The safety, efficacy and quality of herbal mixtures have been an important concern for health authorities and health professional, especially now there is increase in the use of herbal mixtures. This study was aimed at isolation and identification of microorganisms from some liquid herbal mixtures sold in Enugu metropolis, South East of Nigeria. A total of twenty samples of herbal mixture were selected at random, from herbal shops in Enugu metropolis and were analysed in Microbiology laboratory, Godfery Okoye University, samples were inoculated onto Nutrient agar, MaCkonkey agar and Blood agar plates, and incubated at 37^oC for 24 hrs. Potatoe dextrose agar slants were also inoculated for the isolation of fungi. Viable bacterial counts of the samples were also performed using nutrient agar. The organisms isolated were identified using biochemical tests, and the following organisms were identified Streptococcus sp, Escherichia coli, Staphylococcus sp, Proteus sp, Aspergillus sp, Rhizopus sp, Pseudomonas sp Bacillus sp Penicillum sp mucor sp. the herbal medicine were highly contaminated and most of the organisms, isolated were gotten through poor handling, poor manufacturing of this herbal mixtures and raw materials used in preparing this herbal mixtures.

TABLE OF CONTENTS

TITLE:

TITLE PAGE . . . . . . . . . i

APPROVAL PAGE. . . . . . . . . ii

DEDICATION . . . . . . . . . iii

ACKNOWLEDGEMENT . . . . . . . IV

TABLE OF CONTENTS. . . . . . . . V

LIST OF TABLES. . . . . . . . . Viii

ABSTRACT . . . . . . . . . X

CHAPTER ONE: INTRODUCTION . . . . .

INTRODUCTION . . . . . . . . 1

1.1 BACKGROUND OF STUDY . . . . . . 1

1.2 AIM . . . . . . . . . . 3

1.3 OBJECTIVE . . . . . . . . 3

CHAPTER TWO:

LITERATURE REVIEW . . . . . . 4

2.1 ORGISINSM ISOLATED FROM HERBAL PREPARATION . 4

2.2 HISTROY OF HERBAL MEDICINES. . . . . 6

2.3 SOME HERBAL MEDICINE IN USE IN NIGERIA

AND THEIR APPLICATION. . . . . . . 7

2.4 W H O APPROVAL FOR HERBAL MEDICINE . . . 12

2.5 COMMON MICROBIAL CONTAMINAT ASSOCIATED

WITH MEDICINAL PLANT USED IN PRODUCING

HERBAL MEDICINE . . . . . . 13

2.6 DECONTAMINATION OF PLANT MATERIALS

USED IN HERBAL MEDICINE. . . . . . 16

2.7 MORE ON HERBAL MEDICINE . . . . . 17

CHAPTER THREE:

3.0 MATERIAL/METHODS . . . . . . . 21

3.1 SAMPLE COLLECTION . . . . . . 21

3.2 PREPARATION OF MEDIA. . . . . . . 23

3.3 INOCULATION OF MEDIA FOR BACTERIA ISOLATION. . 23

3.4 INOCULATION OF MEDIA FOR FUNGI ISOLATION. . 23

3.5 IDENTIFICATION OF FUNGI ISOLATE. . . . . 23

3.5.1 USE OF SLIDE CULTURE METHOD FOR FUNGI ISOLATE. 23

3.5.2 CELL MOPHORLOGY OF FUNGI ISOLATE. . . . 24

3.6 EXAMINATION OF PLATE CULTURES. . . . . 24

3.7 BIOCHEMICAL TESTS. . . . . . . .24

3.7.1 GRAM STAINING. . . . . . . . 25

3.7.2 CATALASE TEST. . . . . . . . 25

3.7.3 COAGULASE. . . . . . . . . 26

3.7.4 CITRATE TEST. . . . . . . . . 26

3.7.5 INDOLE TEST. . . . . . . . 26

3.7.6 VOGES PRAUSKEUR TEST. . . . . . . 27

3.7.7 METHYL – RED TEST. . . . . . . . 27

3.7.8 OXIDASE TEST. . . . . . . . . 28

3.7.9 UREASE TEST. . . . . . . . . 28

3.7.10 MOTILITY TEST. . . . . . . . 28

3.8 PROCEDURE FOR VIABLE BACTERIA COUNT. . . . 29

CHAPTER FOUR:

4.0 RESULT . . . . . . . . . 30

4.1 GRAMS STAINING . . . . . . 34

CHAPTER FIVE

5.0 DISCUSSION . . . . . . . . 36

5.1 CONCLUSION . . . . . . . . 37

REFERENCES. . . . . . . . . . 38

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF STUDY

Herbal medicines are naturally occurring plant derived substances that are used to treat illness with local or regional healing practices. And these products are complex mixtures of organic chemicals that may come from any raw or processed part of a plant.

Herbal medicine botanically is known as medicine or phytomedicine, is it the process of using plant seeds, Berries roots, leaves, barks, or flower for medicinal purposes, which many of them are believed to have medicinal properties which are used to treat minor illness and disturbances (Snezana, et al., 2012). They are promoted as natural and safe and are therefore the preferred choice. There herbal preparations are used to treat various types of aliment, including diaherea, urinary tract infection, typhoid fever and skin disease (Sofawora 1993).

The world health organization (WHO) defined traditional medicine (TM) as the total combination of knowledge and practices, whether explicable or not, used in diagnosing preventing or eliminating physical mental or social diseases(WHO, 2008) which may rely exclusively on past experience and observation, handed down from generation to generation verbally or otherwise.

Throughout history, all cultures have employed a variety of plant derived material for the prevention and treatment of disease (H.B et al., 1999) these herbal medicines have received official recognition worldwide by different health authorities (R.B,1983;O.Akarale 1987). In developing countries, as much as 80% of the indigenous population depends on traditional plants as their primary source of health care (R.B, 1983). In most African countries including Nigeria herbal medicine is recognized as an important component of health care system, especially among rural dwellers that constitute about 70% of the population (Esinione et al., 2002). Alternative medicine, such as herbal medicine are now gaining popularity, especially because of typically low side effect (Wilt et al., 2000) and high level of acceptance by patient (Ujam et al., 2013).

There appear to be an increase in the production of herbal medicine in the last decade, and there has been an upsurge in the circulation of herbal product in Nigeria (Oyetayo, 2008).With these increased usage, the safety, efficacy and quality of medicine have been an important concern for health authorities and health professional (Oluyege and Adelabu, 2010).Due to the increased widespread use of traditional medicine it has prompted the WHO to promote the integration of traditional medicine and complimentary or alternative medicine into the national health care system of some countries and to encourage the development of national policy and regulation as essential indicators of the level of integration of such medicine within the national healthcare system (WHO, 2011).

Since they are natural products all parts of the plants can be degraded by bacteria and fungi especially molds. Unscientific methods of cultivation and collection, inappropriate harvesting and cleaning, unsuitable transportation, prolonged drying and storages, inadequate hygiene of producers and congenital climatic condition renders the raw plant material prone to infestation and exposed them to many microbe contamination. Raw materials are most often degraded by microorganisms before harvesting during handling and after prolonged storage (Matthew, 1995; Kenneth 1989). The presence sufficient numbers of microbes can be harmful to consumers. As a result of fungal contamination, the risk of mycotoxin production, especially afflatoxin, should be taken into consideration in the manufacturing process because of the proven mutagenic, carcinogenic, tretratogenic, neurotoxic, nephrotoxic, immunosuppressive activities, (Reifei, 1988; Scimca, 1995; FAO, 2000; Hohler, 2000).

The microbial quantity of herbal drugs has to be coordinated with the regulation of the European pharmacopoeia 6^th edition and regulation of medical safety of dietary ingredient. Despite several reports of fungal contamination and aflatoxins production in food stuff, limited research has be carried out on the microbial isolation and identification.

Herbal product purchased was analyzed, by isolating and identifying microbial contaminants. The microbial properties of some liquid herbal infective drugs produced and marketed in Enugu, south east Nigeria, the level of contamination was estimated and also identified. While isolation of pure culture was done based on morphology, difference where elevation forms, pigmentation and size were the major distinguishing factors for the major distinguishing factors for both fungal and bacterial contamination.

1.2 AIM

The overall aim of this work was to culture some selected herbal mixture sold by clinics, chemists, supermarkets, and streets in Enugu metropolis for microbial, contamination.

1.3 OBJECTIVES