Kwame Nkrumah University of Science and
Technology, Kumasi
COLLEGE OF SCIENCE
DEPARTMENT OF CHEMISTRY
The Antimicrobial Activities of the Stem Extract of Strophanthus gratus, Apocynaceae
DANIEL HENNEH
JUNE, 2013 THE ANTIMICROBIAL ACTIVITIES OF THE STEM EXTRACT OF STROPHANTHUS GRATUS, APOCYNACEAE
By
Daniel Henneh
BPharm(Hons), KNUST, Kumasi
A Thesis submitted to the Department of Chemistry, Kwame Nkrumah University of
Science and Technology in partial fulfillment of the requirements for the award of the
degree of
MASTER OF SCIENCE (ORGANIC CHEMISTRY)
College of Science
OCTOBER, 2013 CERTIFICATION
I hereby declare that this submission is my own work towards the Master of Science degree and that to the best of my knowledge, it neither contains material previously published by another person nor material which has been accepted for the award of any degree of the university, except where due acknowledgement has been made in the text.
Daniel Henneh (PG2851708) …………………… ……………………..
Candidate Signature Date
Certified by:
Dr. Sylvester K. Twumasi …………………… ……………………..
Supervisor Signature Date
Certified by:
Mr. R. B. Voegborlo …………………… ……………………..
Head of Department Signature Date
DEDICATION
This thesis is dedicated to my wife, Janet Henneh, and my elder brother, Hon. Kwasi Ameyaw-
Cheremeh, MP (Sunyani East Constituency) whose unflinching encouragement helped me to enrol in the MSc program and also to be able to complete the program. Thank you for your invaluable assistance.
ACKNOWLEDGEMENT
First, I thank God for helping, strengthening and guiding me throughout the program. Whenever the challenges abounded, you were there to take me through. You sent men to encourage and strengthen me. I am grateful to you.
Second, to my dynamic supervisor, Dr. S. K. Twumasi, I say a big thank you for your commitment and support. You were always available to help, guide and advise. Without your unreserved assistance, I would never have completed my MSc work.
I specially thank Dr. S. Osafo Acquaah of the Chemistry Department, College of Science,
KNUST. Your encouragement always echoed in my mind and produced desire and energy for the completion of this work.
I thank all the lecturers of the Department of Chemistry, College of Science, KNUST, for their variety of assistance to me. I also, thank Mr Adu and all lecturers and technicians of the
Microbiology Department, Faculty of Pharmacy, KNUST for their assistance in my laboratory work.
Lastly, I thank my course mates, especially David and Maxwell for their co-operation.
ABSTRACT
The ethanolic and aqueous extracts of the stem of Strophanthus gratus from the Botanical
Gardens, KNUST, were tested for in vitro antimicrobial activities. Traditional herbal practitioners use the decoctions of the stem of this plant to treat gonorrhoea and syphilis.
Phytochemical tests on the extracts showed that they contained saponins, flavonoids, steroids, alkaloids, anthraquinone glycosides, cyanogenetic glycosides, cardiac glycosides and tannins.
Test organisms used included E. faecalis, Pr. vulgaris, Staph aureus, B. subtilis, B. thuringiensis,
S. typhi, Ps aeruginosa and Neisseria gonorrhoeae. The results showed that both the ethanolic and aqueous extracts were active against the test organisms. However, the ethanolic extract recorded lower Minimum Inhibitory Concentrations against the organisms when compared with the aqueous extract. When the activities of the extracts were compared with those of ciprofloxacin under the same experimental conditions, it was realized that the extracts were more active than ciprofloxacin against the test organisms with the exceptions of Neisseria gonorrhoeae, S. typhi and E. coli. Auxiliary tests on the ethanolic extract of the plant showed that the plant has some antioxidant activity in addition to its antimicrobial activity.
TABLE OF CONTENTS
TITLE PAGE
CERTIFICATION……………………………………………………………………….ii
DEDICATION……………………..……………………………………………………iii
ACKNOWLEDGEMENT………….…………………………………………………..iv
ABSTRACT……...……………………………………………………………………….v
TABLE OF CONTENTS……………………………….………………………………vi
LIST OF TABLES……………………………………………….……………………....x
LIST OF FIGURES……………………………………………………………………...x
1 INTRODUCTION……………………………………………………………………....1
1.1 BACKGROUND……………….…………………………………………….…….1
1.2 STATEMENT OF THE PROBLEM…………….………………………………....2
1.3 OBJECTIVES…………………………………………………………………...….3
1.4 JUSTIFICATION OF THE PROJECT………………………………………..……4
2 LITERATURE REVIEW …………………………………………………………...….7
2.1 PHYTOCHEMICAL PRINCIPLES IN PLANTS……………………………...….7 2.1.1 Alkaloids …………………………………………………………...……….7
2.1.2 Flavonoids ……………………………………………………………...…...9
2.1.3 Tannins ……………………………………………………………...……..10
2.1.4 Terpenoids ……………………………………………………………....…11
2.1.5 Steroids from plants……………………………………………………..…11
2.1.6 Anthracene and anthraquinone derivatives…………………………..….…12
2.1.7 Cyanogenetic glycosides ……………………………………………….….12
2.2 STROPHANTHUS …………………………………………………………….…12
2.2.1 Uses of strophanthus gratus ……………………………...……………….13
2.2.2 Phytochemicals from Strophanthus gratus ………………………………..13
2.3 THE PRINCIPLE OF SOXHLET EXTRACTION ……..…………………….….15
2.4 DISEASE CAUSING BACTERIA …………………………………..………..…16
2.4.1 Staphylococci……………………………………...……………………….16
2.4.2 Neisseria ………………………………………………………………...... 16
2.4.3 Bacillus.………………………………………………...……………….…17
2.4.4 Pseudomonas …………………………………………………..………….17 2.4.5 Escherichia………………………………………………………….…...…17
2.4.6 Salmonella …………………………….……………………………..……18
2.4.7 Proteus ……………………………………………………..……….….….18
2.4.8 Klebsiella …………………………………………………...………….….18
2.5 ANTIBIOTICS ………………………………………………………………...... 18
2.5.1 Mechanisms of action of antimicrobial agents ……………………..……..21
2.5.2 Assay of antibiotics……………………………………………….…….…21
2.6 ANTIOXIDANT ACTIVITY OF PLANTS ………………………………….….22
3 MATERIALS AND METHODS ……………………………………………………..25
3.1 Materials ……………………………………………………………………….....25
3.1.1 Collection of plant material …………………………………………….…25
3.1.2 Chemicals…………………………………………………………………..25
3.1.3 General cleaning and sterilization of glassware ……………………….…..26
3.2 METHODS ……………………………………………………………………….....26
3.2.1 Preparation of extracts…………………………………………………..…26
3.2.2 Phytochemical screening……………………………………………….….27 3.2.2.1 Test for saponins ………………………………………………...27
3.2.2.2 Test for general glycosides …………………………………...…27
3.2.2.3 Test for flavonoids ………………………………………………28
3.2.2.4 Test for terpenoids and steroids …………………………………28
3.2.2.5 Test for carotenoids …………………………………………..….28
3.2.2.6 Test for coumarins …………………………………………...….29
3.2.2.7 Test for alkaloids ………………………………………………...29
3.2.2.8 Test for anthraquinones………………………………….…….....29
3.2.2.9 Test for anthraquinone glycosides ……………………………....30
3.2.2.10 Test for cyanogenetic glycoside …………………..……………30
3.2.2.11 Test for cardiac glycosides………………………….…………..30
3.2.2.12 Test for tannins ……………………………………..……….…31
3.2.3 THIN LAYER CHROMATOGRAPHY ….…………………….….…...31
3.2.4 IR METHODOLOGY ……………………………..……………….…...31
3.2.5 PREPARATION OF SOLUTIONS OF EXTRACT ………………..…..32
3.2.6 PREPARATION OF MEDIA …………………………….…………..…32 3.2.6.1 Sterile Distilled Water …………………….……………………..32
3.2.6.2 Nutrient Agar …………………………………………….……...32
3.2.6.3 Nutrient Broth ……………….………………………………..…32
3.2.7 ANTIMICROBIAL ACTIVITY TESTS …………………….……….…33
3.2.7.1 Preparation of broth culture …………………………….…….…33
3.2.7.2 Preparation of nutrient agar culture ……………………………..33
3.2.8 ANTIOXIDANT ACTIVITY TESTS ……………………………….…....34
3.2.8.1 Preparation of solutions………………………………………….34
3.2.8.1 Total Phenol Assay …………………………………………...…35
3.2.8.2 Total Antioxidant Capacity Assay ………………………………35
3.2.8.3 Reducing Power …………………………………………………36
4. RESULTS AND DISCUSSION ……………………………………………...…38
4.1 RESULTS …………..…………………………………………………………...38 4.1.1 Results for phytochemical screening………………………………………38
4.1.2 Results for extraction………………………………………………………39
4.1.3 Results for thin layer chromatography……………………………………..40
4.1.4 Results for antibiotic activity tests………………………………………....40
4.1.5 Results for antioxidant activity tests…………………………………….…47
4.1.6 Results for the IR spectrophotometry…………………………………...…51
4.2 GENERAL DISCUSSIONS ………………………..…………………………...54
4.3 ANTIMICROBIAL ACTIVITY ……………………..…………………………56
4.4 ANTIOXIDANT ACTIVITY ……………………..……………………………60
5. CONCLUSIONS AND RECOMMENDATIONS………………………………62
5.1 CONCLUSIONS………………………………………………………………...62
5.2 RECOMMENDATIONS………………………………………………...…………62
References ……………………………………………………………………………....63
Appendix 1: Anova: Two-Factor Without Replication……………………………….…68
Appendix 2: MIC Exploratory plots……………………………………………………..69
Appendix 3: t-Tests……………………………………………………………………....70
Appendix 4: Correlation of MICs between test drugs……………….....………………..71 LIST OF TABLES
Table 4.1.1: Results for phytochemical screening……………………………………….39
Table 4.1.2 Extraction yields………………………………………………………….…39
Table 4.1.3: Results for thin layer chromatography…………………………………….40
Table 4.1.4a: Antibiotic activity test results for ethanol extract…………………………41
Table 4.1.4b: Antibiotic activity test results for water extract………………………...…41
Table 4.1.4c: Antibiotic activity test results for ciprofloxacin…………………………..42
Table 4.1.4e: Minimum Inhibitory Concentrations (MIC) and Ratios…………………..46
Table 4.1.4f: Minimum Inhibitory Concentrations and Ratios…………………………..46
Table 4.1.5a: Total Phenolic Content test results………………………………………..47
Table 4.1.5b: Total antioxidant capacity test results………………………………….…48
Table 4.1.5c: Reducing Power test results for S. gratus and n-propylgallate…………....50
LIST OF FIGURES
Figure 2.1: Chemical structures of some useful alkaloids ………………………………..8
Figure 2.2: Basic flavonoid rings …………………………………………………………9
Figure 2.3: Chemical structures of some tannins ……………………………………..…10
Figure 2.4: Chemical structures of some terpenoids ………………………………….…11
Figure 2.5: chemical structures of anthracene and anthraquinone ………………………12
Figure 2.6: Chemical structure of Ouabain……………………………………………....14
Figure 2.7: Chemical structures of lignans from S. gratus………………………………15
Figure 4.1.4a: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol
Extract……………………………………………………………………………………43
Figure 4.1.4b: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol
Extract…………………………………………………………………………………....43
Figure 4.1.4c: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract
…………………………………………………………………………………...44
Figure 4.1.4d: Plots of Zone of Growth Inhibition against Log Concentration of Water
Extract……………………………………………………………………………………44
Figure 4.1.4e: Plots of Zone of Growth Inhibition against Log Concentration of
Ciprofloxacin…………………………………………………………………………….45 Figure 4.1.4f: Plots of Zone of Growth Inhibition against Log Concentration of
Ciprofloxacin…………………………………………………………………………….45
Figure 4.1.5a(A): A plot of Absorbance of tannic acid against concentration of tannic acid...... 47
Figure 4.1.5a(B): A plot of the total phenolic content present in S. gratus expressed as tannic acid equivalent (TAE) against concentration of S. gratus...... 48
V VI 7− Figure 4.1.5b(A): A plot of the Absorbance of PMo 4Mo 8O40 (formed in ascorbic acid solutions) against concentration of ascorbic acid...... 49
Figure 4.1.5b(B): A plot of the total antioxidant capacity (TAC expressed as Ascrobic acid equivalent - AAE) of S. gratus against the respective concentrations of S. gratus...49
Figure 4.1.5c: A plot of total phenolic content expressed as tannic acid equivalents (TAE) against total antioxidant capacity (TAC) expressed as ascorbic acid equivalents of S. gratus………………………………………………………………………………….….50
Figure 4.1.5d: Reducing power of S. gratus compared to n-propyl gallate ……………..51
CHAPTER ONE
1 INTRODUCTION
This chapter covers the background of the work, the statement of the problem, objectives of the work and justification of the work.
1.1 BACKGROUND
Plants do exhibit various therapeutic activities (Taleb-Contini et al; Shargel et al, 2007).
There is a worldwide renewed interest in natural products for therapy and health promotion
(Cseke et al, 2006) because from 1981 to 2002, no combinatorial compounds became approved drugs, although several are currently in late-stage clinical trials (Cseke et al, 2006). Meanwhile plant chemical constituents have served as lead compounds for discovery of drugs including aspirin, atropine, belladonna, capsaicin, cascara, colchicines, digoxin (lanoxin), ephedrine, ergotamine, ipecac, opium, physostigmine, pilocarpine, podophyllum, psyllium, quinidine, reserpine, scopolamine, senna, taxol, tubocrarine, viblastine and vincristine (Shargel et al, 2007).
Various plants in Ghana and Africa are traditionally used for their therapeutic benefits (Ghana
Herbal Pharmacopoeia, 2007; Gulla et al., 2001). The challenge however, remains that not enough research has been done into these plants and their preparations used in Ghana. The Food and Drugs Board focuses research on the safety of the use of herbal preparations and not on their therapeutic abilities. An investigation into the therapeutic ability of plants can lead to the discovery of new drug leads (Shargel et al, 2007). A recent survey revealed that 61% of the 877 drugs introduced worldwide can be traced to or were inspired by natural products (Cseke et al,
2006). Some plants in Ghana are used because of their antimicrobial actions. This is evidenced by the labeled uses of the various herbal preparations certified by the Food and Drugs Board.
Many uncertified preparations are also used for their antimicrobial activity. Usually, several phytochemical principles with different pharmacological targets are involved in the medicinal actions of herbal preparations. This characteristic may be advantageous or disadvantageous when compared with single isolated compounds. It is advantageous when the constituents work together to bring about therapy but disadvantageous when they work antagonistically or when some of the components are responsible only for toxic effects (Katzung, 2007).
This work focuses on the antimicrobial activity of the aqueous and ethanolic stem extracts of the indigenous shrub Strophanthus gratus. However, investigations would also be made to ascertain additional benefits of the use of Strophanthus gratus in the area of antioxidant effects.
1.2 STATEMENT OF THE PROBLEM
The stem of Strophanthus gratus is used by traditional herbal practitioners to treat gonorrhea and syphilis (Houghton et al, 2006). These claims are yet to be scientifically verified.
The Ghana Herbal Pharmacopoeia and other books contain literature on indications, pharmacological actions and secondary metabolites of certain plants (Ghana Herbal
Pharmacopoeia, 2007). But Strophanthus gratus is not listed in the Ghana Herbal
Pharmacopoeia. Literature has documented works mostly on the seeds of Strophanthus gratus with a few on the stem and leaves. Literature states that the seeds contain 4 -8% of ouabain (G- strophanthin), a useful cardiac glycoside (Evans, 1989; Burkill, 1985; Cowan et al, 2001). According to literature, the stem also contains minimal quantities of ouabain. Other isolated secondary metabolites from the stems are lignans (Cowan et al, 2001).
Therefore, there are two problems to be solved regarding the use of the stem of
Strophanthus gratus as herbal preparation to treat infections. These are verification of the antimicrobial actions and identification of the phytochemical principles responsible for the antimicrobial actions of the plant; both in vitro and in vivo works are necessary. However, this research work focuses on the in vitro antimicrobial and antioxidant activities of the plant.
1.3 OBJECTIVES
The objectives are:
1. To obtain aqueous and ethanolic extracts of the stem of Strophanthus gratus: Traditional
herbal practitioners use the aqueous decoction in the treatment of the infections.
However, since the solubilities of various metabolites in water differ from the solubilities
of the same metabolites in ethanol, this study would explore the antimicrobial properties
of both extracts.
2. To screen the extracts for phytochemical principles: The extracts would be screened for
the presence or absence of alkaloids, various glycosides, tannins and all major groups of
phytochemical principles.
3. To determine the in vitro antimicrobial activities of the extracts: The antimicrobial
activities of the extracts would be determined using water and methanol as the solvents
since these solvents do not have any antimicrobial actions of themselves.
Dimethylsulfoxide (DMSO) would be used in case of solubility problems 4. To establish Thin Layer Chromatograph of the extracts.
5. To determine the in vitro antimicrobial activities of prototype antibiotic under similar
conditions as (3) above and compare them to the antimicrobial activities of the crude.
Crude herbal antibiotics should not be used unless they have some form of advantage
over orthodox medication
6. To obtain the IR spectra of the extracts. The IR spectrum would be a profile against
which future extracts’ spectroscopic profile can be checked. Depending on the season and
or age of the plant certain specific metabolites may be absent or present and that may
affect the antimicrobial activities of the plant. For instance, a specific alkaloid may be
present or absent depending on the age and or season of the year but the test for alkaloids
in general may always be positive. IR spectrum would assist in discovering any
differences between the extracts used in this study and also between the extracts used in
this study and the extracts of future studies.
7. To determine some antioxidant activities of the extracts if proven to have antimicrobial
activities.
1.4 JUSTIFICATION OF THE PROJECT
Infectious diseases are by far the most important agents of diseases in Africa today.
Millions of children suffer and die from malaria, respiratory infections and diarrheal disease each year. Sexually Transmitted Infections are also common in Africa (Parry et al, 2004).
Approximately 80% of the people in developing countries depend on traditional medicine (Trape et al, 2005). In Ghana, it is estimated conservatively that between 60-90% of the general population rely on medicinal plants either totally or partially for their health care needs (Parry et al, 2004). The major problems normally encountered in the use of orthodox medicines are drug resistance and high cost (Parry et al, 2004). Herbal medicines are usually cheap with lesser incidence of drug resistance (Evans, 1989).
Strophanthus gratus stem is believed to treat a number of infections including gonorrhea and syphilis. However, no experimental data is available on the medicinal uses and phytochemical principles of the stem. In Nigeria, scientific study has been conducted on the leaves of Strophanthus hispidus (a closely related species to Strophanthus gratus) because it was traditionally claimed to cure diabetes. The research established that the leave extracts have in vivo hypoglycaemic effects and therefore the leaves are potential antidiabetic drug. The research also established the following phytochemical principles: alkaloids, flavonoids, saponins, and cardiac and cyanogenic glycosides (Ojiako et al, 2009)
Ciprofloxacin, the first line orthodox drug for the treatment of gonorrhoea in Ghana is currently facing serious resistance challenges from Neisseria gonorrhoeae. A more effective alternative and cheaper drug from a plant would therefore make a great impact on the health of
Ghanaians. Once, Strophanthus gratus allegedly treats gonorrhea, it is important that the extract is assayed with this causative organism. However, antimicrobial agents could have broad spectrum actions and therefore it also becomes important to screen the extract against other organisms (Shargel et al, 2007).
This study is aimed at establishing the groups of phytochemical principles and antimicrobial activities of the stem extracts of Strophanthus gratus. Antioxidant activity work would be done on the extracts if proven to have antimicrobial activity. This is because antioxidant activity may augment antimicrobial activity. This work would create the platform for further research into the stem of Strophanthus gratus to possibly standardize a herbal preparation from it.
CHAPTER TWO
2 LITERATURE REVIEW
The literature review includes phytochemical principles such as alkaloids, tannins, glycosides etc which are responsible for plant action, uses of Strophanthus gratus and phytochemical principles from S. gratus, disease causing bacteria, soxhlet extraction principle, antibiotics: types, mechanisms of action and assay methods, and antioxidant activity of plants and its assay methods.
2.1 PHYTOCHEMICAL PRINCIPLES IN PLANTS
The actions of any herbal medicine is due to certain phytochemicals or secondary metabolites within the herb. Not all the chemical compounds elaborated by plants are of equal interest to medicinal chemists, herbalists and pharmacognosists. The active phytochemicals are usually alkaloids, tannins, saponins, glycosides, etc and such active principles deserve special attention. Other groups of compounds such as carbohydrates, fats and proteins are usually of dietetic importance, and many such as starch and gums are used in pharmacy although lacking any marked pharmacological action (Evans, 1996).
2.1.1 Alkaloids
Alkaloids are chemically heterogenous group of basic nitrogen containing substances found predominantly in higher plants. However, such basic substances also occur in lower plants, animals, microorganisms, and marine organisms (Bhat et al, 2005)
Alkaloids usually contain one or two nitrogen atoms although some like ergotamine may contain up to five nitrogen atoms. True alkaloids meet the following criteria: Nitrogen is part of a heterocyclic ring
The occurrence of the compound is restricted to plant kingdom
The compound has complex molecular structure
The compound manifests significant physiological activity
Many alkaloids possess curative properties and are therefore important in the pharmaceutical industry. Alkaloids display a variety of pharmacological activities: analgesic potentiator (cocaine), antiamebic (emetine), anticholinergic (atropine) antimalarial (quinine), antihypertensive (reserpine), cardiac depressant (quinidine), central stimulant (caffeine), diuretic
(theophylline), gout suppressant (colchicine), and many more (Bhat et al, 2005). Some of these alkaloids are illustrated below:
Figure 2.1: Chemical structures of some useful alkaloids 2.1.2 Flavonoids
Flavonoids are the largest group of naturally occurring phenolic compounds, which occur in different plant parts both in free state and as glycosides. They are also known as plant pigments or co-pigments. The presence of these pigments is responsible for the various colours exhibited by plant parts. They exhibit various biological activities including: antimicrobial, antiulcer, antiarthritic, estrogenic, anticancer. The basic rings upon which the flavonoids are built include flavones, flavonol, flavonone and dihydroflavonol (Bhat et al, 2005; Ojiako et al, 2009).
Figure 2.2: Basic flavonoid rings
2.1.3 Tannins
Tannins are group of polymeric phenolic compounds which have tanning properties.
They are classified into hydrolysable, condensed and pseudotannins. Hydrolysable tannins can be hydrolysed either by acids or enzymes known as tannase. Hydrolysable tannins are formed by combination of several molecules of gallic acid and ellagic acid through ester-linkages to central glucose molecule. Some hydrolysable tannins have antitumour, anti-viral, anti-HIV, antimicrobial or other biological activities (Bhat et al, 2005; Ojiako et al, 2009).
Condensed tannins are polymeric proanthocyanidins and occur commonly in woody and some herbaceous plants. Condensed tannins reportedly have these actions: antioxidant (radical scavenging) and protection against cardiovascular disorders. Pseudotannins are simpler phenolic compounds of low molecular weight co-occuring with tannins e.g. gallic acid (Bhat et al, 2005).
Figure 2.3: Chemical structures of some tannins 2.1.4 Terpenoids
Terpenoids are polyisoprene compounds synthesized by some plants, marine organisms and fungi. They exhibit various biological activities including antimalarial, antihypertensive, anti-tumour and antiglaucoma. Some skeletal types of terpenoids include farnesol, farnesene and labdane (Bhat et al, 2005; Evans, 1989).
Figure 2.4: Chemical structures of some terpenoids
2.1.5 Steroids from Plants
Steroids from plants exist as conjugates with sugars. Such conjugates are known as glycosides. The two main steroidal glycosides are cardiac glycosides and saponins. Cardiac glycosides exhibit characteristic stimulatory effect on mammalian hearts. Saponins lower the surface tension of water causing their aqueous solutions to froth readily (Bhat et al, 2005; Evans,
1989).
2.1.6 Anthracene and Anthraquinone Derivatives
Anthracene consists of three fused benzene nuclei while the anthraquinone is 9,10-oxo- anthracene. Anthraquinones may occur in the free state and as glycosides. Anthracene and anthraquinones are purgatives (Bhat et al, 2005; Evans, 1989).
Figure 2.5: chemical structures of anthracene and anthraquinone
2.1.7 Cyanogenetic glycosides
Cyanogenetic glycosides produce hydrogen cyanide upon hydrolysis. E.g amygdalin.
2.2 STROPHANTHUS
Strophanthus is a genus of 35-40 species of flowering plants in the family Apocynaceae.
The genus includes vines, shrubs and small trees. They are native mainly to tropical Africa extending to South Africa with some few species in Asia (Philippines, Southern China and
Southern India) (Endress et al, 2000). Strophanthus gratus is a scandent shrub which can grow to
25 feet or more. It is glabrous with leaves oblong and can be up to 6 inches long. The leaves are short-acuminate with veins spreading at right angles to midrib. The sepals are broad and the seeds glabrous (Endress et al, 2000).
2.2.1 Uses of Strophanthus gratus
The leaves are used in Sierra Leone for gonorrhea. The leaf-sap is put onto ulcerated sores in Ivory Coast. The leaves are mashed and applied to guinea-worm sores in Ghana. They are used as a dressing for sores in Nigeria. The leaf and stem decoctions are taken for constipation in Ghana and Nigeria. A decoction of the crushed stem is taken in Ghana for severe sickness with weakness poison. The decoction is also used to treat gonorrhea and syphilis in
Ghana and Nigeria. The seeds are used as arrow poisons across various parts of Africa (Burkill,
1985).
2.2.2 Phytochemicals from Strophanthus gratus
Strophanthus gratus contains ouabain, a cardiac glycoside (Cowan et al, 2001). The cardiac glycosides are present mainly in the seeds (4-8% weight by weight) but in minimal quantities in other parts of the plant (Burkill, 1985).
Figure 2.6: Chemical structure of Ouabain
Until Cowan et al’s work, the only known isolated and characterized phytochemical in S. gratus was ouabain, a cardiac glycoside. In 2000, Cowan et al isolated lignans from the stem of
Strophanthus gratus (Cowan et al, 2001). Three lignans; pinoresinol, 8-hydroxypinoresinol and olivil have been isolated from the stem. The seeds contain glycosides, saponins, steroids; tannins, astringents (Burkill, 1985).
Figure 2.7: Chemical structures of lignans from S. gratus
pinoresinol, R = H; 8-hydroxypinoresinol, R = OH
olivil
A related species, Strophanthus hispidus has been worked on in Nigeria. All parts of
Strophanthus hispidus contain alkaloids, flavonoids, saponins, and cardiac and cyanogenic glycosides (Ojiako et al, 2009). A time trend hypoglycemic study of both ethanolic and chloroformic extracts from various parts of the plant showed that it has in vivo hypoglycemic activity. Probably, this is why local folks in Nigeria use it to treat diabetes (Ojiako et al, 2009).
2.3 THE PRINCIPLE OF SOXHLET EXTRACTION
The soxhlet exractor is for the extraction of solids such as dried leaves or seeds. The solid is put in a clean thimble. The solvent vapor rises in the side tube and condensate drops onto the solid in the thimble, leaches out soluble material, and after initiating an automatic siphon, carries it to the flask where nonvolatile extracted material accumulates. Substances of low solubility can be extracted by prolonged operation (Williamson et al, 2007)
2.4 DISEASE CAUSING BACTERIA
Several bacteria are disease causing organisms. The bacteria discussed below are common bacteria in the Ghanaian environment (GNDP, 2004)
2.4.1 Staphylococci
They are Gram-positive cocci. Staphylococci are non-motile and non-sporing and can grow aerobically or anaerobically. Staphylococcus aureus produces a golden yellow pigment. It causes skin lesions such as boils and can affect bone tissues in the case of staphylococcal osteomyelitis. It produces a toxin which, if ingested with food in which the organism has been growing can give rise to food poisoning. A common manifestation of its infection is the production of pus i.e. it is pyogenic. Other common conditions associated with staphylococcal infections are impetigo and conjunctivitis (Hugo et al, 1992; Parry et al, 2004)).
2.4.2 Neisseria
The Gram-negative pathogenic cocci belongs to the genus Neisseria. The cells are slightly curved rather than true spheres and have been likened to a kidney bean in shape. They often occur in pairs and are embedded in pus cells. Neisseria gonorrhoeae is the causal organism of the venereal disease gonorrhea. The organism can also affect the eyes, causing a purulent ophthalmia. Neisseria meningitides is a cause of cerebrospinal fever or meningococcal meniningitis (Shagel et al, 2007; Hugo et al, 1992).
2.4.3 Bacillus
They are Gram-positive rods. Members of this genus are widespread in air, soil and water and in animal products such as hair, wool and carcasses. It occurs characteristically as a large rod with square ends. It is aerobic and spore-forming. Bacillus cerus has been implicated as a cause of food poisoning. Bacillus polymyxa is the source of the antibiotic polymyxin B. Bacillus subtilis and Bacillus licheniformis are the source of bacitracin (Shagel et al, 2007; Hugo et al,
1992).
2.4.4 Pseudomonas
Pseudomonas aeruginosa is a Gram-negative rod and has in recent years assumed the role of a dangerous pathogen. It has long been a troublesome secondary infection of wounds, especially burns, but was not necessarily pathogenic. With the advent of immunosuppressive therapy following organ transplant, systemic infections including pneumonia have resulted from infection by this organism. It has also been implicated in eye infections resulting in the loss of sight. It is resistant to many antibacterial agents and is biochemically very versatile, being able to use many disinfectants as food sources (Shagel et al, 2007; Hugo et al, 1992).
2.4.5 Escherichia
Escherichia are Gram-negative organisms. Escherichia coli and the organisms
(Salmonella, Shigella, Proteus, Serratia marcescens, Klebsiella) are known as enterobacteria, so called because they inhabit the intestines of humans and animals. They are of great significance in public health. Escherichia coli is the cause of enteritis in young infants and the young of farm animals where it can cause diarrhoea and fatal dehydration. It is a common infectant of the urinary tract and bladder in humans, and is a cause of pyelitis, pyelonephritis and cystitis (Shagel et al, 2007; Hugo et al, 1992).
2.4.6 Salmonella
Salmonella are Gram-negative rods. Salmonella typhi causal organism of typhoid fever,
Salmonella paratyphi causes paratyphoid fever whilst Salmonella typhimurium, Salmonella enteritidis and very many other closely related organisms are a cause of bacterial food poisoning
(Shagel et al, 2007; Hugo et al, 1992).
2.4.7 Proteus
These are also gram-negative rods. Proteus vulgaris and morganii can infect the urinary tract of humans. They are avid decomposers of urea producing ammonia and carbon dioxide.
These organisms occasionally cause wound infection (Shagel et al, 2007; Hugo et al, 1992).
2.4.8 Klebsiella
Klebsiella pneumonia subspecies aerogenes is found in the gut and respiratory tract of man and animals, and in soil and water. It can give rise to acute bronchopneumonia in humans but is not a common pathogen (Shagel et al, 2007; Hugo et al, 1992).
2.5 ANTIBIOTICS
An antibiotic is a compound or substance that kills or slows down the growth of bacteria
(Nussbaum, 2006). However, with increased knowledge of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial compounds, including anti-fungal and other compounds (Nussbaum, 2006). The term "antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution
(Waksman, 1947). This definition excluded substances that kill bacteria but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibacterial compounds are relatively small molecules with a molecular weight of less than 2000 atomic mass units.
With advances in medicinal chemistry, most of today's antibiotics chemically are semisynthetic modifications of various natural compounds (Nussbaum, 2006). These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus 'Penicillium'), the cephalosporins(e.g. cefaclor), and the carbapenems. Compounds that are still isolated from living organisms are the aminoglycosides (e.g. amikacin), whereas other antibacterials—for example, the sulfonamides (e.g. sulphamethoxazole), the quinolones (e.g. ciprofloxacin), and the oxazolidinones—are produced solely by chemical synthesis. Accordingly, many antibacterial compounds are classified on the basis of chemical/biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity. In this classification antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.
The chemical structures of some of the named antibiotics are illustrated below.
2.5.1 Mechanisms of action of antimicrobial agents
The penicillins inhibit bacterial growth by interfering with the transpeptidation reaction of bacterial cell wall synthesis. Beta-lactamase production by microbes is the commonest form of resistance to penicillins. Cephalosporins are similar to penicillins, but more stable to many bacterial Beta-lactamases and therefore have a broader spectrum of activity. Tetracyclines are broad-spectrum bacteriostatic antibiotics that inhibit protein synthesis. Macrolide antibiotics such as erythromycin can act as bacteriostatic or bactericidal depending on the concentration. They act by binding to the 50S ribosomal RNA and inhibit protein synthesis. The aminoglycosides are irreversible inhibitors of protein synthesis (Katzung, 2007)
2.5.2 Assay of antibiotics
In microbiological assays, the response of a growing population of microorganisms to the antimicrobial agent is measured. An example is the agar diffusion assay. In that type of assay, the drug diffuses into agar seeded with a susceptible microbial population and produces a zone of growth inhibition. For two and three-dimensional assays, samples to be assayed are applied in some form of reservoir (cup or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of inhibition forms as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is within, limits, related to the concentration of the antibiotic in the reservoir (Hugo et al, 1992).
During incubation, the antibiotic diffuses from the reservoir and that part of the microbial population away from the influence of the antibiotic increases by cell division. The edge of a zone is formed when the minimum concentration of antibiotic which will inhibit the growth of the organism on the plate (critical concentration) reaches for the first time a population density too great for it to inhibit. The position of the edge is thus determined by the initial population density, growth rate of the organism and the rate of diffusion of the antibiotic (Hugo et al, 1992).
2.6 ANTIOXIDANT ACTIVITY OF PLANTS
Polyphenols (electron-rich compounds) have the ability to go into electron-donation reactions with oxidizing agents to form stable species (Kang et al., 2005) and thus inhibit or delay the oxidation of different biomolecules (Amarowicz, 2005; Seidel et al., 2000). Hence various plant phenols such as vitamin E (α-tocopherol), exhibit antioxidant properties (Kang et al., 2005; Ozgova et al., 2003; Seidel et al., 2000). Phenolic antioxidants are potent free radical terminators and this is thought to be due to the ability to donate hydrogen to free radicals and their presence is a good marker of potential antioxidant activity. The high potential of phenolic compounds to scavenge free radicals may be explained by their phenolic hydroxyl groups.
Detection of phenols in an extract is a preliminary evidence of its possible antioxidant activity. The total phenol assay is based on the reduction of phosphomolybdate-phosphotungstate salts to form a blue complex that is detected quantitively at 760 nm. The reagent used is Folin-
Cicoalteu’s phenol reagent (hexavalent phosphomolydic/phosphotunstatic acid complexes) illustrated below:
3H2O•P2O5•13WO3•5MoO3•10H2O
3H2OxP2O5•14WO3•4MoO3•10H2O
Folin-Ciocoalteu’s phenol reagent does not contain phenol. Rather, the reagent will react with phenols and non-phenolic reducing substances to form chromogens that can be detected spectrophotometrically.
The phosphomolybdenum method of assay of the total antioxidant capacity is based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate/ Mo (V) complex with a maximal absorption at 695nm. Prieto et al., 1990 successfully used this method to quantify vitamin E in plant extracts. The study revealed that the antioxidant activity of the extract increased with increasing concentration. This suggests the presence of vitamin E in the plant extract. Other compounds that might contribute to the total antioxidant capacity includes carotenoids, flavonoids and cinnamic acid derivatives (Taga et al.,
1984). In this test, ammonium phosphomolybdate, (NH4)3PMo12O40, is formed. This compound is able to accept more electrons from ascorbic acid or other donors to form a mixed valence complex that can be detected spectrophotometrically.
VI 3− - V VI 7− PMo 12O40 + 4e ⇌ PMo 4Mo 8O40
A number of in vitro models have been used for the assessment of antioxidant properties of pharmacologically active agents. Antioxidants may be classified according to their chemical nature and mode of function. Based on their mode of action, three types have been found to be consistent; enzyme antioxidants; the preventive antioxidants (Cui et al., 2004), and the scavenging or chain-breaking antioxidants (Scheibmeir et al., 2005 Reiter, 1997; Shen et al.,
2002). The reducing power measurement, as described by Oyaizu, investigates the ability of an agent to transform Fe3+ to Fe2+. Other authors have observed a direct correlation between antioxidant activity and reducing power of certain plant extracts (Duh, 1998; Tanaka et al.,
1988). These reducing properties are generally associated with the presence of reductones (Duh,
1998) which have been shown to exert antioxidant action by breaking the free radical chain reaction by donating a hydrogen atom (Gordon, 1990). Reductones are also reported to react with certain precursors of peroxides, thus preventing peroxide formation. The activity of antioxidants has been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity, and radical scavenging (Diplock, 1997). CHAPTER THREE
3 MATERIALS AND METHODS
This chapter addresses two sections; materials and methods.
3.1 MATERIALS
This section encompasses the plant material collection, chemicals and the preparation of glassware for the work.
3.1.1 Collection of plant material
The stem of the plant, Strophantus gratus was collected from the Botanical Gardens,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana in October, 2009. It was then authenticated by Prof. T.C. Fleischer at the Department of Pharmacognosy, Kwame
Nkrumah University of Science and Technology (KNUST), Kumasi.
3.1.2 Chemicals
98% ethanol was from Joseph Mills Ltd (UK). Ascorbic acid, ammonium molybdate, ferric chloride, n-propyl gallate, potassium ferricyanide, tannic acid, thiobarbituric acid (TBA), trichloroacetic acid (TCA) and Folin-Ciocalteau reagent were from Sigma-Aldrich Inc. (St.
Louis, MO, USA). Ciprofloxacin was from Dabur Pharma (New Delhi, India). Nutrient agar and nutrient broth were from Microtrade Ltd (UK). Phytochemical reagents were mostly from
ReAgent Manufacturing Ltd (UK).
3.1.3 General cleaning and sterilization of glassware
All glassware were washed thoroughly with soap solution, rinsed with distilled water and dried before used. Glassware such as pipettes and spatula were disinfected with Dettol antiseptic solution, packed into a canister and autoclaved at 121oC for 15 minutes. Petri dishes were washed, rinsed and packed into suitable canisters to dry. They were then sterilized in an oven at
170oC for 1 hour.
3.2 METHODS
This section covers methods for the preparation of the extracts from Strophanthus gratus, phytochemical screening of raw plant material and extracts, thin layer chromatography, infra red spectroscopy, antibiotic activity and antioxidant activity of the extract.
3.2.1 Preparation of extracts
The stems of the Strophanthus gratus were air-dried for 60 days and powdered. For cold maceration, 50g of the air-dried coarsely powdered material was weighed into a flat bottom flask. The sample was macerated with 1250mL of the solvent for 24hours at room temperature.
The flask was gently shaken at 2hours intervals for the first 6hours and allowed to stand for 12h without shaking.
For Soxhlet extraction, accurately 50g of the air dried coarsely powdered material was accurately weighed into a clean thimble and placed in the column of the soxhlet. 1250ml of water was added in 250ml batches. After each addition the column was covered with the condenser and the heating mantle turned on. The total extraction time was 18 hours. The extracts were concentrated in a rotary evaporator apparatus at approximately 60 °C. The concentrated extracts were kept in a desiccator until analyses. The procedure was repeated for three more 50g batches of the powder. The mass of the dried powdered samples and the extracts were weighed and the percentage yields were calculated as follows.
% yield= mass of extract x 100% Mass of sample
The procedure was repeated using ethanol (98%) as the solvent.
3.2.2 Phytochemical screening
The phytochemical screening was conducted as per Trease and Evans Pharmacognosy
(Evans, 1996).
3.2.2.1 Test for saponins
2g of extract sample was weighed and boiled in 10ml distilled water for 3-5 minutes. It was then filtered hot and shaken vigorously. A separation of froth after shaking, which persisted after some time indicated the presence of saponins, otherwise saponins were absent.
3.2.2.2 Test for general glycosides
0.5g of the extract sample was put into two separate beakers and dried at 600C. 5ml of dilute sulphuric acid was added to one beaker and 5ml of distilled water to the other and heated on a boiling water bath for 3-5 minutes and contents filtered into two separate test tubes.
The two filterates were then cooled and made alkaline with NaOH solution and heated with
Fehlings solution for 3 minutes. The formation of reddish-brown precipitation in the test tube containing the filtrate from H2SO4 treatment and the absence of precipitate in the other test tube indicated the presence of glycosides, otherwise glycosides were absent.
3.2.2.3 Test for flavonoids
(a) A small amount of magnesium ribbon was added to an alcoholic solution of the sample
which was followed by the addition of concentrated HCl dropwise. A brick-red
colouration indicates the presence of flavonoids, otherwise flavonoids are absent.
(b) A small amount of the sample was put on filter paper that has been moistened with dilute
NH3 and viewed under U.V. light. A blue colouration indicated that flavonoids were
present, otherwise flavonoids were absent.
3.2.2.4 Test for terpenoids and steroids
(a) A small amount of the extract was evaporated to dryness in a crucible and redissolved in
chloroform. A few drops of acetic anhydride were added followed by two drops of
concentrated H2SO4. Reddish-pink colouration indicated the presence of terpenoids and
steroids.
(b) A small amount of concentrated H2SO4 was added to a solution of the extract in
chloroform. Red colouration in the chloroform layer indicated terpenoids and steroids.
3.2.2.5 Test for carotenoids a) About 3ml of antimony trichloride was added to 2ml of the extract. Dark-blue colouration is indicative of carotenoids and vice versa b) About 1ml of concentrated H2SO4 was added carefully to 2ml of the extract which formed a layer under the ethereal solution. The presence or absence of an intense dark-blue or blue-voilet or greenish-blue colour in the acid layer showed the presence or absence of carotenoids.
3.2.2.6 Test for coumarin
About 3mL of the extract was put into a test tube. The test tube was then covered with a piece of filter paper moistened with dilute NaOH solution and placed in a hot water bath. After about 15 minutes, the paper was removed and exposed to UV light. Yellow- green fluorescence indicates the presence of coumarins, otherwise coumarins are absent.
3.2.2.7 Test for alkaloids
10ml of 1% HCl was added to about 2mL of the extract and left to stand for about 30mins stirring occasionally. The resulting solution was filtered and some few mL of saturated picric acid was added to 2ml portions of the filtrate. The formation or absence of precipitate indicated the presence or absence of alkaloids respectively.
3.2.2.8 Test for anthraquinones
A small amount of sample was boiled with 25ml of 0.5M KOH and 4ml of H2O2. The mixture was cooled, filtered and acidified with a few drops of acetic acid. The acidulated mixture was extracted with about 15ml of benzene. The benzene was shaken with a small amount of NH4OH.
The formation of red colouration indicated anthraquinone, otherwise anthraquinones are absent.
3.2.2.9 Test for anthraquinone glycosides
20ml of dilute H2SO4 was added to 2mL of the extract and boiled boiled. The mixture was filtered hot and a portion of the cooled filtrate was shaken with equal volume of benzene. The benzene layer was separated and shaken with about half its volume of dilute NH3 solution. A colourless ammoniacal layer indicates the absence of anthraquinone glycoside and vice versa.
3.2.2.10 Test for cyanogenetic glycoside
A sodium picrate paper was prepared by saturating a strip of filter paper in a solution of 0.5g
Na2CO3 and 0.5g of picric acid dissolved in 100ml of water. The paper was then blotted to dry.
2mL of the extract was placed in a test tube. The material was allowed to hydrolyze (with dil.
HCl) in a stoppered test tube. A few drops of chloroform were then added and the piece of moist sodium picrate paper was inserted into the test-tube, taking care that it does not come into contact with the material or touch the inner sides of the tube. The test-tube along with its contents was kept warm at 35oC for about 3 hours. The presence of the red colour of the sodium picrate paper after the 3 hours is taken as a positive test for cyanogenetic glycoside and vice versa.
3.2.2.11 Test for cardiac glycosides
To 2mL of extract was added 2mL of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1mL of concentrated sulphuric acid. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring appeared below the brown ring, while in the acetic acid layer a greenish ring formed just above the brown ring and gradually spread throughout this layer. 3.2.2.12 Test for tannins a) About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black colouration. b) To an aliquot of the extract (dissolved in water), 2ml of sodium chloride (2%) was added, filtered and mixed with 5 ml 1% gelatin solution. The presence or absence of precipitation indicated the presence or absence of tannins respectively.
3.2.3 Thin layer chromatography
The extracts (both water and ethanol extracts) were spotted on silica gel plates 1cm from the bottom and 8cm from the edge. The spots were made concentrated by repeatedly touching the plates but ensuring that they were as small as possible (1mm in diameter). The plates were then developed with ethanol, water, acetone and combinations of the three solvents. The developed plates were exposed first to UV light and secondly to Iodine crystals and measurements taken.
3.2.4 IR spectrophometry
1.7mg of the dry extract and 200mg of spectroscopic-grade KBr were weighed and transferred into a stainless steel capsule containing a ball bearing. The capsule was shaken for 2 min on a Wig-L-Bug. The sample was evenly distributed over the face of a 13-mm die and subjected to a pressure of 14,000psi for 4mins while under vacuum in a hydraulic press to produce a transparent disk which was placed in the IR spectrophotometer and the spectrum run.
3.2.5 Preparation of solutions of extract
2.5g of each extract was weighed and dissolved in 10ml of methanol to obtain 25%w/v solution. By the method of serial dilution, 15%, 10%, 5%w/v extract solutions were also prepared. For the water extract, additional concentrations of 50%, 30% and 20% were later prepared for further investigations. 0.7g of ciprofloxacin powder was weighed and dissolved in
10ml of water to obtain 7%w/v solution. Serial dilution was used to obtain 5%, 3% and 1% solutions.
3.2.6 Preparation of media
Sterile distilled water was used in the preparation of the nutrient agar and nutrient broth as follows.
3.2.6.1 Sterile distilled water
Sterile distilled water was prepared by autoclaving distilled water in sealed glass bottles at 121 degree Celsius for 30 minutes.
3.2.6.2 Nutrient agar
28g of nutrient agar powder was weighed and dissolved in 1 liter of distilled water. It was then allowed to soak for 10 minutes; swirled to mix then sterilized by autoclaving for 15 minutes at
121oC.
3.2.6.3 Nutrient broth
32.5g of nutrient broth powder was weighed and dissolved in 1 liter of distilled water. It was then allowed to soak for 10 minutes; swirled to mix then sterilized by autoclaving for 15 minutes at 121oC. 3.2.7 Antimicrobial activity tests
The tests were done as described by Manual of Microbiology (Sawer, 2002). They are described in the following subsections.
3.2.7.1 Preparation of broth culture
10ml of nutrient broth was inoculated with pure culture of test organism. It was rolled in the palms for even mixing of contents. The process was repeated for the other test organisms. The seeded broth was incubated at 37oC for 24 hours. These were pure broth cultures of the respective test organisms. The following test organisms were used: Enterococcus faecalis, Ps aeruginosa, Proteus vulgaris, Staph aureus, Bacillus subtilis, Enterococcus coli, Bacillus thuringiensis, Salmonella typhi, Neisserria gonnorrhoeae
3.2.7.2 Preparation of nutrient agar culture
The experiment was performed as described by the Manual of Microbiology (Sawer, 2002).
20ml nutrient agar in test tubes were melted in boiling water and stabilized at 45oC in thermostatic water bath for 15 minutes. Each of the molten agar was inoculated with 0.1ml of the
24-hour broth culture of test organism. They were rolled in the palms for even mixing of contents. The seeded agar were poured into separate sterile petri dishes and allowed to set. Using sterile cork borer six (6), 4 equidistant cups were created in each of the set agar and the cups labeled with the four prepared concentrations of the extract. Each cup was filled with the respective concentration of the crude extract or ciprofloxacin (the standard drug) to three-fourth full. The plates were covered and left on the Laminar Horizontal Flow Table for one hour for the extract to penetrate the agar. The plates were then incubated at 37oC for 24 hours. The zones of growth inhibition were measured and the mean zones of growth inhibition calculated. The experiment was done in triplicate for each test organism.
Control: Two control experiments were performed. In one control, the agar was not seeded with test organism. In the other control, the agar was seeded with test organism but the solvent (methanol) was used in place of the extract solutions.
3.2.8 Antioxidant activity tests
The ethanol extract was used for the antioxidant tests because it gave better antimicrobial activity test results. The antioxidant tests done included the total phenol assay, total antioxidant capacity and the reducing power assay.
3.2.8.1 Preparation of solutions
Tannic acid solutions: 3.0003g of Tannic acid powder (99.99%w/w) was weighed and dissolved in 10mL of distilled water to obtain 0.3mg/mL solution. By the method of serial dilution, densities of 0.10mg/mL, 0.03mg/mL and 0.010mg/mL of tannic acid solutions were also prepared.
Ascorbic acid solutions: 3.0303g of ascorbic acid crystals (99.0%w/w) was weighed and dissolved in 10mL of distilled water to obtain 0.3mg/mL solution. By the method of serial dilution, densities of 0.10mg/mL, 0.03mg/mL and 0.010mg/mL of ascorbic acid solutions were also prepared. n-propyl gallate: 1.2245g and 0.9184g of n-propyl gallate crystals (98%w/w) were weighed and each one dissolved in 10mL of water to obtain 0.12mg/mL and 0.09mg/mL solutions respectively. By the method of serial dilution, 0.06, 0.03, 0.01, 0.003 and 0.001mg/mL solutions were also prepared.
3.2.8.2 Total phenol assay
The total soluble phenols present in the extract was quantitatively determined by colorimetric assay using the Folin-Cicocalteu’s phenol reagent (Singleton, 1977). Tannic acid (0.01, 0.03, 0.1 and 0.3 mg/mL) was used as the standard drug and water was used to prepare the blank. 1ml of each extract solution (0.1, 0.3, 1 and 3mg/mL), 1mL each of the tannic acid solutions and 1mL of water (for the blank) were separately added to 1 ml of Folin-Cicocalteu’s phenol reagent
(diluted five fold in distilled water) in test tubes. The content of the test tubes were mixed and allowed to stand for five minutes at 25 o C in the incubator. 1ml of 2 % sodium bicarbonate solution was added to each mixture. The reaction mixtures were then incubated at 25 oC for 2 hours. The mixtures were then centrifuged at 3000 rpm for 10 min to obtain a clear supernatant.
The absorbance of the supernatants were then determined in triplicates at 760 nm using the UV- visible spectrophotometer (LKB Biochrom, Cambridge, England, Model 4050) against the blank solution. Tannic acid absorbances were plotted against tannic acid concentrations to obtain a calibrated concentration absorbance curve using MicroSoft Excel 2007 edition. The absorbances of the extract solutions were used to deduce the tannic acid equivalents (TAE) from the plot.
3.2.8.3 Total antioxidant capacity assay
Ascorbic acid was used as the standard antioxidant drug and water was used to prepare the blank.
3 mL each of the extract solutions (0.1, 0.3, 1 and 3 mg/mL) and 3mL each of the ascorbic acid solutions (0.010, 0.03, 0.10, 0.3mg/mL) and 3mL of water (for the blank) were placed in separate test tubes. 0.3 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was then added to each and the resulting mixtures were incubated at
95°C for 90 min. After the mixtures had cooled to room temperature, the absorbance of each solution was measured in triplicate using the UV-visible spectrophotometer (LKB Biochrom,
Cambridge, England, Model 4050) at 695 nm against the blank solution. The measurements were done in triplicates. The absorbances of the ascorbic acid solutions were plotted against ascorbic acid concentrations to obtain a calibrated concentration absorbance curve using MicroSoft Excel
2007 edition. The absorbances of the extract solutions were used to deduce the ascorbic acid equivalents (AAE) from the plot.
3.2.8.4 Reducing Power
The reducing capacity of the extract was determined using the method of Fe3+ -reduction to
Fe2+(Oyaizu, 1986). n-propyl gallate (0.001, 0.003, 0.01, 0.03, 0.06, 0.09 and 0.12 mg/mL ) was used as the standard antioxidant drug and water was used as the blank. 1mL each of the extract solutions (0.03, 0.1, 0.3, 1, 2, 3 and 4 mg/mL) and n-propyl gallate as well as water (for the blank) were placed into separate test tubes. Each test tube content was mixed with 2.5 ml of 0.2
M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1 % potassium ferricyanide solution. The mixture was incubated at 50oC for 20 min. Following this, 1.5 ml of 10 % trichloroacetic acid solution (TCA) was added to the incubated mixture, and centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) from each mixture was then mixed with 2.5 ml distilled water and 0.5 ml of the 0.1 % ferric chloride solution (FeCl3 (aq)) in a test tube. The absorbance was then measured at
700 nm using the UV-visible spectrophotometer (LKB Biochrom, Cambridge, England, Model 4050). The absorbance measurements were done in triplicates. Data was presented as concentration-absorbance curves with MicroSoft Excel, 2007 edition and the EC50 (concentration that gives 50% of maximal response) determined. CHAPTER FOUR
4 RESULTS AND DISCUSSION
The results obtained for phytochemical screening, extraction, antimicrobial and antioxidant tests are provided in the following subsections. The results for IR spectroscopic measurements and thin layer chromatography are also provided. The results are further discussed.
4.1 RESULTS
The results are presented in the form of tables and graphs in the following subsections.
4.1.1 Results for phytochemical screening
The results obtained for the raw plant material, cold maceration with water and ethanol and hot extraction are presented in table 4.1.1 below.
Table 4.1.1: Results for phytochemical screening
TEST INFERENCE RELATIVE INTENSITY
raw plant cold cold ethanol hot water hot ethanol material water extract extract extract extract
Saponins Present + - + +
General Present - - + + glycoside
Flavonoids Present - + + +++
Steroids and Present + + + + Terpenoids
Carotenoids Absent - - - -
Table 4.1.1: Results for phytochemical screening
TEST INFERENCE RELATIVE INTENSITY
raw plant cold cold ethanol hot water hot ethanol material water extract extract extract extract
Coumarins Absent - - - -
Alkaloids Present + + ++ ++
Anthraquinones Absent - - - -
Anthraquinone Present - - + + glycoside
Cyanogenic Present - - + + glycosides
Tannins Present ++ + +++ +
Cardiac Present - - + + glycoside
4.1.2 Results for extraction The extraction yields obtained are presented in table 4.1.2 below Table 4.1.2 Extraction yields
METHOD cold maceration soxhlet extraction
EXTRACT water extract ethanol extract water extract ethanol extract WEIGHT/g 4.9651 3.2614 12.2312 5.8434
% YIELD 9.93 6.52 24.46 11.69
4.1.3 Results for thin layer chromatography
The results obtained for the thin layer chromatography conducted are presented in table 4.1.3 below. The Solvent system used is ethanol: water: acetone in the ratio of 6:3:1 and the solvent front was 8.4cm.
Table 4.1.3: Results for thin layer chromatography
SEPARATIONS AVERAGE SAMPLE Rf VALUE FRONT/cm
water ethanol water ethanol
A 0.5 0.4 0.06 0.05
B 3.2 3.2 0.38 0.38
C 5.0 5.1 0.60 0.61
D 7.4 7.4 0.88 0.88
E 8.1 8.2 0.96 0.98
4.1.4 Results for antibiotic activity tests
The results obtained for antimicrobial activity tests with both extracts and ciprofloxacin are presented in table 4.1.4a, table 4.1.4b and table 4.1.4c. The results have also been presented graphically in figures 4.1a, 4.1b, 4.2a, 4.2b, 4.3a and 4.3b. The Minimum Inhibitory
Concentrations obtained from the graphs are further presented in tables 4.1.5a and 4.1.5b.
Table 4.1.4a: Antibiotic activity test results for ethanol extract
ZONE OF GROWTH INHIBITION/mm AT DIFFERENT TEST ORGANISM EXTRACT CONCENTRATIONS
25%w/v 15%w/v 10%w/v 5%w/v E faecalis 27.0 25.5 24.0 22.5 Ps aeruginosa 25.0 23.5 21.0 20.0 Pr vulgaris 28.0 26.0 22.0 20.0 Staph aureus 22.0 21.5 19.0 18.0 B subtilis 29.0 25.5 22.5 20.0 E coli 18.0 17.0 15.0 12.5 B. thuringiensis 28.0 27.0 24.0 21.5 Salmonella typhi 26.5 23.0 21.0 16.0 N. gonorrhoeae 22.0 20.0 14.0 11.0
Table 4.1.4b: Antibiotic activity test results for water extract
ZONE OF GROWTH INHIBITION/mm AT DIFFERENT TEST ORGANISM EXTRACT CONCENTRATIONS
50%w/v 30%w/v 20%w/v 10%w/v E faecalis 28.0 26.0 23.5 21.0 Ps aeruginosa 24.5 23.5 20.0 19.0 Pr vulgaris 28.0 25.5 22.0 20.0 Staph aureus 23.0 21.5 19.0 17.0 B subtilis 28.0 25.0 23.0 19.5 E coli 21.5 17.5 15.0 14.0 B thuringiensis 28.0 25.0 22.5 21.0 Salmonella typhi 25.0 24.0 21.5 14.0 Neisseria gonorrhoeae 25.0 21.0 16.0 12.0
Table 4.1.4c: Antibiotic activity test results for ciprofloxacin
TEST ORGANISM ZONE OF GROWTH INHIBITION/mm AT DIFFERENT CIPROFLOXACIN CONC.
7%w/v 5%w/v 3%w/v 1%w/v
E. faecalis 35.0 30.0 26.0 24.0
Pr vulgaris 30.5 26.0 22.0 20.0
Staph aureus 20.0 17.0 15.5 13.0
B subtilis 25.0 22.0 17.5 14.0
E coli 24.5 23.0 19.0 18.5
B. thuringiensis 21.5 16.5 14.0 12.5
S. taphyi 36.0 32.5 28.0 26.5
Ps. Aeruginosa 20.0 17.0 15.0 14.0
Neisseria gonorrhoeae 34.5 30.0 28.5 25.5 Figure 4.1.4a: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol Extract
Zone of Growth 30 E. faecalis Inhibition/mm Ps. aeruginosa 25 Pr. vulgaris Staph aureus 20 B subtilis
2 15 R = 0.9884 2 R = 0.9064 2 10 R = 0.9347 2 R = 0.9480 2 5 R = 0.9716
0 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Log [Ethanol extract]
Figure 4.1.4b: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol Extract
Zone of Growth 30 E. coli Inhibition/mm B. thuringiensis 25 S. typhi 20 N. gonorrhoeae
15 2 R = 0.9617 R2 = 0.9796 10 R2 = 0.9961 5 R2 = 0.9374
0 -2 -1 0 1 2 Log [Ethanol extract]
Figure 4.1.4c: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract Zone of Growth E. faecalis 30 Inhibition/mm Ps. aeruginosa 25 Pr. Vulgaris 20 Staph aureus 2 R = 0.8957 15 2 R = 0.9899 2 R = 0.9884 10 2 R = 0.9593
5
-1.5 -1 -0.5 0 0.5 1 1.5 2 Log [Water extract]
Figure 4.1.4d: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract
Zone of Growth 30 B. subtilis Inhibition/mm 25 E. coli
B. thuringiensis 20
S. typhi 15 2 R = 0.9438 2 N. gonorrhoeae 10 R = 0.9984 2 R = 0.8840 2 5 R = 0.9068 2 R = 0.9797
-1 -0.5 0 0.5 1 1.5 2 Log [Water extract]
Figure 4.1.4e: Plots of Zone of Growth Inhibition against Log Concentration of Ciprofloxacin
E. faecalis Zone of Growth35 Ps. aeruginosa Inhibition/mm 30 Pr. vulgaris Staph aureus 25 B subtilis 20
2 R = 0.9834 15 2 R = 0.9341 10 2 R = 0.9778 2 R = 0.8160 5 2 R = 0.9064 0 -0.8 -0.5 -0.2 0.1 0.4 0.7 1 Log [Ciprofloxacin]
Figure 4.1.4f: Plots of Zone of Growth Inhibition against Log Concentration of Ciprofloxacin
Zone of Growth E. coli 35 Inhibition/mm B. thuringiensis 30 S. typhi 25 N. gonorrhoeae 20 R2 = 0.9747 15 R2 = 0.9245 R2 = 0.8724 10 R2 = 0.9483 5
-1.2 -0.9 -0.6 -0.3 0 0.3 0.6 0.9 Log[Ciprofloxacin]
Table 4.1.4e: Minimum Inhibitory Concentrations (MIC) and Ratios
MIC/ %w/v MIC RATIO TEST ORGANISM Ethanol Extract Water Extract Water Extract: Ethanol Extract B. Subtilis 0.1682 0.2622 1.5589 Pr. vulgaris 0.1204 0.2233 1.8547 Ps. aeruginosa 0.01 0.0652 6.5200 Staph aureus 0.0066 0.1239 18.7727 E. faecalis 0.0020 0.0898 44.9000 N. gonorrhoeae 1.2083 2.4848 2.0564 S. typhi 0.3882 1.1431 2.9446 E. coli 0.1328 0.5853 4.4074 B. thuringiensis 0.0311 0.0948 3.0482
Table 4.1.4f: Minimum Inhibitory Concentrations and Ratios
MIC/ %w/v MIC RATIO TEST ORGANISM Cipro. Cipro :Ethanol Extract Cipro: Water Extract B. Subtilis 0.7335 4.3609 2.7975 Pr. vulgaris 0.3513 2.9178 1.5732 Ps. aeruginosa 0.2123 21.2300 3.2561 Staph aureus 0.3380 51.2121 2.7280 E. faecalis 0.2294 114.7000 2.5546 N. gonorrhoeae 0.0873 0.0723 0.0351 S. typhi 0.1266 0.3261 0.1108 E. coli 0.1119 0.8426 0.1912 B. thuringiensis 0.6090 19.5820 6.4241
4.1.5 Results for antioxidant activity tests
The results of the antioxidant activity tests are detailed in tables 4.1.5a, 4.1.5b, 4.1.5c
Table 4.1.5a: Total Phenolic Content test results DETERMINATION TANNIC ACID S. GRATUS
Concentration in mg/ml 0.0100 0.0300 0.1000 0.3000 0.1000 0.3000 1.0000 3.0000
Mean Absorbance 0.0125 0.0477 0.1261 0.3882 0.0251 0.0751 0.2620 0.7862 Total Phenolic Content in mg TAE/ml 0.0216 0.0606 0.2064 0.6153
Figure 4.1.5a(A): A plot of Absorbance of tannic acid against concentration of tannic acid
A R2 = 0.9970 Mean 0.5 Absorbance 0.4
0.3
0.2
0.1
0 0 0.1 0.2 0.3 0.4 [Tannic Acid]/ mg/mL
Figure 4.1.5a(B): A plot of the total phenolic content present in S. gratus expressed as tannic acid equivalent (TAE) against concentration of S. gratus.
Total B Phenolic 0.8 Content/ 2 R = 1 mgTAEmL-1 0.6
0.4
0.2
- 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
[S. gratus ]/mg /mL
Table 4.1.5b: Total antioxidant capacity test results DETERMINATION ASCORBIC ACID S. GRATUS
Concentration in mg/ml 0.0100 0.0300 0.1000 0.3000 0.1000 0.3000 1.0000 3.0000
Mean Absornbance 0.0214 0.0642 0.2241 0.7422 0.0121 0.0486 0.0593 0.2371 Total Antioxidant Capacity in mg TAE/ml 0.0074 0.0174 0.0548 0.1596
V VI 7− Figure 4.1.5b (A): A plot of the Absorbance of PMo 4Mo 8O40 (formed in ascorbic acid solutions) against concentration of ascorbic acid.
A Mean 0.8 2 Absorbance of R = 0.9991 V VI 7− PMo 4Mo 8O40 0.6
0.4
0.2
0 0 0.1 0.2 0.3 0.4 [Ascorbic Acid]/mg/mL
Figure 4.1.5b(B) A plot of the total antioxidant capacity (TAC, expressed as Ascorbic acid equivalent - AAE) of S. gratus against concentration of S. gratus
B 0.20 Total Antioxidant 2 R = 1 Capacity (TAC) / 0.15 mg AAE/mL
0.10
0.05
0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 [S gratus]/mgmL-1
Figure 4.1.5c: A plot of total phenolic content (TPC) expressed as tannic acid equivalents (TAE) against total antioxidant capacity (TAC) expressed as ascorbic acid equivalents (AAE) of S. gratus.
0.70 Total Phenolic 2 Content/ 0.60 R = 1 mgTAE/mL 0.50
0.40
0.30
0.20
0.10
0.00 0.00 0.05 0.10 0.15 0.20 Total Antioxidant Capacity/mgAAE/mL
Table 4.1.5c: Reducing Power test results for S. gratus and n-propylgallate
S GRATUS n-PROPYL GALLATE Log conc. Mean absorbance of Fe2+ Log conc. Mean absorbance of Fe2+ -1.5228 0.0234 -3.0000 0.0375 -1.0000 0.0656 -2.5229 0.0656 -0.5229 0.1313 -2.0000 0.3125 0.0000 0.4875 -1.5228 0.6750 0.3010 0.5123 -1.2218 0.7522 0.4771 0.5233 -1.0458 0.7530 0.6021 0.5235 -0.9208 0.7540 Figure 4.1.5d: Reducing power of S. gratus compared to n-propyl gallate
0.8 Mean S gratus Absorbance 2+ 0.7 of Fe n-propyl gallate
0.6 EC50 =0.0126 EC50 =0.3981 -1 -1 mgmL 0.5 mgmL
0.4
0.3
0.2
0.1
0 -4 -3 -3 -2 -2 -1 -1 0 1 1 -0.1 Log [Drug]
4.1.6 Results for the IR spectrophotometry The conditions for the ethanol extract IR spectrum are: Resolution: 4.0 cm-1 No. of scan: 40 Detector speed: 2.8mms-1 Remarks: Ethanol extract: KBr disc Similarly, the conditions for the water extract IR spectrum are: Resolution: 4.0 cm-1 No. of scan: 30 Detector speed: 2.8 mms-1 Remarks: Water extract: KBr disc The IR spectra are depicted in figures 4.1.6a and 4.1.6b Figure 4.1.6a: IR spectrum of hot ethanol extract of S. gratus
Figure 4.1.6b: IR spectrum of hot water extract of S. gratus
4.2 GENERAL DISCUSSIONS
There was an immediate change to dark coloration upon the addition of water to the S. gratus sample. However, the addition of ethanol to the sample slightly changed the colour of ethanol.
When subjected to heat (soxhlet extraction), both solvents intensified in colour with that of water forming foam in the process of boiling. As a result, maceration yielded low percentage of extract; 9.93% (water extract) and 6.52% (ethanol extract). Soxhlet extraction on the other hand yielded relatively high percentage of extract; 24.46% (water extract) and 11.69% (ethanol extract).
For cold maceration, the water extract tested positive for saponins, alkaloids and tannins whereas the ethanol extract tested positive for flavonoids, alkaloids and tannins. Both the water and ethanol extracts from soxhlet extraction contained saponins, flavonoids, alkaloids, anthraquinone glycosides cyanogenetic glycosides, tannins and cardiac glycosides. Except for tannins and anthraquinone glycosides, the phytochemical test results are in consonant with earlier research by Ojiako et al, 2009 on a related species, Strophanthus hispidus. However, the intensity of coloration for the tests indicated that the water extract contained more tannins whereas the ethanol extract contained more flavonoids. These differences are obviously due to solubility differences. Tannins, though organic compounds are more polar because they are polyphenolic making them more soluble in water. Flavonoids are also phenolic compounds but the fact that the ethanol could extract more flavonoids than the water indicates that the flavonoids present in S. gratus are less polar making them more soluble in ethanol than in water. The percentage yields indicate that the water was able to extract about two times as ethanol for both cold maceration and soxhlet extraction. Since the phytochemical tests indicate that the water is able to extract more tannins than the ethanol, the differences in yields are more likely to be due to the relative quantities of tannins extracted by the solvents. Even though, the yields were different due to variations in the relative quantities of different phytochemical groups extracted, the phytochemical groups extracted for the soxhlet extraction were the same. This is evidenced by the TLC results – resolved components had similar Rf values (Table 4.1.3). This is further supported by the similar IR profiles of the two extracts (Fig. 4.1.6a and 4.1.6b). The main differences in the IR profiles occur at 2927.7 cm-1 where the ethanol extract had a more intense absorption than the water extract. Such peaks are usually due to aliphatic hydrogens and are not of much importance in antimicrobial activity. However, it indicates that the ethanol is able to extract more non-polar components than the water.
Since the extracts contain several phytochemicals, not much information can be deduced from the IR as there could be several overlapping peaks. However, some observations and deductions may be made. The broad peaks near 3500cm-1 are characteristic of hydroxy functional groups (Williamson et al, 2007) and this confirms that the extracts contain phenolic compounds (tannins and flavonoids). The peaks occurring around 1640cm-1 may be due to aromatic C=C since peaks between 1660 – 1600cm-1 are usually due to C=C bonds (Williamson et al, 2007) and this confirms that the extracts contain tannins and flavonoids which are phenolic compounds having C=C bonds.
4.3 ANTIMICROBIAL ACTIVITY
The antimicrobial activity tests results show that both the water extract and ethanolic extract of the stem of S. gratus possess antimicrobial activity against all the test organisms used
(Table 4.1.4a, b and c). Many plants do possess antimicrobial activity (Evans, 1996). It is therefore not strange that these extracts also possess some antimicrobial activity. The Minimum
Inhibitory Concentration (MIC) of a drug is the least concentration that can inhibit the growth of a particular test organism (Sawer, 2002). Based on the experimental MICs (Table 4.1.4e, and f), the ethanol extract was most potent against E. faecalis (MIC = 0.002) and least potent against
Neisseria gonorrheoae (MIC=1.2083). The order of increasing potency against the test organisms for the ethanolic extract is in the order of N. gonorrheoae < S. typhi < B. subtilis < E. coli < Pr. vulgaris < B. thuringiensis < Ps. aeruginosa < Staph. aureus < E. faecalis. The water extract was however most potent against Ps. aeruginosa (MIC = 0.0652) and least potent against
N. gonorrheoae (MIC = 2.4848). The order of increasing potency against the test organisms for the water extract is in the order of N gonorrheoae < S typhi < E coli < B subtilis < Pr vulgaris <
Staph aureus < B thuriengiensis < E faecalis < Ps aeruginosa.
The orders of potency of the extracts against the test organisms were not the same. This is an indication that the compositions of the extracts are not the same. Earlier, qualitative tests done on the extracts indicate that some of the phytochemicals are more soluble in water than alcohol and vice versa. This is probably the major cause of the different orders of potency. Strikingly, both extracts recorded the least potency against Neisseria gonorrhoeae, the organism which causes gonorrhea, the disease the decoction of S. gratus stem is mostly used to treat in Ghana. The extracts rather show more in vitro activity towards other common disease causing organisms such as Pseudomonas aeruginosa and Staph aureus. Nonetheless, in vitro and in vivo results can be totally different. Some drugs when taken orally are activated or deactivated by the liver in a process called first pass metabolism (Shargel et al, 2007). Even though, the in vitro activities show that the extracts are less potent towards Neisseria gonorrheoae and more potent against the other test organisms, but in vivo activity may prove otherwise.
A two-factor ANOVA without replication (appendix 1) indicates that there were no significant differences between the mean MICs for the various extracts and that the susceptibilities of the organisms to the various extracts were similar. This was so because the
MICs for three organisms (namely Neisseria, E coli and S typhi) deviated from the normal pattern of MICs whereby the MICs of ethanol extract were lesser than those of the water extract which were also lesser than those of ciprofloxacin (Table 4.1.4e). The deviation is clearly seen in an exploratory plot (appendix 2) of individual MICs for the various organisms from the different extracts. The two-factor ANOVA without replication is therefore misleading as a statistical tool in this analysis without considering other statistical analysis.
A paired t-test between the ethanol extract and the water extract (appendix 3) indicates that there was significant differences (tcal>tcrit) between the MICs for the two extracts and the differences are not just attributable to random errors. However, the differences are likely to be due to the variable susceptibilities of the organisms to the extracts. The exploratory plots for both extracts followed similar patterns and that also accounts for the high correlation (R2 = 0.9666) between the MICs for the water and ethanol extracts. Except for the huge deviation of N. gonnorrhoeae, E. coli and S. typhi, the other paired t-tests would have also followed a similar pattern. As a result of the deviations, the correlation of MICs between ethanol extract or water extract and ciprofloxacin (appendix 4) were very poor (R2<0.3). Since an antibiotic is not intended to be effective against all pathogenic organisms, it is very expedient to analyse the susceptibility of the organisms to the extracts and ciprofloxacin, organism by organism.
The ratios of the Minimum Inhibitory Concentration of the aqueous extract to the
Minimum Inhibitory Concentrations of the ethanol extract for the various test organisms ranged from 1.5 to 45 (Table 4.1.4e). Therefore, for any test organism used, the Minimum Inhibitory
Concentration of the water extract was higher than the Minimum Inhibitory Concentration of the ethanol extract. Since the lower the MIC, the more potent a test drug against a test organism, the water extract can be said to be 1.5 to 45 times less potent than the ethanol extract against the various test organisms used. Once again, these are in vitro tests and in vivo tests may sometimes prove otherwise.
Flavonoids and tannins are known to exhibit antimicrobial activity (Bhat et al, 2005). The alkaloids and steroids usually exhibit metabolic activity and in some cases alkaloids exhibit antiprotozoa activity (e.g quinine for the treatment of malaria). The anthraquinones and anthracene glycosides may exhibit antimicrobial activity but they are widely known for their purgative actions (Evans, 1989). It was deduced from the qualitative phytochemical screening that the ethanol extract contained a higher concentration of flavonoids than the water extract, while the water extract contained a higher concentration of tannins than the ethanol extract.
These were the major differences in the phytochemical screening tests. Therefore, the antimicrobial activities of the two extracts may be mainly due to the tannins and flavonoids present. The higher activity of the ethanol extract is most likely due to the higher concentration of flavonoids since that was the major difference between the water and ethanol extracts. Also, the favonoids in the stem of S. gratus exhibit more profound antimicrobial activity than the tannins against the test organisms because the ethanol extract depicted higher in vitro antimicrobial activity.
The MICs revealed that both extracts were least potent against Neisseria gonorrheae, the organism whose infections folks use the decoctions of the stem extract to treat (Burkill, 1985).
The folkloric use of S. gratus for gonorrhea treatment is therefore not wrong but folks use water as the solvent for extraction. Meanwhile, the tests have proved that the water extract is two times less potent than the ethanol extract against Neisseria gonorrheae. Therefore, it would be better if folks used ethanol for the extraction.
Apart from its use in gonorrhea, the plant is used traditionally in the treatment of wounds and constipation (Burkill, 1985). The usefulness of S gratus in wound treatment may result from its antimicrobial actions and also its antioxidant actions. The antimicrobial action would assist in the prevention of wound sepsis and the antioxidant would help in the prevention of cell degeneration in the wound. The plant’s ability to treat constipation must be due to the presence of anthraquinone glycosides. Anthraquinone and its glycosides are known to induce peristalsis of the gastrointestinal tract (Evans, 1989). Before any drug would be accepted for use, pharmacological and toxicological (safety) tests must be carried out (Shargel et al, 2007). In both tests, the results are usually compared with a drug or drugs already in use for indications of interest and whose pharmacological and toxicological profiles are well-known. When the MIC of the extracts were compared with those of Ciprofloxacin under similar conditions, it was realized that Ciprofloxacin was 4 to 115 times less potent than the ethanol extract and 1.5 to 6.5 times less potent than the water extract against all the test organisms with the exception of Neisseria gonorrheae, S. typhi and E. coli. For these three organisms, the ethanol extract was 1.2 to 14 times less potent and the water extract 5 to 28 times less potent than ciprofloxacin. So based on MICs in vitro, ciprofloxacin is a better antimicrobial agent than S gratus for gonorrhea, typhoid fever and E coli infections. Therefore the extracts cannot be better than ciprofloxacin in the treatment of the named infections. The side effects of ciprofloxacin (nausea, constipation, abdominal pains etc) are tolerable within therapeutic doses (BNF, 2009). The toxicology of the extracts must be studied to ascertain their effects and compared with the conventional antibiotics. It could be that the extracts have less side effects and are more potent when used in vivo, in which their use may be further considered.
4.4 ANTIOXIDANT ACTIVITY
The phenol content of tannic acid increased with increasing concentration (Table 4.1.5a).
The extract also showed a concentration dependent increase in phenolic content expressed as tannic acid equivalent (Table 4.1.5b). The total antioxidant capacity of ascorbic acid increased with increasing concentration. The extract also depicted a concentration dependent increase in total antioxidant capacity expressed as ascorbic acid equivalent. This implies that, the higher the concentration the better the capacity of the extract to reduce Mo(VI) to Mo(V). The antioxidant capacity of S. gratus (TAC) was strongly dependent on the total phenolic contents (TPC) as revealed by the high correlation between TPC and TAC (Fig. 4.1.5b and 4.1.5c). The extract and the reference antioxidant n-propyl gallate increasingly reduced Fe3+ to Fe2+ with increasing concentration. This resulted in concentration dependent increase in absorbance. From the EC50 values, S. gratus was found to be about 31 fold less potent than n-propyl gallate, the reference antioxidant (Table 4.1.5c and Fig 4.1.5d). The detection of phenols in the S. gratus extract was a preliminary evidence of its possible antioxidant activity. Phenolic compounds commonly responsible for antioxidant effects are tannins and flavonoids. S gratus contains both groups of antioxidant compounds.
The antioxidant study indicates that the ethanolic extract of Strophanthus gratus possesses antioxidant effects. This property may augment the use of the plant for its antimicrobial effects as in vivo bacteria activity may lead to the production of oxidants (Salawu et al, 2006). CHAPTER FIVE
5 CONCLUSIONS AND RECOMMENDATIONS
This section has two subsections namely conclusions and recommendations.
5.1 CONCLUSIONS
The aqueous and ethanolic stem extracts of Strophanthus gratus possess antimicrobial activities against N. gonorrheoae, S. typhi, B. subtilis, E. coli, Pr. vulgaris , B. thuringiensis, Ps. aeruginosa,, Staph. aureus and E. faecalis. The ethanolic extract was more active than the aqueous extract against the test organisms. When the activities of the extracts were compared with those of ciprofloxacin under the same in vitro experimental conditions, it was discovered that the extracts were more potent than ciprofloxacin with the exceptions of N. gonorrhoeae, S. typhi and E. coli. The ethanolic extract of S. gratus has antioxidant properties that may augment its antimicrobial properties.
5.2 RECOMMENDATIONS
The experiment focused on only in vitro tests. Since in vitro results may not reflect in vivo results, it is recommended that later works be extended to that area. The work did not also reach separation of groups of phytoconstituents and individual phytoconstituents. It is recommended that later works also consider such areas to clearly ascertain which phytoconstituents are responsible for the antimicrobial activities. Later works may also focus on toxicological studies to determine the safety of the use of extracts in man. REFERENCES
Abad M.J., Bermejo P., Carretero E., Martinez-Acitores C., Noguera B., Villar A. (1996). Antiinflammatory activity of some medicinal plant extracts form Venezuela. J Ethnopharmacol 55(1): 63-68.
Amarowicz R, Troszynska A., Shahidi F. (2005). Antioxidant Activity of Almond Seed Extract and its Fractions. J Food Lipids 12: 344-358.
Attaguile G., Russo A., Campisi A., Savoca F., Acquaviva R., Ragusa N., Vanella A. (2000). Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L. Cell Biol Toxicol 16(2): 83-90.
Auddy B., Ferreira M., Blasina F., Lafon L., Arredondo F., Dajas F., Tripathi P.C., Seal T., Mukherjee, B. (2003). Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative diseases. J Ethnopharmacol 84(2-3): 131-138.
Bhat S.V., Nagasampagi B.A., Sivakumar M. (2005). Chemistry of Natural Products. New Delhi: Narosa Publishing House. Pp 103-104, 115-116, 237-243, 584-594, 605-609.
BNF. (2009). British National Formulary, London: BMJ Group. Pp 328-329, 592.
Burkill, H.M. (1985). The Useful Plants of West Tropical Africa. Vol. 1, 2nd edn. Royal Botanical Gardens Kew. Pp 50-75.
Cowan S., Stewart M., Abbiw D.K., Latif Z, Sarker S.D, Nash R.J.(2001). Lignans from Strophanthus gratus. Phytochemical communication. Fitoterapia 72 (2001).80 – 82.
Coyle J.T., Puttfarcken P. (1993b). Oxidative stress, glutamate, and neurodegenerative disorders. Science 262(5134): 689-695. Cseke J.C., Kirakosyan A., Kaufman P. B., Warber S.L., Duke J.A., Brielmann H.L. (2006). Natural Products from Plants, Second edn. CRC Press, Florida pp 2, 3.
Endress A., Bruyns T. (2000). "A revised classification of the Apocynaceae". Botanical Review 66: 1–56
Evans W.C. (1996). Trease and evans Pharmacognosy, 13th edition, Bailliere Tindall, London Pages 512
Evans W.C. (1996). Trease and evans Pharmacognosy, 14th edition, ELBS Imprint, Great Britain
Forestieri A.M., Monforte M.T., Ragusa S., Trovato A., Iauk L. (1996). Antiiflammatory, Analgesic and Antipyretic Activity in Rodents of Plant Extracts used in African Medicine. Phytother Res 10: 100-106.
Ghana Herbal Pharmacopoeia. (2007). Science and Technology Policy Research Institute and Council for Scientific and Industrial Research. Accra, Ghana: QualiType Ltd
GNDP. (2004). Standard Treatment Guidelines, Ghana National Drugs Programme, Accra: Yamens press. Pp 1-15
Govindarajan R.., Rastogi S., Vijayakumar M., Shirwaikar A., Rawat A.K., Mehrotra, S., Pushpangadan, P. (2003). Studies on the antioxidant activities of Desmodium gangeticum. Biol Pharm Bull 26(10): 1424-1427.
Gulla J., Singer A.J., and Gaspari R., (2001), Herbal use in ED patients, Acad Emerg Med. 8, 450.
Houghton P.J., Mensah A.Y., Herbal practitioners and pharmacists in Ghana. The Pharmaceutical Journal, Vol 271 No 7258. 93-94
Idu M., Onyibe H.I. (2007). Medicinal Plants of Edo State, Nigeria. Research Journal of Medicinal Plants 2: 32-41. Kang H., Mansel R.E., Jiang, W.G. (2005). Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells. Int J Oncol 26(5): 1429-1434.
Katzung B.G. (2007). Basic and Clinical Pharmacology, 10th Edn, USA: McGraw Hill Companies Inc. Pages 11 - 15, 725-759
LeBel C.P., Ischiropoulos H., Bondy S.C. (1992). Evaluation of the probe 2',7'- dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5(2): 227-231.
Miller J.R. (2003) GraphPad Version 4.0. Step-by-Step Examples. GraphPad Software Inc., San Diego, CA.
Mitsuda H., Yasumoto K., Yamamoto A. (1967). Inhibition of lipoxygenase by saturated monohydric alcohols through hydrophobic bondings. Arch Biochem Biophys 118(3): 664-669.
Mshana N.R., Abbiw D.K., Addae-Mensah I. (2000). Traditional Medicine and Pharmacopeia. Scientific , Technical and Research Commission of the Organization of African Union.
Nunoo L. (2004). Development of ―Finger Prints‖ and Evaluation of Efficacy and Safety of Herbal Blood Tonics, Unpublished Thesis. Pp 10-13
Nussbaum V.F. (2006). "Medicinal Chemistry of Antibacterial Natural Products – Exodus or Revival?". Angew. Chem. Int. Ed. 45 (31): 5072–5129.
Ojiako O.A, Igwe C.U. (2009). A Time-Trend Hypoglycemic Study of Ethanol and Chloroform Extracts of Strophanthus hispidus. Journal of Herbs, Spices & Medicinal Plants, Volume 15, Issue 1 January 2009: 1 – 8
Oyaizu M. (1986). Studies on products of browning reaction: Antioxidative activities of browning reaction prepared from glucosamine. Jpn J Nutr 44: 307-315. Ozgova S., Hermanek J., Gut I. (2003). Different antioxidant effects of polyphenols on lipid peroxidation and hydroxyl radicals in the NADPH-, Fe-ascorbate- and Fe-microsomal systems. Biochem Pharmacol 66(7): 1127-1137.
Parry E., Godfry R., Mabey D., Gill G. (2004). Principles of Medicine in Africa. 3rd Edn, Cambridge, UK: Cambridge University Press. Pp 114-115, 515
Prieto P., Pineda M., Aguilar M. (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 269(2): 337-341.
Salawu S.O., Akindahunsi A.A., Comuzzo P. (2006). Chemical composition and in vitro antioxidant activities of some Nigerian vegetables. J. Pharmacol. Toxicol. 1(5): 429-437
Sawer B. (2002). Manual of Microbiology, 10th edition, New York: Lippincott Williams and Wilkins. Pp 17-35.
Seidel V., Verholle M., Malard Y., Tillequin F., Fruchart J.C., Duriez P., Bailleul F., Teissier E. (2000). Phenylpropanoids from Ballota nigra L. inhibit in vitro LDL peroxidation. Phytother Res 14(2): 93-98.
Shargel S., Mutnick A.H., Souney P.F., Swanson L.N. (2007). Comprehensive Pharmacy Review, 6th edn, New York: Lippincott Williams and Wilkins Pp 35, 171 – 186
Singleton E.B. (1977). Robert Shaw MacIntyre. 1912-1976. Radiology 122(3): 847.
Taleb-Contini S.H., Salvador M.J., Watanabe E., Ito I.Y.,Oliveira D. (2003). Antimicrobial activity of flavonoids and steroids isolated from two Chromolaena species, Brazilian Journal of Pharmaceutical Sciences 39 (4):403 Trape J.F., Pison G., Speigel A., Enel C., Rogier C. (2002). Combating Malaria in Africa. Trends Parasitol, 18, 224-230
Waksman S.A. (1947). "What Is an Antibiotic or an Antibiotic Substance?". Mycologia 39 (5): 565–569.
Williamson K.L., Minard R.D., Masters K.M. (2007). Macroscale and Microscale Organic Experiments, fifth edn, Boston: Houghton Mifflin Company. Pp 136-140.
Appendix 1: Anova: Two-Factor Without Replication
Ethanol Water Test Organism Extract Extract Cipro. B. Subtilis 0.1682 0.2622 0.7335 Pr. vulgaris 0.1204 0.2233 0.3513 Ps. aeruginosa 0.01 0.0652 0.2123 Staph aureus 0.0066 0.1239 0.338 E. faecalis 0.002 0.0898 0.2294 N. gonorrhoeae 1.2083 2.4848 0.0873 S. typhi 0.3882 1.1431 0.1266 E. coli 0.1328 0.5853 0.1119 B. thuringiensis 0.0311 0.0948 0.609
Anova: Two-Factor Without Replication
SUMMARY Count Sum Average Variance B. Subtilis 3 1.1639 0.387966667 0.091753963 Pr. vulgaris 3 0.695 0.231666667 0.013381203 Ps. aeruginosa 3 0.2875 0.095833333 0.010935123 Staph aureus 3 0.4685 0.156166667 0.028237343 E. faecalis 3 0.3212 0.107066667 0.013151293 N. gonorrhoeae 3 3.7804 1.260133333 1.439016583 S. typhi 3 1.6579 0.552633333 0.278596803 E. coli 3 0.83 0.276666667 0.071550103 B. thuringiensis 3 0.7349 0.244966667 0.100404623
Ethanol Extract 9 2.0676 0.229733333 0.149653688 Water Extract 9 5.0724 0.5636 0.639993145 Cipro. 9 2.7993 0.311033333 0.05121868
ANOVA Source of Variation SS df MS F P-value F crit Rows 3.178469707 8 0.397308713 1.791467132 0.152724869 2.5910962 Columns 0.545599687 2 0.272799843 1.230055965 0.318482241 3.6337235 Error 3.548454393 16 0.2217784
Total 7.272523787 26
Appendix 2: MIC Exploratory plots
MIC PLOTS - EXPLORATORY
0 1 2 3 4 5 6 7 8 9 10 Ps. aeruginosa E. faecalis 0 B. thuringiensis Pr. vulgaris Staph aureus B. Subtilis 0.5 E. coli
Ethanol Extract 1 Water Extract S. typhii Cipro.
1.5
2
2.5 N. gonorrhoeae
3
Appendix 3: t-Tests
t-Test: Paired Two Sample for Means
Ethanol Extract Water Extract Mean 0.229733333 0.5636 Variance 0.149653688 0.639993145 Observations 9 9 Pearson Correlation 0.983163506 Hypothesized Mean Difference 0 df 8 t Stat -2.353550478 P(T<=t) one-tail 0.023211625 t Critical one-tail 1.859548033 P(T<=t) two-tail 0.046423249 t Critical two-tail 2.306004133
t-Test: Paired Two Sample for Means Ethanol Extract Cipro. Mean 0.229733333 0.311033333 Variance 0.149653688 0.05121868 Observations 9 9 Pearson Correlation -0.405734757 Hypothesized Mean Difference 0 df 8 t Stat -0.467728434 P(T<=t) one-tail 0.326227909 t Critical one-tail 1.859548033 P(T<=t) two-tail 0.652455818 t Critical two-tail 2.306004133
t-Test: Paired Two Sample for Means Water Extract Cipro. Mean 0.5636 0.311033333 Variance 0.639993145 0.05121868 Observations 9 9 Pearson Correlation -0.514859662 Hypothesized Mean Difference 0 df 8 t Stat 0.808794351 P(T<=t) one-tail 0.221010183 t Critical one-tail 1.859548033 P(T<=t) two-tail 0.442020365 t Critical two-tail 2.306004133
Appendix 4: Correlation of MICs for between test drugs
Ethanol extract against water extract 3 Ethanol y = 2.0332x + 0.0965 2 Extract 2.5 R = 0.9666 2
1.5 1 Water Extract
0.5
0 0 0.5 1 1.5 Water Extract
ciprofloxacin against water extract
0.8 y = -0.1457x + 0.3931 2 0.7 R = 0.2651 0.6 0.5 Cipro. 0.4 0.3 0.2 0.1 0 0 0.5 1 1.5 2 2.5 3 Water extract
Appendix 4: Correlation of MICs for between test drugs
Ethanol Extract Ethanol Extract against Ciprofloxacin 0.8 0.7 y = -0.2374x + 0.3656 2 0.6 R = 0.1646 0.5 0.4 0.3 0.2 0.1 0 0 0.5 1 1.5 Ciprofloxacin