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ABSTRACT

The antibacterial and anti-inflammatory activities of the leaf extract of Hibiscus asper Hook F.
(Malvaceae) were investigated. Solvent extraction of the leaves yielded the crude methanol extract
(CME), while solvent-guided fractionation of CME yielded a n-hexane fraction (HF), chloroform
fraction (CF), ethyl acetate fraction (EF), acetone fraction (AF), butanol fraction (BF), methanol
fraction (MF) and water fraction (WF). TheCME and the solvent fractions were subjected to
phytochemical analysis using standard procedures. The acute toxicity test (LD50) of oral doses of
CME was conducted using mice. CME and the solvent fractions were screened for antibacterial
activity using agar well diffusion method against Gram-positive bacteria: Staphylococcus aureus,
Bacillus subtilis and Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and
Klebsiellapneumoniae (clinical strains). The crude extract, CME was also screened for antiinflammatory
activities using egg albumen-induced rat paw oedema as a model for acute
inflammation, while formaldehyde-induced rat paw oedema was used as a model for chronic
inflammatory states. Results of the phytochemical analysis of CME revealed the presence of various
substances including alkaloids, tannins, flavonoids, saponins, sterols, terpenoids, carotenoids and
glycosides.The solvent fraction HF contains mainly steroids, terpenoids and carotenoids, CF contains
alkaloids, carotenoids, flavonoids, terpenoids and glycosides, EF contains flavonoids and glycosides,
AF contains tannins and glycosides,while BF, MF and WF contains saponins and glycosides. The
acute toxicity test for CME revealed that it has a high safety profile (LD50>5000 mg/kg) as the
extract was well tolerated by the animals. Result of the antibacterial screening showed that CME and
the solvent fraction EF inhibited the growth of Staphylococcus aureus, Bacillus subtilis,
Pseudomonas aeruginosa and Escherichia coli, with their minimum inhibition concentrations (MIC)
determined to be 11.34, 10.11, 6.78 and 7.61 mg/mL for CME and 5.75,3.05,9.54 and 3.37 mg/mL,
for EF respectively. Klebsiellapneumoniae was not susceptible to CME and EF. The five test
organisms were prominently susceptible to MF with MIC values as 6.25, 9.59,10.84,9.63 and 5.90
mg/mL for Staph. aureus, B. subtilis, E. coli,P. aeruginosa and K. pneumoniaerespectively. Result of
the anti-inflammatory studies showed that the crude extract, CME elicited a dose-related inhibition
of egg albumen-induced (acute) oedema in rats. At the dose level 200 mg/kg (i.p.), CME showed
moderate inhibition of rat paw oedema (% oedema inhibition = 54.10), while 300 mg/kg (i.p.)
showed high activity (% oedema inhibition = 73.77) at 4 h (P<0.05 and P<0.01). The oedema
inhibition by CME at doses of 200 and 100 mg/kg were significant (P<0.05) at 1 h and 4 h after
treatment respectively. The crude extract, CME also ameliorated chronic inflammation (arthritis)
induced by formaldehyde in rats. At 100 mg/kg (i.p.), CME showed small but significant (P<0.05)
inhibition of rat paw oedema (% inhibition of arthritis = 14.79). At dose levels of 200 and 300 mg/kg
(i.p.), CME showed fair and moderate activities (% inhibition of arthritis = 26.06 and 40.14
respectively). This work indicates that Hibiscus asper leaf has both antibacterial and antiinflammatory–
activities.

 

 

TABLE OF CONTENTS

TITLE PAGE……………………………………………………………………………….. i
CERTIFICATION…………………………………………………………………………. ii
DEDICATION……………………………………………………………………………… iii
ACKNOWLEDGEMENTS…………………………………………..…………………….. iv
TABLE OF CONTENTS……………………………………………………………………. v
LIST OF PLATES…………………………………………………………………………… viii
LIST OF TABLES…………………………………………………………………………… ix
LIST OF FIGURES…………………………………………………………………………. x
LIST OF APPENDICES…………………………………………………………………….. xi
ABSTRACT …………………………………………………………….………………….. xii
CHAPTER ONE
INTRODUCTION ……………………………………………………………………………. 1
1.1 Background…………………………………………..……………………………… 1
1.1.1 Medicinal and herbal/traditional medicines…………………………………………. 1
1.1.2 Discovery of plant-derived drugs……………………………………………………. 2
1.2 Literature Review………………………….………………………………………… 6
1.2.1 Pharmacognostic profile ofHibiscus asper………………………………………….. 6
1.2.2 Origin and geographical distribution of the plant…………………………………… 6
1.2.3 Botany of the plant……………………………………………………………………. 6
1.2.4 Description of the plant…………………………………………………………….. 7
1.2.5 Ethnomedicinal uses of Hibiscus asper………………………………………………. 10
1.3 Antimicrobial Agents………………….……………………….…………………… 10
1.3.1 Bacteriostatic and Bactericidal drugs………………………………………………… 10
1.4. Major Groups of Antimicrobial Compounds from Plants…………………………… 11
1.4.1 Alkaloids……………………………………………………….…………………… 18
1.4.2 Phenolics and Polyphenols………………………………………………………….… 21
1.4.2.1 Simple phenols and phenolic acids……………………………………………… . 21
1.4.2.2 Quinones………………………………………………………………………… .. 21
1.4.2.3 Flavonoids, flavonones and flavonols…………………………………………… … 22
1.4.2.4 Coumarins…………………………………………………………………………… 25
1.4.2.5 Tannins……………………………………………………………………………… 27
1.4.3 Terpenoids…………………………………………………………………………….. 27
1.4.4 Peptides and Lectins………………………………………………………………… … 29
1.5 Principles of Inflammation………………………………….…..…………………….. 30
1.5.1 Classification of inflammation ……………………………………………………….. 34
1.5.1.1 Acute inflammation………………………………………………………………….. 34
1.5.1.2 Chronic inflammation………………………………………………………………… 34
vi
1.5.2 Macrophage role in inflammation………………………………………………………. 34
1.5.3Cyclooxgenase pathway……………………………………………………………….. 35
1.5.4 Lipoxygenase pathway……………………………………………………………… … 37
1.5.6 Anti-inflammatory Drugs……………………………………………………………… 37
1.5.6.1 Steroidal anti-inflammatory drugs (SAIDs)…………………………………………. 37
1.5.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs)………………………………….. 39
1.5.6.3 Aspirin………………………………………………………………………………. 39
1.6 Plants with Anti-Inflammatory Activities…………………………………………. … 40
1.7 Experimental Approach for the Study of the invitro Antimicrobial and
Anti-Inflammatory Activities…………………………………………………..……….. 44
1.7.1 In vitro anti-microbial susceptibility testing (AST)……………………………………. 44
1.7.2 Agar well diffusion test………………………………………………………………. 44
1.7.3 Anti-inflammatory models…………………………….. ……………………………… 44
1.8 Chromatographic Separations……………………………….………………………. 45
1.8.1 Classification of Chromatographic methods………………………………………….. 46
1.8.1.1 Adsorption chromatography………………………………………………………… 46
1.8.1.2 Partition chromatography……………………………………………………………. 47
1.9 Rationale, Aims and Objectives…………………………………………………… 47
1.9.1 Rationale ……………………………………………………………………………. 47
1.9.2 Aims and objectives………………………………………………………………… 48
1.9.3 Specific objectives …………………………………………………………………. 48
CHAPTER TWO
2.0 MATERIALS AND METHODS………………………………………………………. 49
2.1 Materials ……………………………………………………………………………… 49
2.1.1 Chemicals, drugs and reagents…………………………………………………………. 49
2.1.2Instrumentation …………………………………………………………………….. . 49
2.1.3 Plant materials.……………………………………………………………………….49
2.2 Methods…………………………………………………………………………….. 49
2.2.1 Extraction…………………………………………………………………………. … 49
2.2.2 Fractionation of the crude extract…………………………………………………….. 50
2.3 Phytochemical methods……………………………………………………………… 50
2.3.1 Phytochemical analysis of crude extract and fractions………………………………. 50
2.3.1.1 Test for alkaloids………………………….…………………. …………………….. 50
2.3.1.2 Test for terpenoids and steroids……………………………………………………… 50
2.3.1.3 Test for tannins……………………………………………………………………… 52
2.3.1.4 Test for carotenoids………………………………………………………………….. 52
2.3.1.5 Test for flavonoids…………………………………………………………………… 52
2.3.1.6 Test for saponins……………………………………………………………………… 52
vii
2.3.1.7 Test for glycosides………………………………………………………………….. 52
2.3.1.8 Test for cyanogenic glycosides……………………………………………………… 52
2.3.1.9 Test for cardiac glycosides………………………………………………………….. 52
2.4Microbiological methods…………………………………………………………………….. 53
2.4.1 Preparation of media………………………………………………………………… 53
2.4.2 TestOrganisms…………………………………………………………………………. 54
2.4.3 AntibacterialScreening for susceptibility to crude extract and solvent fractions of Hibiscus
asper………………………………………………………………………………… 54
2.5 Pharmacological methods…………………………………………………………… 54
2.5.1 Experimentalanimals…………………………………………………………………. . 54
2.5.2Acute Toxicity Test……………………………………………………………………. 54
2.5.3Anti-inflammatory Tests……………………………………………………… ………. 55
2.5.3.1 Acute inflammation…………………………………………………………………. 55
2.5.3.2 Chronic inflammation ……………………………………………………………… 55
2.6 Statistical analysis…………………………………………………………………… 56
CHAPTER THREE
3.1 Chemical Classes Present in Crude Extract of Hibiscus asper and Solvent Fractions…. 57
3.2 Acute Toxicity Test (LD50)……………………………………………………………… 58
3.3 Antibacterial Screening and Minimum Inhibition Concentration………………………. 59
3.4 Effect of Hibiscus asper CME on Egg Albumen-Induced (Acute) Oedema…………… 61
3.5 Effect of Hibiscus asper CME on Formaldehyde-Induced Oedema in Rats…………… 64
CHAPTER FOUR
4.1 Discussion………………………………………………………………………………. 69
4.2 Conclusion………………………………………………………………………… . 72
4.3 Suggestions for further work……………………………………………………………. 72
APPENDIX………………………………………………………………………………….. 73
REFERENCES………………………………………………………………………………. 93

 

 

CHAPTER ONE

INTRODUCTION
1.1 Background
Nature has been a continuous source of pharmacologically active molecules and medicinal plants
which have been used by human generations (Iriti et al, 2010). Plants have also been one of the
important sources of medicine since the dawn of civilization. Of the250,000-500,000 known plant
species on earth, more than 80,000 are medicinal(Increase-Coker, 2005).
The tropics are naturally endowed with about 150,000 seed plants, with 120,000 found in the tropical
rain forests alone; an indication of the rich ethnomedicinal attributes of the rain forest plants(Increase-
Coker, 2005; Lewis, 2001).In Brazil alone, about 80,000 species of plants were described, which offer
enormous prospects for discovery of new compounds with therapeuticproperty (Adebolu and
Oladimeji, 2005).In spite of a rapidly expanding literature on phytochemistry, only a small percentage
of the total plant species have been examined chemically and it is still a vast field for research
(Gyang, 2001).
The plant genome, paradoxically is much larger than other natural sources (Bacteria: 1000 genes,
fungi: 10,000 genes, plants: more than 100,000 genes) and this offers a broader biochemical network
for the generation of unique and novel chemical structures. A prime example of this scenario is the
natural product anticancer drugs paclitaxel. Paclitaxel produced by Taxus brevifolia is a
cytotoxicagent that is most likely produced to prevent organisms from feeding on the plant. Thus, for
this and other reasons, drug discovery from plant, and hence research in the area has gotten better
appreciation and concern, nowadays (Silverman, 2008).
1.1.1 Medicinal and herbal/traditional medicines
Medicinal plants are used against ailments of several microbial and non-microbial origins due to their
valuable effects in health care. The affordability, reliability,availability and low toxicity of medicinal
plants in therapeutic use has made them popular and acceptable by all religions for implementation in
medicinal health care all over the world (Akharaiyi, 2011). Plants are indeed the first materials used in
alternative medicine as a type of remedy against manydiseases. In Nigeria, Ghana, Mali and Zambia,
the first line of treatment for 60% of the children with high fevers, resulting from malaria, is the use of
herbal medicines at home(WHO, 2001). Although some herbs had been priced for their medicinal,
flavouring and aromatic qualities for centuries, the synthetic products of the modern age surpassed
their importance, for a while especially in the western world. However, with the emergence of chronic
and incurable diseases, there is a tremendous renaissance and consequently, the absolute dependence
on synthetics seems to be over. People are returning to herbal products with hope for safety. The
herbal products today symbolize safety in contrast to the synthetics that are regarded as unsafe
2
tohumans and the environment (Joy et al, 1998). For this reason, the use of plants as medicines has
been patronized morevigorously and has therefore resulted in an increase in the amount of herbal
products traded within and across countries (Suresh et al, 2008). For example, Farnsworthet al,
in1985 reported that the World Health Organization estimated that about 80 % of the world
population relied on herbs for primary health care needs.More than 30% of the entire plant species at
one time or otherwere used for medicinal purposes. In India, drugs of herbal origin have been used in
traditional systems of medicine such as Unani (Islamic) andAyurveda (Hindu) (Ali, 2008). Since
ancient times,the Ayurveda system of medicine uses about 700 species, Unani 700, Siddha600,
Amchi600 and modern medicine around 30 plant species (Evans, 2002; Agrawal and Paridhavi, 2007).
Ayuvedic and other ethnomedical healers in South Asia use at least 1800 different species in
treatments and are regularly consulted by some 800 million people. In China where medicinal plants
usage goes back at least four millennia, healers have employed more than 5000plant species.
It has also been estimated that in developed counties such as United States, plants drugs constitute as
much as 25% of the total drugs, while in fast developing countries such as China and India, the
contribution is as much as 80% (Joy et al, 1998). The market for herbal medicine is quite huge in
North America, Europe and Asia (excluding China), spending an estimated $12.6 million on it
annually (Unekwe et al, 2006). That plants are veritable sources of scientific bioactive drug discovery
is attested to by the fact that many species of these plants are important condiments of observed
efficacious traditional medicine and concoctions in developing countries (Increase-Coker, 2005).
About 121 (45 tropical and 76 subtropical) major plant drugs have been identified and are still utilized
in modern medicine (Kumar et al, 1997; Ali, 2008) (Table 1.1).
Traditional systems of medicine continue to be widely practiced on many accounts. Due to population
rise, inadequate supply of drugs, prohibitive cost of treatments, side effects of several allopathic drugs
and development of resistance to currently used drugs for infectious diseases, emphasis have shifted
towards the increased use of plant materials as a source of medicines for a wide variety of human
ailments (Mathur et al, 2011; Joy et al, 1998). The affordability of herbals has also drawn the
attraction towards their use.
1.1.2 Discovery of plant-derived drugs
The discoveries made on plants for the treatment of many human diseases in folk medicine have been
associated with the study of traditional pharmacopoeia, beliefs, wisdom from the village elders and
traditional healers (Sofowora, 1982; Nwude and Ibrahim, 1980; Aliu and Nwude, 1982; Ibrahim et al,
1983). Similarly, observation of the natural instinct and progression of wild primates to
3
Table 1.1: Somemajor plant drugs still utilized in modern medicine (Kumar et al, 1997: Ali,
2008).
DRUG NAME OF PLANT USE
Vinblastine Catharanthus Anticancer
Aimalacine Catharanthus roseus Anticancer, vasodialator
Rescinnamine Rauwolfia serpentine Tranquilizer
Reserpine Rauwolfia serpentine Tranquilizer
Quinine Cinchona sp Anthimalarial,Antiarrhythmic
Pilocarpine Pilocarpus jaborandi Antiglucoma
Cocaine Erythroxylum coca Topical anaesthetic
Morphine Papaver somniferum Painkiller
Codeine Papaver somniferum Anticough
Atropine Atropa belladonna Spasmolytic
Atropine Hyoscyamus niger Spasmolytic
Cardiac glycosides Digitalis sp. For congestive heart failure
Taxol Taxus baccata T. brevifolia Antitumour
Berberine Berberis aristata Antidiarrhoeal, antiprotozoal
Pristimerin Celastrus paniculata Antimalarial
Quassinoids Ailanthus Antiprotozoal
Plumbagin Plumbago indica Antimalarial, antifungal
Diospyrin Diospyros Montana Anticancer
Gossypol Gossypium sp. Antispermatogenic
Allicin Allium sativum Antifungal, amoebicide
Ricin Ricinus communis Antitumour
Emetine Cephaelis ipecacunha Amoebicide
Glycrrhizin Glycyrrhizia glabia Antiulcer
Nimbidin Azadirachta indica Antiulcer
Catechin Acacia catechu Antiulcer
Sophoradin Sophora subrostrata Antucler
Magnolol Magnolia bark Peptic ulcer
Forskolin Coleus forskohlii Heart disease
Digitoxin,Digoxin Digitalis, Thevetia Cardio-tonic
4
Table 1.1: Some major plant drugs still utilized in modern medicine (Kumar et al, 1997: Ali,
2008) contd.
Thevenerin Thevetia Cardio-tonic
Nerrifolin Thevetia Cardio-tonic
Podophyllin Podophyllum emodi Anticancer
Indicine N-oxide Heliotropium indicum Anticancer
Elipticiine Ochrosia Anticancer
Homoharringtonine Cephalotaxus Anticancer
utilize medicinal plants in the wild and same also in some domestic animals have often led to the
discovery of medicinal plants with medicinal efficacies. (Mbaya and Ibrahim, 2011).
It is estimated that almost 75% of the useful bioactive plant derived pharmaceuticals used worldwide
were discovered by systematic investigation of “lead” molecules from traditional medicine
(Silverman, 2008). The author also reported that 20 leading drugs in1999, nine of them were derived
from natural productsand that almost 40% of the 520 new drugs approved for the drug market
between 1993 and 1994 were natural products (including plants). Greater than 60% of the anticancer
and anti-infective agents that are in the market or in clinical trials are of natural products origin
(Silverman, 2008).
The increase or prevalence of multiple drug resistance has resulted in the development of new
synthetic antimicrobial, antioxidant and anti-inflammatory drugs;moreso the new drug is necessary to
search for new antimicrobial, antioxidant and anti-inflammatory sources (Govindappa et al, 2011).
Many drugs used presently for the management of inflammatory conditions have known side and
toxic effects (Srinivas et al, 2011).
The severe side-effects of these drugs therefore made it imperative to search for anti-inflammatory
agents from plant source which have minimal drawbacks. Secondary metabolites from medicinal
plants eliciting antimicrobial, antioxidant and anti-inflammatory activities have the potential of filling
this need because their structures are different from those drugsalready studied, while those with
greater activity may likely differ in structure (Fabricant and Farnsworth, 2001). In this growing
interest, many of the phytochemical bioactive compounds from medicinal plants have shown many
pharmacological activities (Turker and Usta, 2008). Screening of various bioactive compounds from
plants has led to the discovery of new medicinal drugs exhibiting efficient treatment roles against
various diseases (Kumar et al, 2004; Sheeja and Kutta, 2007).
5
The study of pharmacological activities including antibacterial and anti-inflammatory activities of
medicinal plants is based on the investigation of active principles such as alkaloids, saponins, tannins,
flavonoids, glycosides, terpenes, phenolics, vitamins and volatile oils (Iwu, 1993; Evans, 2002,
Harborne, 1998; Ali, 2008). These active principles reside in different parts of plants including leaves,
stem, barks, roots, fruits, seeds and flowers. However, certain substances (lignin, starch, cellulose and
chitin) could modify or inhibit these activities of medicinal plants making it imperative to carry out
extraction, characterization and identification of the active principles (Ofokansi et al, 2011). A
directed (preselected) screening offers better chances of finding interesting metabolites than an
undirected (blind) screening. Such a directed screening could be based on ecological observations of
traditional experiences.
The phytochemical research based on ethno-pharmacological information is generally considered an
effective approach in the discovery of new bioactive agents from higher plants. Knowledge of the
chemicals constituents of plants is desirable not only for the discovery of therapeutic agents, but also
because such information may be of value in disclosing new sources of economic phytochemical
substances, which could serve as precursors for the synthesis of complex chemical substances,
(Sofowora, 1993).
Many researchers have conducted studies on the antimicrobial and anti-inflammatory activities of
different plant materials and have documented their findings. These workers:Adedapo et al,
2009;Takhiet al,2011; Sharma et al, 2010; Singh et al, 2010; Narayanet al, 2010; Akroum et al, 2009;
have identified different plants mostly used in folkloric medicine, which have proven antimicrobial
activities.
In the same vein these authors: Osadebe et al, , 2008; Lee et al, 2011; Ibrahim et al, 2011; Ayoola et
al, 2011; have in recent years used different animal models to identify numerous natural products with
anti-inflammatory principles and hence development of new anti-inflammatory therapeutics
(Sannigrahi et al, 2010). These plants could therefore serve as potential sources for discovery of novel
drugs.
6
1.2 Literature Review
1.2.1 Pharmacognostic profile of Hibiscus asper
Kingdom – Plantae
Division – Angiosperms
Phylum – Magnoliophyta
Class – Magnoliopsida
Subclass – Archichlamydaes
Order – Malvales
Family – Malvaceae
Subfamily – Malvoideae
Tribe – Hibisceae
Genus – Hibiscus
Species – H. asper
Bionomial name: Hibiscus asper Hook F.
Common names:
English: Wild sorrel, bush roselle, false roselle
French: Roselle savage
Hausa: Yakuwar daji, Yakuwar kaya, Yakuwar kwado,Ramar ruwa, Ramar rafi,
Jan yakuwa
Yoruba: Kekeke,Ahon ekun
Ibo: Agbaba okobolu; Ile-agu
(Ugwuozor 2010, personal communication; Schippers and Bosch, 2004;
Blench and Dendo, 2007)
1.2.2 Origin and geographical distribution of the plant
Hibiscusasper is widely distributed throughout tropical Africa.It is occasionally cultivated as a
vegetable e.g. in Senegal and Democratic Republic of Congo (Quattrocchi, 2012). It is found in fallow
fields, grassland and edges of gallery forest. As a weed, it is not considered harmful in its area of
origin. It is also mostly anthropogenic in the neighbourhood of habitation, occasionally
subspontaneous in the bush, throughout the drier Savanna (Schippers and Bosch, 2004).
1.2.3 Botany of the plant
The genus Hibiscus of the family Malvaceae comprises of 200-300species mainly in the tropics
andsub-tropics (Schippers and Bosch; 2004) Evans, 2002). The Hibiscussection Furcaria, a natural
group of about 100 species of which 30 are in tropical Africa, have in common pergamentaceous
calyx (rarely fleshly) with 10 strongly prominent veins, 5 running to the apices of the segments and 5
7
to the sinuses (Wilson, 2006). The leaves of several other species of theHibiscus section Furcaria the
more well-known economically important vegetables namely:Hibiscus sabdariffaL.,
Hibiscuscannabinus L., Hibiscus surattensis L., and Hibiscus acetosella Welw. Ex Hiern, Hibiscus
mechowii Garcke, arecultivated in Democratic Republic of Congo and used as cough medicine
(Schippersand Bosch, 2004). Hibiscus asper is very similar to H.cannabinus, but can also be
distinguished from related Hibiscus species by its stem with fine prickles, poorly developed
vegetative branches, narrow epicalyxes, lobes which are not bifurcated and a calyx with nectary,
white wooly hairs and curved prickles or bristles (Schippers and Bosch, 2004).
1.2.4 Description of the plant
The plant, H.asper, (Plate 1.1) is a perennial erect herb, of limited branching growing up to 2.5m tall;
stems having fine prickles and simple or stellate hairs. The leaves are alternate and simple and have
thread like stipules up to 6mm long. The leaf petioles are 0.5-18cm long, leaf blade is lanceolate to
ovate and unlobed or shallowly to deeply palmated 3-5 (-7)-lobed, up to 18cm x 14cm, margin
serrated or slightly sinuate, with 2-fid stellate hair on ribs and veins, palmately veined with a distinct
nectary at base of midrib.The leaves are covered with bristly hairs on the lower surface and along the
main nerves. The inflorescence is of 1 or 2 or 3 sessile flowers in the axils of the leaves. (Akobundu
and Agyakwa, 1987)
The flowers are yellow with a purple centre. They are actinomorphic, hermaphrodite or rarely
unisexual but bisexual. Sepals are valvate and with or without an epicalyx of bracteoles. The petals
number five and free from each but often adnate at the base to the staminal column, contorted or
imbricated. The stamens are numerous and monodelphous; anthers are one-celled ovary is syncarpous
and the carpels are rarely in vertical rows; the style is one, and ovules are a axile placentas (Saunders,
1966). The fruits are (Plate 1.2) ovoid or globose capsules (breaking into separate compartments)
about 3cm in diameter and up to 2cm long, hairy often speckled red and covered by calyx with bristles
sparsely and finely appressed-pubscent and many seeded. The seeds are brown and about 3mm long,
usually contain some endosperm and with straight or curved embryo. (Akobundu and Agyakwa,
1987)
8
Plate 1.1: Hibiscus asper plant
9
Plate 1.2: Hibiscus asper fruits
10
1.2.5 Ethnomedicinaluses of H.asper
The leaves ofH.asper have a wide range of usage in folkloric or ethno-medicine. The leaves dried
over a fire are applied to eczematous sores or other skin problems in human or domestic animals, in
Senegal, Guinea and Mali (Kesharo and Adam, 1974; Schippers and Bosch, 2004).The plant is
mucilaginous in water, the juice slightly acidic and a decoction is used as a remedy for urethritis,
tertiary syphilis, venereal diseases or as a vehicle for other medicines in Northern Nigeria (Dalziel,
1948; Quattrocchi, 2012). It has also been reported by Schippers (2000) that the leaves are used to
treat stomach ache and rubbed on joints of children to make them walk.
Schippers and Bosch, 2004) the plant is used as a poison antidote for venomous stings, bites, etc and
used for the treatment of cutaneous and subcutaneous parasitic infections. It has been documented
thatH.asper is used in the treatment of painful and irregular menstruationin Benin (Adjanohoun et al,
1989). The authors, Mapi (1988) and Quattrocchi (2012) observed that the plant is used to treat
leucorrhea in Cameroon, malaria in Mali and Benin, and angina in Central Africa Republic.
In addition, it is used as a depurative and diuretic in Mali and also applied on boils to reduce
swellinginflammation and hasten pus formationin Nigeria (Quattrocchi 2012). In some tropical
regions of Africa, it is also used as a potent sedative, tonic, restorative and antidepressive drug (Foyet
et al, 2011).
The leaves of H.asper are eaten as a boiled vegetable and widespread in the Sahel region. In Nigeria,
the fruits are used to thicken soup (Schippers and Bosch, 2004). The authors also reported that it is
considered an important fodder plant in the Sahel and although eaten by all livestock, if eaten in
excess, it can cause bloat in cattle, while the seeds form a main part of the diet of wild guinea fowls in
Northern Cameroon.
1.3 Antimcrobial Agents
Antimicrobial drugs are effective in the treatment of infections because of their selective toxicity; that
is, they have the ability to injure or kill an invading microorganism without harming the cells of the
host. In most instances, the selective toxicity is relative rather than absolute, requiring that the
concentration of the drug be carefully controlled to attack the microorganism while still being
tolerated by the host (Howland and Mycek, 2006).
1.3.1 Bacteriostatic and Bactericidal drugs: Naturally occurring and synthetic antibiotic are
classified as either bacteriostatic or bactericidal. Bacteriostatic drugs inhibit the growth and
replication of bacteria at serum levels achievable in the patient, thus limiting the spread of infection
while the body’s immune system attacks, immobilizes, and eliminates the pathogens. If the drug is
removed before the immune system has scavenged the organisms, enough viable organisms may
remain to begin a second cycle of infection.
11
Bactericidal drugs kill bacteria at drug serum levels achievable in the patient. Because of their
aggressive antimicrobial action, these agents are often the drugs of choice depending on the clinical
outcome (Pankey and Sabath, 2004)
The effects of bacteriostatic and bactericidal drugs on bacterial are shown in Figure 1.1.
Figure 1.1 Effects of bactericidal and bacterostatic drugs on the growth of bacteria in
vitro(Howland and Mycek, 2006)
By contrast, addition of a bactericidal agent kills bacteria, and the total number of viable organisms
decreases. For example, chloramphenicol is bacteriostatic against most gram-negative rods and is
bactericidal against other organisms, such as S. pneumonia, Haemophilus influenza, Neisseria
meningitidis and some strains of Bacteroides (Sharma, 2004; Howland and Mycek, 2006).
1.4:Major Groups of Antimicrobial Compounds from Plants
Substances involved in essential metabolic processes within an organism are identified as “primary
metabolites”. For example carbohydrates, lipids, porphyrins, amino acids, nucleic acids, polyacids
(e.g, citric, tartaric, and the like), etc. are among this class (Eastwood, 2001). The term “secondary
metabolites” is used to represent those products of plant metabolism that are associated with some
12
readily detectable properties (e.g, taste, color, and odor), a biological activity (e.g, toxicity, medicinal
or agrochemical use) or simply novel chemical structure (Evans, 2002).
In many cases, these substances serve as plant defense mechanisms against predation by
microorganisms, insects, and herbivores. Some, such as terpenoids, give plants their odors (De Groot
and Rauen, 1998); others (quinones and tannins) are responsible for plant pigment (Siame et al,
1994). Many compounds are responsible for plant flavor (e.g., the terpenoids capsaicin from chili
peppers), and some of the same herbs and spices used by humans to season food yield useful
medicinal compounds. (Cowan, 1999).
The secondary metabolites are derivatives produced by various enzymatic processes that generate
chemical compounds distinct from the primary metabolic products. Structures of some secondary
metabolites are shown in Figure 1.2. Discovery scientists in the chemical, pharmaceutical or
agrochemical industries have little interest in primary metabolites except their obvious uses in
nutrition and dietary supplements. Hence, interest is, by and large, given to secondary metabolites
such as alkaloids, flavonoids, coumarins, etc. Most secondary metabolites possess antimicrobial
properties (Wallace, 2004) and some of these are successful in getting access into the arsenal of
antibiotic therapy (Eastwood, 2001). Representative examples of groups of compounds from plant
having antimicrobial activity are given below.
13
ALKALOIDS
Figure 1.2: Structures of some secondarymetabolites of plants
14
COUMARINS
Figure 1.2: Structures of some secondary metabolites of plants contd.
15
FLAVONOIDS
Figure 1.2: Structures of some secondary metabolites of plants contd.
16
(b) Condensed Tannins
TANNINS
Figure 1.2: Structures of some secondary metabolites of plants contd.
17
PHENOLS
Figure 1.2: Structures of some secondary metabolites of plants contd.
18
1.4.1 Alkaloids
Alkaloids constitute a major class of chemical group present in plant drugs. Originally, it means
“alkali like” which was applied indiscriminately to all the organic bases (Finar, 2002). Alkaloids may
be described as naturally occurring organic substances, having heterocyclic nitrogenous nucleus
exhibiting basic properties and having a pronounced physiological action (Ali, 2008). In the plant
kingdom, the alkaloids appear to have a restricted distribution in certain families and genera. Among
the angiosperms, the Apocynaceae, Papaveraceae, Ranunclaceae, Rubiaceae, Solanaceae and
Berberidaceae are outstanding as alkaloid yielding families (Kar, 2007; Ali, 2008).Alkaloids are
found to have a range of pharmacological activities such as antimalarial, anti-tumor, relaxing heart
and respiratory muscle, anesthetic, analgesic etc.
The first medically useful example of an alkaloid was morphine, isolated in 1805 from the opium
poppy Papaver somniferum (Atherden, 2006), whilethe name morphine comes from the Greek
Morpheus, god of dreams. Codeine and heroin are both derivatives of morphine. Diterpenoid
alkaloids, commonly isolated from the plants of the Ranunculaceae, or buttercup family, are
commonly found to have antimicrobial properties. Solamargine, a glycoalkaloid from the berries of
Solanum khasianum, and other alkaloids are useful against HIV infection as well as intestinal
infections associated with AIDS. While alkaloids have been found to have microbiocidal effects
(including against Giardia and Entamoeba species), the major antidiarrheal effect is probably due to
their effects on transit time in the small intestine (Cowan, 1999).
There are significantly many works done on the antimicrobial activity of alkaloids. Berberine, a
constituent of many plants is a prototype in the group and has been tested against many strains of
microorganisms. This alkaloid has a long history of medicinal use in both Ayurvedic and Chinese
medicine (Taddese, 2004). It is present in Hydrastiscanadensis (goldenseal), Captischinesis (Coptis or
golden thread), Berberisaquifolium, (Oregon grape), Berberisvulgaris (Berbery) and Berberisaristata
(Tree turmeric). Currently, the predominant clinical uses of berberine include bacterial diarrhoea,
intestinal parasite infections and ocular trachoma infections (Ali, 2008).
Berberine is also potentially effective against trypanosomes and plasmodia. The mechanism of action
of highly aromatic planar quaternary alkaloids such as berberine and harmine is attributed to their
ability to intercalate with DNA (Cowan 1999).
Berberine was also derivated to enhance its antimicrobial activity. A series of compounds bearing 9-
O-acyl- and 9-O-alkyl-subsituents were synthesized and tested against gram-negative, gram-positive
bacteria and fungi. Octanoyl, lauroyl derivated among the acyl analogs and hexyl, heptyl, octyl,
nonyl, decyl, undecyl derivated among the alkyl analogs showed strong antimicrobial activity against
gram-positive bacteria and fungi (Taddese, 2004). Useful alkaloids such as isopteropodine,
19
pteropopin, isomitraphylline which help the white blood cells dispose harmful microorganisms and
cell debris have been reported (Ogunwenmo et al, 2007).
Screening of plant alkaloids for their antimicrobial effect is quite common in the literature but
representative sample of alkaloids that are found to have activity are listed in Table 1.2.
20
Table 1.2: List of exemplary plant alkaloids found to have antimicrobial effect. (Taddese, 2004)
Plant source Parts Antimicrobial alkaloid Test organisms
Polyalthia longifolia Root Pendulamin A and B Escherichia coli
Salmonella typhi”,
Pseudomonas
aeruginosa; Klebsiella spp
Erythrina latissima Seed pod Eryostrine (+) – 10, 11- G+& G- bacteria,
dioxyerystrine yeast, spores
Guatheria multivenia Root Liriodemne C.albicans,
C.neoformans, Staph.aureus
Schizozygia Root Isoschizogaline Bacillus subtilis
Zanthoxylum Stem bark Acetonyldihyomnitidine, 8- Anthibacterial
tetraspermum acetonyldihydroavicine
Pergularia pallida Root Pergularinine, Tylophorinidine Candida species
Murraya koenigii Fresh leaves Mahinimbine, Murrayanol, Staph.aureus, E.coil,
Mahinine Bacillus cereus
Bocconia alborea Leaves Dihydrocholerythrine, G+& G- bacteria and
Dihydrosanguinarine C. albicans
Alangium lamarekii Leaves, wood Deoxytubulosine Pseudomonas cepacia
Mitracarpus scaber Arial part Benz-[g]-isoquinoline-5, 10- AIDS related pathogens
dione
Cryptolepis Root Cryptolepine Saccharomyces
sanguinolenta cerevisiae. E. coli, C.
albicans
Clausena heptaphylla Leaves Clausenal G+& G- bacteria
21
1.4.2 Phenolics and Polyphenols
1.4.2.1 Simplephenolsandphenolicacids
Some of the simplest bioactive phytochemicals consist of a single substituted phenolic ring. Cinnamic
and caffeic acids are common representatives of a wide group of phenylpropane-derived compounds
which are in the highest oxidation state. The common herbs tarragon and thyme both contain caffeic
acid, which is effective against viruses and fungi (Cowan, 1999). The author also reported
thatCatechol and pyrogallol both are hydroxylated phenols, shown to be toxic to microorganisms.
Catechol has two-OH groups, and pryogallol has three. The site(s) and number of hydroxyl groups on
the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence
that increased hydroxylation results in increased toxicity (Geissman, 1963). In addition, some authors
have found that more highly oxidized phenols are more inhibitory (Scalbert, 1991; Urs and Dunleavy,
1975). The mechanisms thought to be responsible for phenolic toxicity to microorganisms include
enzyme inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or
through more nonspecific interactions with the proteins (Cowan, 1999).
Phenolic compounds possessing a C3 side chain at a lower level of oxidation and containing no
oxygen are classified as phenylpropanoids (Harborne, 1998) and often cited as antimicrobial as well.
Eugenol is a well-characterized representative found in clove oil. Eugenol is considered bacteriostatic
against both fungi and bacteria (Nascimento et al, 2000).
1.4.2.2 Quinones
Quinones are aromatic rings with two ketone substitutions. They are unbiquitous in nature and are
characteristically highly reactive. These compounds, being colored, are responsible for the browning
reaction in cut or injured fruits and vegetables and are an intermediate in the melanin synthesis
pathway in human skin (Yamaguchi et al, 2010). The range in colour from pale yellow to almost
black (Harborne, 1998).The individual redox potential of the particular quinone-hydroquinone pair is
very important in many biological systems; witness the role of ubiquinone (coenzyme Q) in
mammalian electron transport systems. Vitamin K is a complex naphthoquinone. Its antihemorrhagic
activity may be related to its ease of oxidation in body tissues.
In addition to providing a source of stable free radicals, quinones are known to complex irreversibly
with nucleophilic amino acids in proteins, often leading to inactivation of the protein and loss of
function. For that reason, the potential range of quinone antimicrobial effects is great. Probable targets
in the microbial cell are surface-exposed adhesions, cell wall polypeptides, and membrane-bound
enzymes. Quinones may also render substrates unavailable to the microorganism.
An anthraquinone from Cassiaitalica, has been described, which was bacteriostatic for Bacillus
anthracis, Corynebacterium pseudodiphthericum, and Pseudomonas aeruginosa and bactericidal for
22
Pseudomonas pseudomonalliae (Kamzi, 1994). Hypericin, an anthraquinone from St. John’s Wort
(Hypericumperforatum), has received much attention lately as an antidepressant, and has general
antimicrobial properties (Cowan, 1999).
1.4.2.3Flavonoids, Flavonols and Flavones
Flavonoids or bioflavonoids are hydroxylated phenolic substances which occur as a C6-C3 unit linked
to an aromatic ring (Kar, 2007). They are ubiquitous in photosynthesizing cells and are commonly
found in fruits, vegetables, nuts, seeds, stems, flowers, tea, wine, propolis and honey (Cushnie and
Lamb, 2005). Flavones are also phenolic structures containing one carbonyl group on the aromatic
ring and the addition of a 3-hydroxyl group to it yields a flavonol. Lackeman et al (1986) reported
thatpreparations containing these compounds as the principal physiologically active constituents have
been used to treat human diseases and have low toxicity in mammals. Some of them are widely
employed in medicine for various activities such as ateriosclerosis (Lackeman, et al, 1986),
antiulcerogenic and hepatoprotective activities (Evans, 2002). Flavonoids have also been
demonstrated to have antioxidant activity and are reported to elicit several beneficial effects, such as
anti-inflammatory, anti-allergic, antiviral as well as anticancer activities (Maitreyi and Sunita, 2010;
Taddese, 2004; Kar, 2007).
Some flavonoids are known to be synthesized by plants in response to microbial infection and are
generally referred to as phytoalexins. Therefore, it is expected that they should have effective
antimicrobial activity against a wide array of microorganisms (Taddese, 2004). Catechins, the most
reduced form of the C3 unit in flavonoid compounds, have been shown to inhibitinvitroVibrio
cholerae, Streptococcus mutans, Shigella, and other bacteria and microorganisms (Sakanaka et al,
1992). The catechins inactivated cholera toxin in Vibrio and inhibited isolated bacterial
glycosyltransferases in Streptococcus, possibly due to complexing activities described for quinones.
This latter activity was borne out in vivo tests of conventional rats. When the rats were fed, a diet
containing 0.1% tea catechins, fissure caries (caused by Streptococcus mutans) was reduced by 40%
(Cowan, 1999).
An isoflavone found in a West African legume, alpinumisoflavone, prevents schistosomal infection
when applied topically. Phloretin, found in certain serovars of apples, and may have activity against a
variety of microorganism. Galangin (3,5,7-trihydroxyflavone), derived from the perennial herb
Helichrysum aureonitens, seems to be a particularly useful compound, since it has shown activity
against a wide range of gram-positive bacteria as well as fungi and viruses, in particular HSV-1 and
coxsackie B virus Type 1 (Cowan, 1999).
Many research groups have isolated and identified the structures of flavonoids possessing antifungal,
antiviral and antibacterial activities (Evans 2002; Ali, 2008). Several investigators have examined the
23
relationship between flavonoid structure and antibacterial activity and these are in close agreement
(Saravanakuma et al, 2009; Alvarez et al, 2008).
Flavonoids lacking hydroxyl groups on their B-rings are more active against microorganisms than
those with the –OH groups; hence lipophilic compounds would be more disruptive (Cowan, 1999).
However, several authors have also found the opposite effect, i.e, the more hydroxylation, the greater
the antimicrobial activity (Sato et al, 1996; Ogunwenmo et al, 2007). Therefore, it is safe to say that
there is no clear predictability for the degree of hydroxylation and toxicity to microorganisms.
In seeking to elucidate the antibacterial mechanisms of action of selected flavonoids, the activity of
quercetin, for example has been partially attributed to inhibition of DNA gyrase (Cushnie and Lamb,
2005). The authors reported that it has been proposed that sophoraflavone G and (-)-epigallocatechin
gallate inhibit cytoplasmic membrane function, and that licochalcones A and C inhibit energy
metabolism. Other flavonoids whose mechanisms of action have been investigated include robinetin,
myricetin, apigenin, rutin, galangin, 2,4,2-trihydroxy-5-methylchalcone and lonchocarpol A (Cushnie
and Lamb, 2005). The activity of others has been proposed due to their ability to complex with
intracellular soluble nucleophilic amino acids in proteins and also with bacterial cell walls leading to
enzyme inactivation and loss of function (Ogunwenmo et al, 2007). Many lipophilic flavonoids may
also disrupt microbial membranes (Taddese, 2004).Some of the flavonoids that are active against
different organisms are given in Table 1.3
24
Table 1.3 Antimicrobialactivity of various flavonoids (Favaron et al, 2009, Evans, 2002;
Taddese, 2004)
Flavonoids Organisms tested
Quercetin Staphylococcus aureus, Escherchia coli,
Candida tropicals, Rabies virus, Herpes
Simplex virus type 1, Respiratory syncytial virus
infection
Naringin Staphylococcus aureus, Shigelia boydii
Pseudomonas aeroginosa, Respiratory syncytial
virus infection
Apigenin Strepltococcus pyogens, Streptococcus viridans,
Alternacia tennissima, immune deficiency viral
infection
Rutin Staphylococcus aureus, Shigella boydii
Pseudomonas aeroginosa, Bacilus anthracis,
Rabies virus, Parainfluenza virus
Hespertinn Staphylococcus aureus, Shigella boydii
Baicalin Staphylococcus aureus, Pseudomonas
aeroginosa
Chrysin Streptococcus.jaccalis, Streptococcus baris.
Streptococcus pneumoniae
Fistein Staphylococcus aureus, Staphylococcus albus
Quercetogetin Staphylococcus aureus
Hydroxyethylrutinose Pseudomonas aeroginosa, Clostridium
purfringens
Chloroflavonin Candida albicans
Datiseten Proteus vulgaris
Echinaelin Alternacia tennisima
Quercetrin Rabies virus
Allixin Helicobacter pylori
Resveratol Botrytis cinerea
25
1.4.2.4Coumarins
Coumarins are another class of phenolic compounds that owe their class name from “coumarou”, the
vernacular name of the tonka bean(Dypterix odorata Wild,), from which coumarineitself was isolated
(Bruncton,1999). Coumarins belong to a group of compounds known as benzopyrones made of fused
benzene and alpha-pyrone rings. This group of compounds arises from the metabolism of
phenylalanine via a cinnamic acid or p-coumarin acid synthesis and the primary site of synthesis is
suggested to be the young actively growing leaves with stems and roots playing a comparatively
minor role (Ali, 2008).
The distribution of biologically active coumarins in a wide range of plants seems to correlate with
their ability to act as phytoalexins e.g., capsidol (Kar, 2007). They accumulate on the surface of the
plant parts, in response to traumatic injury during the wilting process, and inhibit the growth and
sporulation of fungal plant pathogens(Kar, 2007). The substituents on the ester or carboxylic acid
functional group on the coumarin ring give it a potent inhibitory activity against both Gram-positive
and Gram-negative bacteria (Kawase et al, 2001). Kayser and Kolodziej (1999) reported that while
coumarins with a methoxy function at C-7 and, if present an OH group at either the C-6 or C-8
position are invariably effective against the spectrum of tested standard bacteria (Gram-negative
microorganisms including the Gram-positive bacterium Staphylococcusaureus), the presence of an
aromatic dimethoxy arrangement is apparently favourable against those microorganisms which
requirespecial growth factors (beta-hemolytic Streptococcus, Streptococcuspneumonia and
Haemophilusinfluenza). A combination of these structural features, two methoxy functions at least
one phenolic group as reflected by the highly oxygenated coumarins, identify promising candidates
with antibacterial broad-spectrum activity.
There are reports on the efficacies of pure coumarins and their analogs against Gram-positive, Gramnegative
bacteria and fungi. Scopletin, for instance, obtained fromAgyreia aspeciosa roots, was found
to be highly potent against Alternaria alternata (Shukla et al, 1999).
As a group, coumarins have been found to stimulate macrophages, which could have an indirect
negative effect on infections. Hydroxycinnamic acids, related to coumarins, seem to be inhibitory to
gram-positive bacteria. Also, phytoalexins, which are hydroxylated derivatives of coumarins, are
produced in plants (including vines, Ulmus glabra, Gosypium, carrots) in response to fungal infection
and can be presumed to have antifungal activity (Evans, 2002).
Table 1.4 lists some of the coumarins that have been tested for antimicrobial activity.
26
Table 1.4: Examples of coumaric compounds reported to have antimicrobial properties (Ali,
2008; Taddese, 2004).
Coumarin Microorganisms
Psoralen Staphylococcus aureaus, Candida species
6,8-dihhydroxy-5-7- dimethoxy Streptococcus pneumonia
coumarine Staphylococcus aureus
Hemiarin Acaligenes faccalis, Aspergillus species, candida species
Scopletin Staphylococcus aureaus, Klebsella pneumoniae, Proteus
Mirabilis,Pseudomonas aeroginosa, Alternaria alternata,
Pencillium chrysogenum
Umckalin Staphylococcus aureaus, Streptococcus pneumonia
5,6,7-trimethoxy coumarine Streptococcus Pneumoniae, Hemophylus influenza
Agelicin Aspergillus species, Candida species, Cryptococcus
pneoformans, Saccharomyes species
5,8-di (2,3-dihydroxy -3- Aspergillus species
methyl butoxy)- psoralen
Byankangelicin Cladosporium species
Oxypeucedanin Cladosportum species
27
1.4.2.5Tannins
Tannin is a general descriptive name for a group of polymeric phenolic substances capable of tanning
leather or precipitating gelatin from solution, a property known as astringency (Akiyama et al, 2001).
Their molecular weights range from 500 to 3,000, and they are found in almost every plant part: bark,
wood, leaves, fruits, and roots and occurs in cell sap often in distinct vacuoles (Akiyama et al, 2001;
Ali, 2008). They are divided into two groups, hydrolysable and condensedtannins. Hydrolysable
tannins are based on gallic acid, usually as multiple esters with d-glucose; while the more numerous
condensed (non-hydrolysable) tannins (often called proanthocyanidins) are derived from flavonoids
monomers. Tannins may be formed by condensations of flavan derivatives which have been
transported to woody tissues of plants. Alternatively, tannins may be formed by polymerization of
quinone units. This group of compounds has received a great deal of attention in recent years, since it
was suggested that the consumption of tannin-containing beverages, especially green teas and red
wines, can cure or prevent a variety of ailments (Serafini et al, 1994).
The antimicrobial activity of tannins has been reported for a number of plants. For example,
comparison in the antimicrobial activity of plant extracts from Syzgium jambor(L) Alston, Hamamalis
virginiana, Kranetia triandra, Alchemilla vulgaris and Rubus fruitlosus have correlated with their
tannin content (Taddese, 2004). Elimination of tannins in these plants totally suppressed the
antimicrobial activities (Djipa, 2000). These compounds were found to form complexes with proteins
through the so-called non-specific forces such as hydrogen bonding and hydrophobic effects, as well
as by covalent bond formation (Stern et al, 1996). Thus, the mode of antimicrobial action of many
types of tannins may be related to their ability to inactivate microbial adhesions, enzymes, and cell
envelope transport proteins.(Ogunwenmo et al, 2007).
1.4.3Terpenoids
Thefragrance of plants is carried in the so called ‘quinta essentia’, or essential oil fraction. These oils
are secondary metabolites that are highly enriched in compounds based on an isoprene structure. They
are called terpenes, their general chemical structure is C10H16, and they occur as diterpenes,
triterpenes, and tetraterpenes (C20, C30, and C40), as well as hemiterpenes (C5) and sesquiterpenes
(C15). When the compounds contain additional elements, usually oxygen, they are termed terpenoids
(or isoprenoids) a subclass of the prenyllipids (terpenes, prenylquinones and sterols) (Finar, 2002).
Terpenoids are synthesized from acetate units, and as such they share their origins with fatty acids.
They differ from fatty acids in that they contain extensive branching and are cyclized. Examples of
common terpenoids are menthol and camphor (monoterpenes) and farnesol and artemisin
(sesquiterpenoids) (Ajali, 2004)
28
As the largest class of natural products, these compounds have a variety of roles in mediating
antagonistic and beneficial interactions among organisms. They defend many species of plants,
animals and microorganisms against predators, pathogens and competitors, and they are involved in
conveying messages to conspecifics and mutualists regarding the presence of food, mates and enemies
(Gershenzon and Dudareva, 2007). They also play vital roles in the plant physiology as well as
important functions in all cellular membranes.
Terpenenes or terpenoids are active against bacteria, fungi, viruses and protozoa and in 1977, it was
reported that 60% of essential oil derivatives examined to date were inhibitory to fungi while 30%
inhibited bacteria (Chaurasia and Vyas, 1977).
The volatile oils (essential oils) are very complex mixture of compounds whose constituents’ oils are
mainly monoterpenes and sesquiterpenes. Other compounds include phenylpropenes and specific
compounds containing sulfur and nitrogen. Basil (Ocimum basilicum L.) is a popular culinary herb
and its essential oils have been used extensively for many years in food products, perfumery, dental
and oral products. Its essential oils and principal constituents were found to exhibit antimicrobial
activity against a wide range of Gram-negative and Gram-positive bacteria, yeast and mold.
(Suppakul et al, 2003).
The triterpenoid betulinic acid is just one of several terpenoids which have been shown to inhibit HIV.
Increasing the hydrophilicity of kaurene diterpenoids by addition of a methyl group drastically
reduced their antimicrobial activity. A terpenoid constituent of chile peppers, capsaicin, although
possibly detrimental to the human gastric mucosa is bactericidal to Helicobacterpylori, while another
hot-tasting diterpene, aframodial, from a Cameroonian spice, is a broad-spectrum antifungal (Ayafor
et al, 1994). A comparable antimicrobial activity to that of cephatoxim against Gram-positive bacteria
was observed for diterpenes obtained from oleoresins of Copaiferapauper (Tincusi, 2002).
Trichorabdal, a diterpene from a Japanese herb directed inhibited Helicobacter pylori. The terpenoid
petalostemumol, obtained from the ethanol-soluble fraction of purple prairie clover showed excellent
activity against Bacillus subtilis and Staphylococcus aureus and lesser activity against Gram-negative
as well as Candidaalbicans (Cowan, 1999).
Terpenoids are widely distributed in nature and are found in abundance in higher plants, fungi, marine
organisms and insect pheromones as well as in insect defense secretions. The antimicrobial activity of
terpenoids has been documented for many plant products (Evans, 2002). A comparable activity to that
of cephatoxim against Gram-positive bacteria was observed from diterpenes obtained from oleoresin
of Copaifera paupera, (Tincusi, 2002).
29
The antimicrobial activity of the terpenoids has been reported to be mostly dependant on the
interaction with the cell membranes of microorganisms. These compounds interact with the lipids
component of cell membranes, easily penetrate into the interior of the cell and kill the cells by
interacting with the intracellular materials (Ahameethunisia and Hopper, 2012). Hence results in
membrane disruption by the liphophic compounds (Jasmine et al, 2007).
1.4.4Peptidesand Lectins
Antimicrobial peptides are found in all kingdoms of life, ranging from plants through insects to
animals (Taddese, 2004). These peptides are termed antimicrobial because they display an unusually
broad activity spectrum. Peptides which are inhibitory to microorganisms are often positively charged
and contain disulfide bonds (Zhang and Lewis, 1997). Their activities may include an ability to kill or
neutralize not only Gram-negative and Gram-positive bacteria but also fungi, parasites, cancer cells,
and even enveloped viruses like HIV and Herpessimplex virus. Moreover, most of the antimicrobial
peptides known today are quite selective for microbes over eukaryotic cells (Kmysz et al, 2003).
The endogenous antimicrobial peptides of plants and animals are typically cationic amphipathic
molecules consisting from 12 to well over 100 residues (Peters et al, 2010). This amphipathic
structural arrangement is believed to play a key role in the antimicrobial mechanism of action. The
hydrophilic and strongly positively charged domain is proposed to initiate peptide interaction with the
negatively charged bacterial surface. Many cationic antimicrobial peptides have been described as
membrane active agents (Kmysz et al, 2003). Despite their vast diversity most antmicrobial peptides
(AMPs) work directly against microbes through a mechanism involving membrane disruption and
pore formation, allowing efflux of essential ions and nutrient (Peters et al, 2010). AMPs are proposed
to the cytoplasmic membrane, creating micelle-like aggregates, leading to a disruptive effect.
Mounting body of evidence indicates the presence of additional or complementary mechanisms such
as intracellular targeting of cytoplasmic components crucial to proper cellular physiology. Thus, the
initial interaction between the peptides would allow them to penetrate into the cell to bind intracellular
molecules resulting in the inhibition of cell wall biosynthesis and DNA, RNA, and protein synthesis
(Peterset al, 2010).However, it is not very certain whether membrane disintegration is the only
mechanism by which these peptides kill bacteria or the peptides have various intracellular targets, or
both mechanisms play roles in the killing process.
One of such antimicrobial from plants is Zeamatin produced by Zea mays and is active against C.
albicans with a minimum inhibitory concentration of 0.5 μg/ml. This peptide permeabilizes fungal
membrane to cause death. Others include amphibine H. frangufoline, nummularine, and rugosanine,
which are active at the level as low as 5 μg/ml (Pandey and Devi, 1990).Recent interest has been
focused mostly on studying anti-HIV peptides and lectins, but the inhibition of bacteria and fungi by
30
these macromolecules, such as that from the herbaceous Amaranthus, has long been known (Cowan,
1999). Lectins are carbohydrate-binding protein that are highly specific for sugar moieties and
typically play a role in biological recognition phenomena involving cells and proteins (Van Damme et
al, 1998).
Thionins are peptides commonly found in barley and wheat and consist of 47 amino acid residues.
They are toxic to yeasts and gram-negative and gram-positive bacteria. Thionins AX1 and AX2 from
sugar beet are active against fungi but not bacteria. Fabatin, a newly identified 47-residue peptide
from fava beans, appears to be structurally related to gammar thionins from grains and inhibits E.coli,
P. aeruginosa, and Enterococcus hirae but not Candida or Saccharomyces (Zhang and Lewis, 1997).
A lectin, Banlec, from banana has been shown to inhibit HIV-1 invitro (Swanson et al, 2010).The
larger lectin molecules, which include mannose-specific lectins from several plants, MAP30 from
bitter melon, GAP31 from Gelonium multiflorum, and jacalin, are with critical host cell components.
It is worth emphasizing that molecules and compounds such as these whose mode of action may be to
inhibit adhesion will not be detected by using most general plant antimicrobial screening protocols,
even with the bioassay-guided fractionation procedures used by natural-products chemists (Cowan,
1999)
1.5 Principles of Inflammation
Inflammation is the complex biological response of vascular tissues to harmful stimuli including
physical agents (burns, radiation trauma, pathogens) noxious chemicals (toxins, caustic substances),
necrotic tissue/cells and/or immunological reactions (Singh et al, 2008; Howland and Mycek, 2006).
It is complex pathophysiological process characterized by four fundamental symptoms: redness, pain,
heat and swelling. A fifth symptom can be organ decreased function or the loss of function of the
injured area (Banning, 2005).
Inflammation is a protective attempt by the organism to remove the injurious stimuli and initiate the
healing process of the tissue (Singh et al, 2008). When healing is complete, the inflammation process
usually subsides. However, if the inflammation is inappropriately triggered by an innocuous agent,
such as pollen, or by an autoimmune response, such as in asthma or rheumatoid arthritis, the defence
reactions themselves may cause progressive tissue injury, and anti-inflammatory or
immunosuppressive drugs may be required to modulate the inflammatory process (Howland and
Mycek, 2006).
Inflammatory responses occur in three distinct phases, each apparently mediated by different
mechanisms:
· Acute or transient phase: characterized by local vasodilation and increase capillary
permeability,
31
· Sub-acute phase: characterized by infiltration of leukocytes and phagocytic cells
· Chronic proliferation phase: Tissue degeneration and fibrosis occur (Agrawal and Paridhavi,
2007; Okpako, 2002).
Inflammation is triggered by the release of chemical mediators from injured tissues and migrating
cells (neutrophils, monocytes/macrohages, mast cells, platelets and lymphocytes) as well as by the
activation of the complement cascade which brings about oedema formation as a result of
extravasation of fluids and proteins and acculumation of leukocytes at the inflammation site (de las
Heras et al, 2003). Howland and Mycek (2006) reported that the specific chemical mediator vary with
the type of inflammation process and includes amines, such as histamine and 5-hydroxytryptamine;
lipids, such as prostaglandins; small peptides, such as bradykinin; and larger peptites, such as
interleukine-1(1L-1).
Inflammation is both a free-radical generated and free-radical producing process. The enzymes
cyclooxygenase (COX) and lipoxygenase act on arachidonic and in cell membranes, oxidizing
arachidonic acid and forming potent pro-inflammatory metabolites, including prostaglandins,
leukotrienes, and thrombroxanes(Figures 1.3 and 1.4) (Foegh and Ramwell, 2001).
According to Chippada et al (2011), lysosomal enzymes released during inflammation produce a
variety of disorders which leads to tissue injury by damaging the macromolecules and lipid
peroxidation of membranes which are assumed to be responsible for certain pathological conditions as
heart attacks, septic shocks and rheumatoid arthritis, etc. The extra-cellular activity of these enzymes
is said to be related to acute or chronic inflammation.
32
Figure 1.3: Summary of cyclooxygenase and lipoxygenase pathways (Howland and Mycek, 2006).
33
Figure 1.4 Pathways of arachidonic acid release and metabolism (Foegh and Ramwell, 2001)
34
1.5.1 Classification of inflammation
Inflammation may be classified as acute or chronic inflammation
1.5.1.1 Acute inflammation: The acute inflammatory reaction is rapid, short-lived and characterized
by classical signs: heat, pain, oedema (resulting from accumulation of fluids,plasma proteins and
leukocytes) erythrema and above all loss of function (Singh et al, 2008; Villarreal et al,2002). At the
site of inflammation, the injured vascular endothelial cells and the emigrated leukocytes release a
large number of soluble mediator (such as cytokine, growth factors) which modulate and maintain the
inflammation (Rosenburg and Gallin, 1999), by acting between inflammatory cells and non
haematopoietic cells such as fibroblasts and vascular endothelial cells, within the traumatized tissue.
Under normal circumstances these cytokines and growth factors cooperate in exquisitely coordinated
networks to sequester and/or eliminate the injurious agent and then restore and maintain homeostasis
in the tissue. If an acute response cannot be resolved, it becomes chronic (Villarreal et al, 2002).
1.5.1.2Chronic inflammation: Chronic inflammation may result from failure to eliminate an acute
inflammatory irritant, from an autoimmune response to a self antigen, or may be caused by an
innately chronic irritant of low intensity that persists (Wakefield and Kumar, 2002).
It results in a progressive shift in types of cells present at site of inflammation and it is characterized
by simultaneous destruction and healing of the injured tissue from incidence of inflammation (Ibrahim
et al, 2011). In acute inflammation, the host response leads to elimination of the irritant, followed by
recovery involving tissue regeneration or repair. Repair is always a feature of chronic inflammation
because it is associated with irritants that cause destruction of tissue architecture, and is typically
achieved by in-growth of granulation tissue, which includes macrophages, fibroblast and new blood
vessels (Wakefield and Kumar, 2002).
In chronic inflammation if the irritant fails to be eliminated (either because of its innate characteristics
or because of an ineffective host response) it may cause continuing tissue damages. In addition, most
persistent irritants are recognized as foreign antigens by the host immune response, which contributes
to the chronic inflammatory process and may add to tissue destruction.
In contrast to acute inflammation, which is usually characterized by recruitment of large numbers of
neutrophil leukocytes, the dominant infiltrating cell in all forms of chronic inflammation is the
macrophage (Wakefield and Kumar, 2002).
1.5.2 Macrophage role in inflammation
Macrophages have three major functions: antigen presentation, phagocytosis and immunomodatation
through production of various cytokines and growth factors.
Macrophages play a crucial role in modulating the initiation, maintenance and resolution of the
inflammatory response. They are activated and deactivated in the inflammatory process.Activation of
35
these cells with pro inflammatory cytokines and bacterial cell components (lipopolysaccharides)
promote the synthesis and release of large amounts of nitric oxide, eicosanoids and bioactive lipids,
mediators involved in the inflammatory onset, as well as a cytokine tumor-necrosis factor (TNFalpha)
interferon-gamma, granulocyte-monocyte colony stimulating factor and interleukin -1 (1L-1B)
(de las Heras et al, 2001). The oxidative metabolism of arachidonic acid (AA) leads to the synthesis
of prostaglandins (PGs) and leukotrienes (LTs) (de las Heras et al, 2003).
Inhibitors of inflammation by removal or deactivation of mediators and inflammatory effects cells
permit the host to repair damaged tissues. Activated macrophages are deactivated by antiinflammatory
cytokines (interleukin-10 and transforming growth factor-beta) and cytokine antagonists
that are mainly produced by macrophages.
Because macrophages produce a wide range of biologically active molecules participating in both
beneficial and detrimental outcomes in inflammation, therapeutic interventions targeted macrophages
and their products may open new vistas for controlling inflammatory diseases. (Fujiwara and
Kabayashi, 2005).
1.5.3 Cyclooxgenase Pathway:All eicosanoids with ring structures including the prostaglandins
(PGs), thromboxanes (TXs) and prostacyclins (PTs) are synthesized via the cyclooxygenase (COX)
pathway(Figure 1.5).Two relatedforms of the cyclooxygenase enzymes; COX-1 (constitutive) which
is responsible for the physiologic production of prostanoids, while COX-2 (inducible) causes the
elevated levels of prostanoids that occur in site of disease and inflammation.COX-1 regulates normal
cellular processes such as gastric cytoprotection, vascular homeostasis, platelet aggregation, and
kidney function.COX-2is constitutively expressed in some tissue, such as the brain, kidney, and bone
(Howland and Mycek, 2006). Nitric oxide (NO) and prostaglandin-E2 are critical mediators produced
by two inducible enzymes (iNOS or NOS-2) and COX-2, respectively and are thought to be
responsible for the massive production of NO and PGs, which have been implicated in the tissue
destruction and pathogenesis of a number of immunological and inflammatory diseases, including
septic shock, rheumatoid arthritis, diabetes, among others (de las Heras et al, 2003).
36
Figure 1.5: Prostaglandin and thromboxane biosynthesis. Compound names are enclosed in boxes.
The asterisks indicate that both cyclooxygenase and peroxidase steps are catalyzed by the single
enzyme prostaglandin endoperoxide (PGH) synthase. (Foegh and Rawell, 2001).
37
1.5.4 Lipoxygenase Pathway:Severallipoxygenases can act on arachidonic acid to form the
hydroperoxyeicosatetraenoic acids(5-HPETE, 12-HPETE, and 15-HPETE)which are unstable
perioxidated derivated that are converted to the corresponding hydroxylated derivatives (the HETEs)
or to leukotrienes or lungs lipoxins depending on the tissue(Figure 1.6).
The cysteinyl-(containing glutathione side chain) – leukotrienes (LTC4, LTD4 and LTE4) are potent
pro-inflammatory agents with the following properties: oedema formation, bronchoconstriction,
airways mucous secretion, proliferation of various cell types. They play a role in bronchial asthma.
LTB4 is a potent inflammatory mediator, inducing adhesion, diapedesis, chemotaxis of leucocytes and
increased proliferation of various cell types including the growth of new blood vessels (angiogenesis)
in chronic inflammation. The lipoxins cause many leukotriene-like effects, e.g. leucocyte chemotaxis,
bronchoconstriction, activation of protein kinase C. (Okpako, 2002).
Arachidonic acid can also be oxygenated by the isoprostane pathway to give rise to free radicals
1.5.6Anti-Inflammatory Drugs
Anti-inflammatory refers to the property of a substance or treatment that reduces inflammation. The
treatment of patients with inflammation involves two primary goals; first, the relief of pain, which is
often the presenting symptom and the major continuing complaint of the patient; and second, the
slowing or-in theory-arrest of the tissue-damaging process (Furst and Munster, 2001).
Anti-inflammatory drugs are available in two major categories: steroids anti-inflammatory drugs
(SAIDs) and non-steroidal anti-inflammatory drugs (NSAIDs).
1.5.6.1 Steroidal anti-inflammatory drugs (SAIDs): Many steroids, specifically glucocorticoids,
reduce inflammation or swelling by binding to cortisol receptors. They are often referred to as
corticosteroids and have powerful anti-inflammatory effects. Examples include; cortisol, cortisone,
triamcinolone, paramethasone, dexamethasone (Decadrone), prednisone (Deltasone), prednisolone,
methylprednisolone (Medrol)(Okpako, 2002; Anonymous, 2012). Unfortunately, the toxicity associated
with chronic corticosteroid theraphy inhibits their use except in the control of acute flare-ups of joint
disease (Furst and Munster, 2001). Some of the adverse side effects include loss of bone (risk of hip
fracture), problems with the bone marrow, cataracts, swelling or weight gain, mood changes, high
blood pressure, problem with one’s ability to fighting infection bronchial asthma, eye problems and
skin disease (Okpako, 2002, Anonymous, 2012).
38
Figure 1.6: Leukotriene biosynthesis. The asterisks indicate that both the lipoxygenase and dehydrase
reactions are driven by the single enzyme 5-lipoxygenase.(GGTP, y-glutamyltranspeptidase) (Foegh
and Ramwell, 2001)
39
1.5.6.2 Non-steroidal anti-inflammatory drugs (NSAIDs):The Non-steroidal anti-inflammatory
drugs (NSAIDs) include a variety of anti-inflammatory agents of different chemical classes. Most of
them have three major clinical effects which are:
– Antipyretic effect: lowering of a raised temperature
– Analgesic effect: reduction of certain types of pain
– Anti-inflammatory effect: modification of the inflammatory reaction.
The reduction of inflammation with the NSAIDs often results in relief of pain for significant period,
and they act by counctracting the cyclooxygenase (COX) enzymes. On its own COX enzymes
synthesizes prostaglandins, creating inflammation. In whole the NSAIDs prevent the prostaglandins
from being synthesized, reducing or eliminating the pain. Various NSAIDs have additional possible
mechanisms of action, including inhibition of chemotaxis, down-regulation of interleukin-1
production, decreased production of free radicals and superoxide, and interference with calciummediated
intracellular events (Furst and Munster, 2001).
The NSAIDs are classed based on their pharmacological interations with two isoenzymes of
cyclooxygenase: the constitutive enzyme which normally exercises physiological function in various
organs, andan inducible enzyme which becomes active in inflammatory situations. Constitutive and
inducible enzymes are referred to as COX-1 and COX-2 respectively. It is proposed that the unwanted
side effects of NSAIDs come from their inhibition of COX-1 (constitutive) enzyme thereby
interfering with normal functions of prostanoids (Vane and Botting, 1998). COX-2 is responsible for
the production of the prostanoid mediators of inflammation (Vane and Botting, 2001).
Long-term treatment with COX-2 specific inhibitors has been shown to increase the risk of heart
attack and strokes, and several of these drugs have been withdrawn (Howland and Mycek, 2006).
1.5.6.3Aspirin
Aspirin is the prototype of traditional NSAIDs. It is the most commonly used and is the drug to which
other anti-inflammatory agents are compared. It irreversibly acetylates and blocks platelet
cyclooxygenase, but the other NSAIDs, including salicylate, are all reversible inhibitors of
cyclooxygenase.
Aspirin is effective as an analgesic, antipyretic and anti-inflammatory drug. It prevents the
aggregation of platelets, and there is some evidence that it can prevent stroke. It is the preferred drug
for the treatment of rheumatoid arthritis, and it has been used in the treatment of osteoarthritis
(Samter, 1997). Aspirin is effective at low doses as an antithrombotic agent by irreversibly inhibiting
cyclooxygenase (COX-1) in platelets and does not interfere with PGI2 induced vasodilation
(Williamson et al, 1996). It modifies the enzymatic activity of COX-2 COX-2 normally
40
producesprostanoids, most of which are pro-inflammatory. Aspirin-modified COX-2 produces
lipoxins, most of which are anti-inflammatory.
Aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) cause ulcers or gastro-in- testinal
bleeding, and some of the ulcerogenic effects is brought about by their inhibiting production of the
cytoprotective prostaglandins.
1.6 Plantswith Anti-Inflammatory Activity
Inflammatory diseases including different types of rheumatic diseases are very common throughout
the world. Although rheumatism is one of the oldest known disease of mankind and affects a large
population of the world, no substantial progress has been made in achieving a permanent cure.In view
of the universal requirement for NSAIDs very many plants have been utilized for the purpose in
traditional medicine and in recent years considerable research effort has been expended on their
investigation (Karawya, et al, 2010; Lee, et al, 2011).
The search, screening and development of drugs for anti-inflammatory activity (AIA) are an unending
problem. There is much hope of finding active antirheumatic compounds from indigenous plants as
these are still used in therapeutics despite the progress made in conventional chemistry and
pharmacology for producing effective drugs (Ali, 2008).
Herbal drugs like holy basil (Ocimum sanctum), turmeric (Curcuma longo), Indian olibanum tree
(Boswellia serrata), ginger, etc are widely used for the treatment of various inflammatory disorders.
They are not only found to be safer and have fewer side-effects, but they also cover a large domain of
mechanisms involved in inflammation thus proving to be more beneficial than synthetic drugs
(Agrawal and Paridhavi, 2007).
41
Table 1.5: Some plants with Anti-inflammatory activity (Ali, 2008; Evans, 2002)
Plant Source Plant part Active compound
Acacia farnesiana Linn Unripe pod Glycoside fruit
(Mimocaceae)
Acanthopana& chisanensis Leaves, stem bark Chiisanoside
nakai (Araliaceae)
Achyrocline satureiodes Flower inflorescence Quercetin, Inteolin
Lam DC (Aseraceae)
Aegle marmelos Correa Roots Marmin (Coumarin)
(Rutaceae)
Aesculus hippocastanum L. Seed Aescin (derivative, B-amyrin)
(Hippocastanaceae)
Anacardium occidentale Seed coat Epicatechin (Condensed tannin)
Linn (Anacardiaceae)
Azadirachia indica .A. Juss Seed oil Nimbidin
(Meliaceae)
Balduina augustifolio Whole plant Helenalin
(Asteraceae)
Boesenbergia pandurata Rhizome 5,7-Dimerhoxyflavone
Roxb Schltr. (Zingiberaceae)
Bougainvillea gabra DC Leaves Steroids
(Nyctaginaceae)
Calophyllum inophyllum Fruit Calophylloide
Linn (Clusiaceae)
Capparis Spinosa .L. Buds Polyprenols
(Capparidaceae)
Capsicum annuum .L Ripe fruits Capsaicin
(Solanaceae)
42
Table 1.5: Some plants with Anti-inflammatory activity (Ali, 2008; Evans, 2002) contd.
Cocculus trilobus Roots Trilobine and Isotrilobin
(Menispermaceae)
Cryptoeria japonica D. Leaves Cis-communi (diterpendoid)
Don (Taxodiaceae)
Curcuma longa Linn Rhizome Curcumin
(Zingiberaceae)
Cyperus rotundus Linn Nut B-sitosterol
(Cyperaceae)
Dianthus barbatus C.V. Aerial parts Barbatosides A and B
(Caryophilaceae)
Dioscorea Mexicana Tuber- Cryptogenin
(Dioscoreaceae)
Echinacea angustifolia DC Aerial parts Polysaccharide
(Asteraceae)
Terminalia ivorensis Stem bark Terminolic acid, quercetin, etc
(combretaceae)
Garcinta mangostana Fruit rind Magnostin
Linn (Clustaceae)
Glycyrrhiza Species Root and Rhizome Glycyrrhizin (triterpenesaponin)
(papilionaceae)
Hedychium Spicatum Lam Rhizomes Hedychenone
(Zingiberaceae)
Hibiscus vitifolius Linn Leaves Gossypin
(Malvaceae)
Ipomoea pessaprae L. Leaves Lapachol
(Convoluulaceae)
Lavandula latifolia Medic Aerial part Coumarin
(Lamiaceae)
43
Table 1.5: Some plants with Anti-inflammatory activity (Ali, 2008; Evans, 2002) contd.
Mandevilla Velutina Tubers Velutinol
(Apocynaceae)
Mentha Piprita Linn Leaves, flowers Polyphenols
(Lamiaceae)
Ochrocarpus longifolius Flowering buds Vitexin (flavonoid)
L. (Clusiaceae)
Bryophyllum pinnalum Lam Leaves B-sitosterol
(Grassulaceae)
Randia dumetorum Lam Seeds Oleandic acid-3-glycoside
(Rubiacea)
Rhamnus infectora Seed Xanthorlamnin
Populus tremuloids Mich Leaves Tremulacin (salicin derivative)
(Salicaceae)
Quercus itex L Leaves Kaempferol derivative
(Papilionaceae)
Sechium edule SW Fruits b-sitosterol beta-D-
(Cucurbitaceae) glucopyranoside
Stephania tetrandra S. Moore Root Tetranidrine (alkaloid)
(Menispermaceae)
Wrightia tinctoria R. Br. Flowers Quercetin-3-0-rhamnoglycoside
(Apocynaceae)
Alpinia officinarum Rhizome Galangin (flavonoid)
(Zingiberaceae)
Echinacea purpurea ` Root Isobutylarnide
(Compositae)
44
1.7Experimental Models for the in vitro Antimicrobial and Anti-inflammatory Studies
1.7.1In vitro antimicrobial susceptibility testing (AST). The ability of bacterial to resist the
damaging effects of antimicrobials may be a normal characteristic of a particular species or may be
acquired by a variety of genetic changes. Testing of bacterial isolates to confirm susceptibility to
chosenantibacterial agents is called susceptibility testing.The most frequent reasons for carrying out
such tests is to monitor antibiotics to confirm that they are suitable for treatment of bacteria isolated
from individual patients (Williams, 2002). There are several variations in methodologies, techniques,
and interpretive criteria currently being used. The choice of an AST methodology may be based on
numerous factors such as ease of performance, flexibility, adaptability to automation or semiautomated
systems, cost, reproducibility, reliability, accurancy. There is no single method that
satisfies all possible bacteria anti-microbial interactions, and methods which particularly suit the
determination being undertaken need to be chosen (Williams, 2002). The most common among the
various methods are the agar diffusion assay (e.g., agar well diffusion) and diluted assay (e.g., broth
dilution and agar dilution) (Hankon, 2002).
1.7.2Agar well diffusion tests.
This is the most widely used method for antibiotics. It is essentially a modification of agar cup
diffusion test, which involves formation of evenly distributed holes by removing the plugs with a
sterile cork borer (Ochei and Kolhatkar, 2008). The antimicrobial agent is then added to each hole so
that it does not have any contact with the agar, earlier seeded with test organisms.
Results of the assays are not affected by thepresence of turbidity or organic matter in the solution of
the antibiotic. Thus, this method can be used to assay antibiotics in blood, urine, fermentation broth,
creams and formulations (Du-Toit and Rautenbach, 2000).
This technique depends on the ability of the antimicrobial agent to diffuse through seeded agar at such
concentration levels than can inhibit growth of the organism and, thereby produce a clear zone of
growth inhibition, whose diameter, inhibition zone diameter(IZD),is measured (Okore, 2009).
1.7.3Anti-inflammatory models
Drug preventing acute and sub-acute inflammation can be tested using the following models; paw
oedema in rats, croton oil ear oedema, pleurisy tests, UV-erythema in guinea pigs, oxazolone-induced
ear edema in mice, granuloma pouch technique and vascular permeability (Williamson et al, 1996;
Agrawal and Paridhavi, 2007).
The effectiveness of drugs which work at the proliferative phase can be measured by methods for
testing granuloma formation, such as the cotton pellet granuloma, adjuvant arthritis, glass rod
granuloma and PVC sponge granuloma. (Lewis, 1989).
45
Paw Oedema technique: This technique is based upon the ability of anti-inflammatory agents to
inhibit the edema produced in the hind paw of the rat after injection of a phlogistic agent (irritant).
Many irritants have been used, such as brewer’s yeast, formaldehyde, dextran, egg albumin, kaolin,
Acrosil and sulphated polysaccharides like carrageenan. The animals are fasted overnight. The control
animals receive distilled water while the test animals receive drug suspension orally. Thirty minutes
later, the animals are subcutaneously injected with 0.1 ml of 1% solution of anirritant in the foot pad
of the left hind paw. The paw is marked with ink and immersed in the water cell of a plethysmometer
up to this mark. The paw volume is measured plethysmographically immediately after injection, 3 and
6 h after injection, and eventually 24 h after injection. The paw volumes for the control groups are
then compared with those of the test group(Agrawal and Paridhavi, 2007) and the % inhibition
determined.
1.8 Chromatographic Separations
Chromatography is a broad range of physical methods employed for the separation and analysis of
complex molecular mixtures. It is based on the concept of partition coefficient. Any solute partitions
between two immiscible solvents or phases. When one solvent is immobilized (by adsorption on a
solid support matrix) and another mobile, which moves through or over the surface of the solid or
stationary phase, it results in most common applications of chromatography. If the matrix support is
polar (e.g. paper, silica, etc) it is a normal phase chromatography, and if it is non-polar, it is a reverse
phase chromatography. In other words, separation is based on the characteristic way in which
compounds distribute themselves between these two phases (Ali, 2008, Agrawal and Paridhavi,
2007). For a soluteA, this can be described in terms of its distribution coefficient, KD:
[A]stationary phase
[A]mobile phase
This is characteristic for a molecule independent of the amount of solute. As the phase carrying the
solute passes over the stationary phase, the solutes are in constant, dynamic equilibrium between the
two phases. For any given compound, the position of this equilibrium is determined by the strength of
interaction of the compound with the stationary phase and the competition for the stationary phase
between the compound and the mobile phase. (Ali, 2008).
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to
separate the components of a mixture for more advanced use (and is thus a form of purification).
Analytical chromatography is done normally with smaller amounts of material and is for measuring
the relative proportions of the analytes in a mixture.
KD=
46
1.8.1 Classification of chromatographic methods
Chromatographic methods can be classified first according to the nature of the stationary and mobile
phase thus:
i Liquid-solid types: e.g., adsorption chromatography thin layer chromatography; ion
exchange chromatography.
ii. Liquid- liquid types: e.g., partition chromatography, paper chromatography.
1.8.1.1 Adsorption chromatography
Adsorption chromatography involves partitioning molecules between the surfaceof a solid stationary
phase on which the sample is adsorbed. The mobile phase may be either a liquid (solid-liquid
chromatography) or a gas (gas-solid chromatography). The technique is based on the interaction
between the solute molecules and active sites on the stationary phase. This attachment or interaction
depends on the polarity of solutes. The dynamic equilibrium of the solutes as they switch between the
stationary and mobile phases, (the processes of sorption and desorption, respectively) is specific for
each molecules and is affected by competition that exist between solutes and solvents for sites on the
stationary phase (Ali, 2008).
This technique proves the statement that “polar like polar”, because if the stationary phase is more
polar than the mobile phase then high polar compounds in the mixture will tightly be bound to the
stationary phase whereas less polar compound will be lightly (or less tightly) bound to the stationary
phase. Less tightly bound compounds will be eluted out by the mobile phase earlier than the tightly
bonded ones.
Stationary Phases: The interaction between the solutes and the solvent for sites on the stationary
phase is purely physical process involving the formation of no chemical bonds, but only the relatively
weak forces of hydrogen bonds, Vander Waals forces and dipole-dipole interactions. For this reason,
almost any inert material can in theory be used as an adsorbent (Ali, 2008). Consequently, such
materials should not react either with the sample or the mobile phase and should be insoluble in the
mobile phase. Common examples include: silica gel, alumina, kieselguhr, celite, cellulose powder,
calcium carbonate, magnesia, talc, fullers earth, charcoal, sucrose, starch, polyamide powder, ionexchange
resin, and Sephadex (Finar, 2002, Agrawal and Paridhavi, 2007).
Elution methods
There are three methods of elution, namely isocratic, gradient and extrusion.
(i) Isocratic elution technique: This technique involves the use of a single solvent or a
solvent mixture of constant combination throughout the process of elution. The major
disadvantages of this technique is that it takes a long time to elute and there is poor
resolution for complex mixtures. (Agrawal and Paridhavi, 2007)
47
(ii) Gradient elution or solvent programming technique: This method is the modified form of
the isocratic technique. The composition of the mobile phase is varied during elution
(Skoog et al, 2005). Two eluting solvents, one weak and one strong are used. The elution
is started with the low affinity solvent (for the solute), later the concentration of the high
affinity solvent is increased gradually until the final mobile phase has a composition
approaching that of the strong solvent. The weak solvent elutes the weakly retained
solutes fast, and the high affinity solvent elutes the strongly retained solutes, which
results in short retention and good resolution.
(iii) Extrusion elution technique: In this method, the passage of eluting solvent is stopped
when reasonable resolution of bands has occurred. After the passage of solvent is stopped,
the column is drained and extruded carefully. It is then placed horizontally and the bands
separated with a scalpel or knife. The solutes from the different bands are then extracted
with a solvent and the resulting substance estimated by a suitable technique (Agrawal and
Paridhavi, 2007).
1.8.1.2 Partition chromatography
In this method, the stationary phase consists of a liquid substance strongly adsorbed on a column of
inert material (as support) such as silica gel. The mixture of substance is dissolved in a solvent (the
mobile phase) which is immiscible with the adsorbed solvent (the stationary phase). This solution is
allowed to pass slowly down the column, and is then followed by eluting solvent. The solutes become
distributed between the stationary and mobile phases, and those solutes with partition coefficients in
favour of the stationary phase will be left behind; whilst those more soluble in the mobile phase will
move further down the column. Thus, the solutes can be separated in this way and the separate solutes
obtained by elution. The column may be extruded and cut up, and the separated solutes recovered
(Finar, 2002; Atherden, 2006).
1.9Rationale, Aim and Objectives
1.9.1Rationale
The recognition and validation of ethnomedicinal practices and the search for plant-derived
antibacterial and anti-inflammatory agents could lead to new strategies in the treatment of bacterial
infections and anti-inflammatory therapeutics. Infectious diseases continue to be the major concern
for health institutions, pharmaceutical companies and governments all over the world (accounting for
over 50,000 deaths everyday) especially with the current increasing trends of multidrug resistance
among emerging and re-emerging bacterial pathogens to the available modern drugs or antibiotics
(Doughari and Manzara, 2008). It is therefore very necessary to search for newer antimicrobial
substances from other sources including plants, which are the cheapest and safer alternative sources of
48
antimicrobials (Sharif and Banik, 2006; Doughari et al, 2007).The side effects of steroidal and nonsteroidal
anti-inflammatory drugs currently used for the management of chronic diseases may be
difficult to manage than the disease itself (Karawya et al, 2010), whereas many medicines of plant
origin had been used without any adverse effects (Paramaguru et al,2011).
There is also paucity of empirical data on the antibacterial and anti-inflammatory properties of
Hibiscus asper. Since there is a wide folkloric reputation of the efficacious use of Hibiscus asper in
the management of bacterial-based diseases and cases involving inflammatory conditions among
others, it becomes necessary to investigate some of these claims, as well as ascertain their potentials
as a source of new lead drugs.
1.9.2 Aim and objectives
The aim of this work is to evaluate the phytochemical constituents and the antibacterial activities of
the extract and fractions of Hibiscus asper leaf, and to study the in vitro anti-inflammatory activities
of the extract only.
1.9.3 Specific objectives:
· To evaluate the phytochemical constituents (qualitatively) of the extract and fractions.
· To fractionate the extract by successive extracting with different solvents.
· To determine the antibacterial activities of the extract and fractions.
· To determine the in vitro anti-inflammatory activities of the extract.
49

 

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