antibacterial activity against four microbes, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus. The aqueous extract of this plant showed minimal level of inhibition on E.coli and S.aureus at the highest concentration (2000µg/ml),with no effect at all on P.aeruginosa and S. typhi. The ethanol extract exhibited more inhibitory effect on Staphylococcus aureus, E. coli, and Salmonella typhi with no effect on Pseudomonas aeruginosa.  The minimum inhibitory concentration of the aqueous extract (cold and hot water extract) was at 500µg/ml, while that of ethanol was at 62.5µg/ml. The qualitative determination of chemical constituent of the plant confirmed the presence of certain metabolites such as Alkoloids, Saponins, Tannins, Oleoresin and Glycosides. The results obtained show that the extract of Piper guineense possess some level of antibacterial activity against some common pathogens.







Cover page                                                                                                                  i

Certification                                                                                                                ii

Dedication                                                                                                                  iii

Table of contents

Acknowledgements                                                                                                    iv

Abstract                                                                                                                      v


1.0       INTRODUCTION                                                                                         1

1.1        OBJECTIVE OF THE WORK                                                                      3

1.2       LITERATURE REVIEW                                                                              4

1.2.1    Morphology and Description                                                                          4

1.3       ECOLOGY AND METHOD OF CULTIVATION                                                 5

1.3.1    Vegetative propagation:                                                                                  5

1.3.2    Diseases Of Plant                                                                                            7

1.4       USES OF PIPER GUINEENSE (UZIZA)                                                    9

1.4.1    Medicinal Uses                                                                                              9

1.4.2    Culinary Uses                                                                                                  9

1.4.3    Pharmacological, toxicological and clinical applications:                               10

1.4.4    Bioavailability enhancement:                                                                          10

1.4.5    As Antioxidant:                                                                                              10

1.5       CHEMICAL CONSTITUENTS OF PIPER GUINEENSE                          11

1.5.1    Vitamin Content                                                                                             13

1.5.2    Mineral Content                                                                                              13

1.6       ROLES OF TEST ORGANISMS IN RELATION TO DISEASES                        13

1.6.1    Staphylococcus aureus                                                                                    13

1.6.2    Pseudomonas aeruginosa                                                                               13

1.6.3    Escheriachia coli                                                                                             14

1.6.4    Salmonella  typhi                                                                                             15


2.0       MATERIALS AND METHODS                                                                  16

2.1       COLLECTION OF PLANT MATERIAL                                                    16

2.2       PREPARATION OF PLANT MATERIAL                                                  16

2.3       ETHANOL EXTRACT  PREPARATION                                                   16

2.4       AQUEOUS EXTRACT PREPARATION                                                    16

2.5       CULTURE MEDIA USED                                                                           17

2.6        SOURCE AND MAINTENANCE OF TEST ORGANISMS                    17

2.6.1    Gram Staining                                                                                                 17

2.6.2    Catalase Test                                                                                                   18

2.6.3    Coagulase Test                                                                                                18

2.6.4    Oxidase Test                                                                                                   19

2.6.5    Indole Test                                                                                                      19

2.7      ANTIBACTERIAL TESTING                                                                       19

2.8       DISC DIFFUSION METHOD                                                                      20

2.9       DETERMINATION OF MINIMUM INHIBITORY                                 

CONCENTRATION (MIC)                                                                          20


CONCENTRATION (MBC).                                                                                    21


CONSTITUENTS                                                                                          21

2.11.1  Test for Tannins                                                                                              21

2.11.2  Test for Saponnin                                                                                            21

2.11.3  Test for flavonoids                                                                                          22

2.11.4  Test for Alkaloid                                                                                             22

2.11.5 Test for glycoside                                                                                            22


3.0       RESULTS                                                                                                       23

3.1       Qualitative Phytochemical Screening                                                             29


4.0    DISCUSSION                                                                                                   30

4.1    CONCLUSION                                                                                                31

4.2    RECOMMENDATION                                                                                    31









Title                                                                                                                            page

Table 1: Antibacterial activity of cold water extract  of Piper guineense                  24

Table 2:  Antibacterial activity of hot water extract of Piper guineense                    25

Table 3: Antibacterial activity of ethanol extract of Piper guineense            26

Table 4:  shows the minimum inhibitory concentration                                              27

Table 5 :  shows the minimum bactericidal concentration                                          28

Table 6: Qualitative Phytochemical Screening                                                           29



There is a growing interest in exploiting plants for medicinal purposes especially in Africa. This stems from the fact that micro-organisms are developing resistance to many drugs and as such created situation where some of the common and less expensive anti-microbial agents are losing effectiveness (Montefore et al., 1989). Herbal medicine which uses medicinal plants primarily presents as an alternative to such situation (Sofowora, 1993). These medicinal plants have immensely contributed to the development of human health and welfare. Concomitantly, there is an increase in data and huge patronage to herbal products round the world (Elsenberg et al., 1990, Omoseyindemi, 2003).

Studies on the use of plant extracts for control of diseases have shown the importance of natural chemicals as possible source of non-phytotoxic and easily biodegradable alternative fungicides and antibiotics (Akueshi et al., 2002)and the advantages of these natural plant products include local availability, little or no toxicity to humans and simple preparation procedures and plant herb biological control generally favoured as a method of disease management because it does not have the disadvantages of chemicals and tends to be more durable in its effect.

Piper guineense (uziza) common referred to as African black pepper or Ashanti pepper is very similar to Piper nigrum which is the true pepper of commerce from which black and white peppers are processed (Isawumi, 1984). P. guineense belongs to the family piperaceae. It has more than 700 species throughout the tropical and subtropical regions of the world. It is known with different vernacular names in Nigeria, Igbo (Uziza), and Yoruba (Iyere). P. guineense has culinary, medicinal, cosmetic and insecticides uses (Dalziel, 1995, Okwute, 1992). P. guineense insecticidal activity against Zonocerus variegatus is attributed to the piperine-amide composed by the plant. It is widely consumed in some part of West Africa especially Nigeria and Ghana on account of its nutritional and medicinal properties. (Negbenebor et al., 1999). P. guineense berries, like other types of pepper, yield an aromatic essential oil on steam distillation. Nothing is known about the chemical  composition of the volatile oil of this plant in Nigeria until in 1988 when Ekundayo et al., (1988) first reported chemical composition of this plant. It was reported that phenyl propanoid-rich essential oil was comprised of myristicin, Sarison, Safron and elemicin as a dominant component and fifty-one mono and sesquiterpenoids as a minor component, also preliminary toxicity of the volatile oil was also evaluated.

In tradition herbal medicine, the seed of Piper guineense are put into variety of uses for instance, in some parts of Nigeria, the seeds are consumed by women after childbirth to enhance uterine contraction for the expulsion of placenta and other remains from the womb (Udoh et al., 1999). Extracts of P. guineense has been reported to stimulate digestion of food by stimulating secretion of digestive enzymes, pancreatic amylase, trypsin and chymotrypsin and is thus used for the treatment of digestive disorders (Platel and Srinivasan, 2000).

Piper guineense can also be used as a food preservative. Food poisoning is still a concern for both consumers and the food industry despite the use of various preservation methods. Food processors, food safety researchers and regulations agencies are continuously concerned with the high and growing number of illness out breaks caused by some pathogenic and spoilage microorganisms in food. (Meng et al., 1998). Consequently there is considerable interest in ways to stop this upward trend and reduce the incidence of food poisoning.

Spices and herbs have been added to food since ancient times, not only as flavoring agents, but also as folk medicine and food preservatives (Nakatani, 1994).

Organisms such as Escherichia Coli and Staphylococcus aureus are associated with the gastro intestinal tract of man and animals. They occur in other environments. S. aureus is among the invasive gram positive known as pyogenic coci implicated in several disease of human. It has been found to be normal flora of upper respiratory tract and vagina.

S. aureus has been known to produce heat stable toxins which are implicated in illness and stomach upset (Mosset et al., 1990). From literatures, S. aureus has shown to be very resistant to a wide variety of antibiotics (Nwinyi et al., 2008). E. Coli is a facultative anaerobe of wide distribution in the environment, it has been implicated in the cause of diarrhea, Urinary tract infection, meningitis, sepsis, wound-infections, nosocomial pneumonia and arthritis. Salmonella typhi is of intestinal origin and infection is spread by consumption of food contaminated by faeces. The clinical syndrome is gastroenteritis.

Many researchers have reported the plant to possess antimicrobial activity therefore, this work reports on the study of the anti-bacterial activity of extracts of Piper guineense against some common human pathogen.

1.1   Objective of the work

·         To determine the antibacterial activity of aqueous (cold and hot water) and ethanol extract of Piper guineense against some common pathogens

·         To identify some of the chemical constituents present in the plant.


1.2.1    Morphology and Description

Piper guineense is a spices that belong to the piperaceae family. It is a climbing perennial forest liana plant with gnarled branch lets spiraling on shrubs to about 10m (Iwu, 1993). Piper guineense is a perennial plant that is characterized by heart-shaped leaves and oval petiolate, alternate 12cm long. The inflorescence is a pedicelled flower spike between 3 and 6cm long, flowers are greenish yellow and arranged in a spiral along the spine (Ayodele, 2012). The fruits from P. guineese have some common and native names in which it is identified in different areas as locations.

The common names include; West African black pepper, Uziza, Ashanti pepper, Pepper Benin, False cubeb, Guinea cubeb, Guinea peper, Dooje. The fruits of piper guineense are a drup mesocarp or fleshy oval 3-5mm in diameter (Iwu, 1993).

Uziza is highly medicinal. It is found to have a stimulating property like pepper and excellent flavouring properties. The seeds are used in preparing pepper soup, especially for lactating mothers. It is traditionally believed that uziza functions as an ointment for amounting corpses. It cannot be eaten or consumed raw, rather the goodness in uziza and the worldwide demand for it is derived from certain of its attributes, which have been found to be very useful as additives blending of food and pharmaceutical industries. The attributes are the aroma, the extraction of which is the sole object of processing uziza either into oleoresin, essential oil, uziza powder or similar concentrates which can later be used in the manufacture of confectionery, beverages and pharmaceutical products (oletunji,1980).



Piper guineense plant has a pan tropical distribution and it is most commonly found in the low land tropical rainforests, but can also occur in higher elevation life zones such as cold forests. It is native to tropical regions of central and western Africa and are semi—cultivated in countries such as Nigeria where the leave is used as a flavoring for stews (Ayodele, 2012). The trees are widely distributed in humid forest zones of West Africa. It thrives well in humus rich virgin soil, fertile, well drake loamy soil. Soil pH should be between 5.5 to 7.0. It is susceptible to water logging and hence is planted by preparing heap of soil on field. High humidity helps for luxuriant crop growth and better yield. Very high or low temperature is not conducive for growth. Temperature ranges between 10-40c. The crop is pollinated through rain drops and hence frequent showers. It is either propagated vegetatively or by seed (Oletunji et al., 1980), propagated by seed

Propagation by seed is carried out by soaking fully ripened barriers in water for 2-3days them removing the flesh and drying them in the shade afterwards, they are planted out in moist, shaded beds filled with a mixture of humus and lots of sand, at a distance of one hand-width to one-another. They will begin to germinate after 30 days and can be transplanted to their final sites after further 6 months-when they have produced 4 leaves (Udo et al, 2001).

1.3.1    Vegetative propagation:

The most widely spread form of propagation is with the use of cuttings. They should be selected from the area of the main shoot of a strong, healthy and highly productive parent plant. Before the shoot is cut from the main shoot, the vegetative apex, as well as the leaves and lateral shoots from the 3rd to the 7th knots, should be removed. As soon as the plants apex has regenerated, the shoot underneath the 7th knot is removed and planted in a seedling bed. The seedling should be set in the soil at an angle of 450 with 3-4 knots. The shoots bed must be kept moist and shaded. The shoots will have taken root after 2 months, whereby only a 30% rate of success should be expected. They can then be planted at their final sites (Udo et al., 2001).

1.3.2    Diseases of Piper Guineense



Phytophtera palmivora

Leaf wilt, yellow discoloring with lot of leaves, shoots and finally entire plants.

Fusariu Solani

Leaf Wilt

Ganoderma lacidum

Leaf Wilt

Colletotriclum, Rhizoctonia

Leaf Flecks

Pseudomonas (Bacteria)

Leaf Flecks


Nematode infestation by meloidogyne Spp. Causes the main problem on conventional pepper cultivation greater damage, especially in Indonesia are caused by various bugs, green flies, beetles as well as butterfly, caterpillars.


The use of Uziza is very rampant though little is known of its importance in our daily cure of sickness and prevention of allergy and running nose. Based on the usefulness and importance, among all the spices, black pepper is commonly referred as “The king of spices”. It is spicy, aromatic and carminative. It is a natural antioxidant. It acts as anti-inflammatory, anti-cancer, anti-periodic and anti-pyretic. It is rubefacient in nature and can help to lower body cholesterol levels. It contains mainly vitamins A, C, E, K, niacin and B-Carotene and traces of minerals such as iron, calcium, phosphorus. The amino acids contained by black pepper works for enhancing the bioavailability of nutrients (Deluxe et al., 2013). These acid constituents can be used in anti-microbial, anti-bacterial applications.

1.4.1    Medicinal Uses

Seeds of Piper guineense have been used internally as medicinal agents for the treatment of worm infestation, tonsillitis, bronchitis and gastrointestinal diseases.

In traditional herbal medicine, the seeds are put into variety of uses for instance, in some parts of Nigeria, the seeds are consumed by women after child birth to enhance uterine contraction for expulsion of placenta and other remains from the womb (Okigbo et al., 2005). Also the powder from the dried fruits mixed with honey acts as carminative and relieves stomach aches.

1.4.2    Culinary Uses

In some parts of Nigeria it is used for preparing pepper soups and different kinds of traditional soups. The leaves are used as flavouring for stew. This is as a result of spicy and aromatic properties.

1.4.3    Pharmacological, toxicological and clinical applications:

Black pepper is described as a drug which increases digestive power, improves appetite, cures cold, cough, diseases of the throat, intermittent fever, Colic, dysentery system. It possesses a broad spectrum antimicrobial activity. (Srimivasan, 2005).

1.4.4    Bioavailability enhancement:

Black pepper and its volatile oil are used in food and food items to aid digestion, relieve gas, Treat food poisoning, Stomach chills, cholera, Dysentary.  Piperine increases the bioavailability of valuable phytochemicals present in food items and can boost the activity of biochemically active compounds contained in it. (Agbor et al., 2006).

1.4.5    As Antioxidant:

An antioxidant is defined as any substance which is capable of delaying, retarding or preventing the development in food of rancidity or either flavour deterioration due to oxidation. Antioxidant compounds in food items play import roles as health protecting factors. Antioxidants are widely used as additives in fats, oils and in food processing to prevent or delay spoilage of foods. Black pepper is a centre of attraction as source of effective antioxidants. It contains several powerful antioxidants and it thus one of the most spices for preventing and curtailing oxidative stress. In addition to their direct antioxidant properties, several of these compounds work indirectly by enhancing the action of other antioxidants. Saturated fats in food are one of the main causes of oxidative stress and black pepper minimizes it.



It was reported that piper guineense contains phenylpropanoid, rich essential oil which was comprised of myristicin, sarisan, safrone and elemicin as a dominant component and fifty-one mono and sesquiterpenoids as a minor component (Ekundayo et al., 1998).


A chemical substance of plant origin composed of carbon, hydrogen, nitrogen and (usually) oxygen. The alkaloids are organic basis similar to the alkalies (Inorganic basis); the name means alkali like. Most alkaloids have pronounced effects on the nervous system of humans and other animals. Many are used as drugs. Some similar alkaloids are caffeine, nicottine, quinine, cocaine and morphine. Alkaloids occur mostly in various genera of seed plants.


Flavonoids are naturally occurring biochemical substances found throughout plants. They are often referred to as bioflavonoids. There are thousands of different plant flavonoid. Generally speaking they are all chemically related because they have polyphenol structures in laboratory research tests, flavonoids tend to prevent other substances from undergoing oxidation reaction. People often refer to flavonoids as antioxidants for this reason, and there is no shortage of people expounding the benefits of flavonoid supplements.


Saponins are glycosides with foaming characteristics. Saponins consists of a polycyclic aglycones attached to one or more sugar side chains. The aglycone part, which is also called sapogenin, is either steroid or a triterpene. The foaming ability of saponins is caused by the combination of a hydrophobic (fat-soluble) sapogenin and hydrophilic (water-soluble) sugar part. Saponins have a bitter taste. Some saponins are toxic and are known as sapotoxin.


A tannin is an astringent, bitter plant polyphenolic compounds binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids. The term tannins refers to the use of wood tannins from oak in tanning animal hide into leather; hence the words “Tan” and tannin for the treatment of leather; however, the term tannins by extension is widely applied to any large polyphenolic compound containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with various macromolecules. The tannin compounds are widely distributed in many species of plants.  Where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation.


Piper guneense also contains Oleoresin which is a mixture of a resin (volatile) and essential oil (Oletunji, 1999). Oleoresins are obtained by solvent extraction of the spice. Oleoresins consist of the volatile essential oil and the non-volatile resinous fraction comprising of taste components, fixatives, anti-oxidants, pigments and fixed oils that are naturally present in the spice, in a highly concentrated form. Oleoresins, the true essence of the spice represents the over all flavor profile of the spice. They serve as convenient and hygienic substitutes for spice powders in food products without impairing any flavor characteristics.


1.5.1    Vitamin Content

Choline, folic acid, Niacin, Pyridoxine, Ruboflavim, Thiamin, Vitamin A and Vitamin C. Vitamin E, Vitamin K is the major Vitamin found in black pepper .

1.5.2    Mineral Content

Calcium, copper, iron, magnesium, manganese, phosphorus, zinc are the main mineral found in the black pepper.


1.6.1    Staphylococcus aureus

Staphylococcus aureus is a gram positive coccus that produces a wide variety of toxins. It is a facultative anaerobe which appears as grape like cluster when viewed through a microscope and has large round, golden-yellow colonies, often with haemolysis when grown in blood agar plate (Ryan and Ray, 2004). Each year about 500,000 patients are affected by staphylococcus infection in American hospitals. Staphylococcus aureus may occur as a commensal on human skin sometimes it infects other tissue when normal barrier have been reached, this leads to furuncle (boils) and carbuncles (a collection of furuncles). In infants staphylococcus infection can cause a severe disease staphylococcus scaled skin syndrome. (Curran and Al-Salihi, 1980). It also causes some diseases like atopic dermatitis, Toxic shock syndrome, and mastitis in cow.

1.6.2    Pseudomonas aeruginosa

Pseudomonas aeruginosa is a member of the Gamma proteobacteria class of bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. Pseudomonas is a Gram-negative rod measuring 0.5 to 0.8µm by 1.5 to 3.0µm. Almost all strains are motile by means of a single polar flagellum. Pseudomonas aeruginosa is an opportunistic pathogen. It causes urinary tract infection, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, dermatitis, soft tissue infection, bacteremia bone and joint infections, gastrointestinal infections and a variety of systematic infections, particularly in patients with  severe burns and in cancer and AID patients who are immune suppressed. Pseudomonas aeruginosa infections is a serious problem in patients hospitalized with cancer, Cystic fibrosis, and burns.

A significant number of pseudomonas can produce exopolysaccharides that are known as slime layers. Secretion of exopolysaccharide makes it difficult for pseudomonads to be phagocytosed by mammalian white blood cells (Ryan and Rey, 2004).

1.6.3    Escheriachia coli

E.Coli is a gram negative, facultative aneaerobic, rod-shaped and non-sporulating organism. The cells are about 2µ long and 0.5µ in diameter with a cell volume of 0.6 to 0.7µm3. The optimal growth of E.Coli occurs at 370 C but some laboratory stains can multiply at temperature of up to 490C (Fotader et al., 2005). Some strains of E. Coli  possess flagella with peritrichus arrangement (Darnton, 2007). Some virulent strains of  E.Coli causes Gastroenteritis, Urinary tract infections, and neonatal meningitis, in rare cases, virulent strains are also responsible for Hemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and Gram negative pneumonia (Toder, 2007).

Bacteria infections are usually treated with antibiotic, Antibiotic resistance is a growing problem, some of this is due to overuse of antibiotics in humans but some of it is probably due to the use of antibiotics as growth promoters in food of animals (Johnson et al., 2006).

1.6.4    Salmonella  typhi

This gram-negative enteric bacillus belongs to the family enterobaceriaceae. It is a motile, facultative anaerobe that is susceptible to various antibiotics. Currently, 107 strains of this organism have been isolated, many containing varying metabolic characteristics, levels of virulence, and multi-drug resistance is prevalent. Diagnostic identification can be attained by growth on MacConkey and the bacteria is trickly non-lactose fermenting. It also produces no gas when grown in TSI media which is sued to differentiate it from other enterobacteriaceae (Dennis et al., 2003). Infection of S.typhi lead to the development of typhoid or enteric fever.


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