ANTIBACTERIAL ACTIVITY OF EXTRACTS OF PIPER GUINEENSE ON SOME COMMON PATHOGENS

ABSTRACT

 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.

TABLE OF CONTENTS

Cover page                                                                                                                  i

Certification                                                                                                                ii

Dedication                                                                                                                  iii

Table of contents

Acknowledgements                                                                                                    iv

Abstract                                                                                                                      v

CHAPTER ONE

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

CHAPTER TWO

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

2.10     DETERMINATION OF MINIMUM BACTERIAL

CONCENTRATION (MBC).                                                                                    21

2.11     QUALITATIVE DETERMINATION OF THE CHEMICAL

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

CHAPTER THREE

3.0       RESULTS                                                                                                       23

3.1       Qualitative Phytochemical Screening                                                             29

CHPTER FOUR

4.0    DISCUSSION                                                                                                   30

4.1    CONCLUSION                                                                                                31

4.2    RECOMMENDATION                                                                                    31

REFERENCES                               

APPENDICES

LIST OF TABLES

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

CHAPTER ONE

1.1       INTRODUCTION

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       LITERATURE REVIEW

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).

1.3         ECOLOGY AND METHOD OF CULTIVATION

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 3^rd to the 7^th knots, should be removed. As soon as the plants apex has regenerated, the shoot underneath the 7^th 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

PEST

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.