ISOLATION AND CHARACTERIZATION OF BIOACTIVE NATURAL PRODUCTS IN JUSTICIA SPICIGERA (ACANTHACEAE)

ABSTRACT

The Phytochemical evaluation of the secondary metabolites in Justicia spicigera using n-hexane, methanol, chloroform and ethanol extracts revealed the bioavailability of bioactive compounds comprising alkaloids (3.048 - 8.265 mg/100 g), tannins (0.080-0.254 mg/100 g), phenols (0.014 - 0.029 mg/100 g), saponins (0.564-0.744 mg/100 g), flavonoids (0.780-9.820 mg/100 g) and steroids (0.667-1.277 mg/100 g). Minerals content revealed the presence of magnesium (1.094 -1.277 mg/100 g), calcium (3.707-4.509 mg/100 g), potassium (1.2-1.3 mg/100 g), sodium (0.325 - 0.55 mg/100 g), phosphorous (0.689-0.708 mg/100 g)., manganese (5.80-7.98 mg/100 g) and the Trace element analysis revealed - zinc (11.13-15.10 mg/100 g), copper (4.88-4.98 mg/100 g), iron (51.30-53.08 mg/100 g) and lead (1.60-2.46 mg/100 g). The vitamins content was ascorbic acid (92.40-57.20 mg/100 g), β-carotenoid (312.50-474.54 mg/100 g), riboflavin (2.63-4.13 mg/100 g), thiamin (0.38-0.75 mg/100 g) and niacin (0.84-1.41 mg/100 g). Extracts of Justicia spicigera prepared with solvents of different polarities (n-Hexane, Chloroform, Methanol, and Ethanol) were used to analyze the anti-bacterial properties using the test microorganisms Streptococcus pneumonia, Salmonella typhi, Escherichia coli and Aspergilus niger with the positive control as Ciprofloxacin. Minimum inhibitory concentration (MIC) was attained at 125-250 mg/ml for E.coli, S. typhi, and S. pneumonia and (MIC) was attained at 250-500 mg/ml for Aspergilus niger. Chemical investigation of the leaves of a herbaceous medicinal plant Justicia spicigera resulted in the isolation of two natural products – Compound S18 and Compound S21 from the crude methanol extract of the leaves. Isolation of Compound S18 and Compound S21 was carried out by Column Chromatography using silica gel as the adsorbent. The composition of the crude mixture was examined using thin layer chromatography. The combined Infra-red, Proton and DEPT Nuclear Magnetic Resonances, Mass Spectroscopy, 1H-1H COSY and 1H-13C-HMBC were used as spectral techniques to propose the structures of the compounds. The High Resolution Electro-Spray Impact Mass Spectroscopy analysis of Compound S18 showed a molecular ion (M+) peak at m/z C35H58O8 and Compound S21 of molecular ion (M+) peak at m/z C31H47O16. The characterization of the isolated natural products from Justicia spicigera revealed Compound S18 is a triterpenoidal glycoside and Compound S21 is confirmed to be a δ-lactone glycoside. The presence of bioactive natural products may be a contributor to the antimicrobial, antioxidant, anti-inflammatory, liver protector, analgesic, anti-cancer, anti-diabetic and antacid properties exhibited by Justicia spicigera. Justicia spicigera offers a wide scope for pharmaceutical industries to use in drug formulations.

TABLE OF CONTENTS  

Title page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi-ix

List of Tables                                                                                                               x

List of Figures                                                                                                             xi

List of Plate(s)                                                                                                            xii

List of Acronyms                                                                                                        xiii

Abstract                                                                                                                      xiv

chapter 1: INTRODUCTION             

1.1       Background of the Study                                                                               1-3

1.2       Secondary metabolites                                                                                   3

1.2.1    phenolic compounds                                                                                       3-4

1.2.2    flavonoids                                                                                                      4-8

1.2.3    lactones                                                                                                          9

1.2.4    xanthones                                                                                                       9-10

1.2.5    Glycosides                                                                                                      10-11

1.2.6    terpenoids                                                                                                      11-13

1.2.7    Alkaloids                                                                                                         13-14

1.2.8    tannins                                                                                                            14-15

1.2.9    saponins                                                                                                          15-16

1.2.10  steroids                                                                                                          16-19

1.3       bioactive phytochemicals                                                                               19-21

1.4       traditional medicine                                                                                       21-23

1.5       Objectives of the Study                                                                                  23

1.6       Scope of the Study                                                                                                     24

1.7       justification of the Study                                                                               24-25

CHAPTER 2: LITERATURE REVIEW                                                             

2.1       Ethno- Botanical Studies of Justicia Plant Species                                        26-35

2.2       Distribution of Justicia Plant Species                                                             35-42

2.3       Description of the Study Plant                                                                       42-43

2.4       Chemical Constituents of Justicia Plant Species                                            44-57

2.5       Ethno-Medical uses of Justicia Plant Species                                     57-59

2.6       Biological Activities of Justicia Plant Species                                                59-64

chapter 3: MATERIALS AND METHODS                                                              

3.1       General Experimental Procedure                                                                    65

3.1.1    Principles of chromatographic techniques                                                      65

3.1.2   Thin layer chromatography                                                                              65-66

3.1.3    Column chromatography                                                                                66-67

3.2       Principles of Spectroscopic Techniques                                                          67

3.2.1    Nuclear magnetic resonance spectroscopy                                                      67-68

3.2.2    One dimensional nuclear magnetic resonance                                                68

3.2.3    One dimensional (ID) proton nuclear magnetic resonance                             68-69

3.2.4    Distortionless enhancement by polarization transfer (DEPT)                         69-70

3.2.5    Two dimensional (2D) nuclear magnetic resonance                                       70

3.2.6    Two-Dimensional ¹H-¹H COrrelated SpectroscopY (COSY)                        70-71

3.2.7    Heteronuclear multiple bond correlation (HMBC)                                         71

3.2.8    Infra-red (IR) spectroscopy                                                                            72-74

3.2.9    Gas chromatography/mass (GC/MS) spectroscopy                                        74-76

3.3       Collection and Identification of Plant Sample                                               76

3.3.1    Preparation of Plant Sample                                                                           76-77

3.3.2    Solvent extraction                                                                                           77

3.3.3    Partitioning                                                                                                     77-78

3.3.4    Isolation of Bioactive Natural Products from the Plant Sample                    78-79

3.4       Mineral Elements Analysis                                                                             79

3.4.1   Determination calcium and magnesium                                                           79-80

3.4.2   Determination potassium and sodium                                                             80

3.4.3   Determination of phosphorous by Vanado-Molybdate

yellow method                                                                                                80-81

3.4.4    Determination of trace metals                                                                         81

3.5       Vitamins Analysis                                                                                           81

3.5.1    Determination of ascorbic acid                                                                       81

3.5.2    Determination of vitamin A                                                                            81-82

3.5.3    Determination of riboflavin                                                                            82

3.5.4    Determination of thiamin                                                                                82-83

3.5.5    Determination of niacin                                                                                  83

3.6       Quantitative Phytochemicals Determination                                                  83

3.6.1    Alkaloids determination                                                                                 83

3.6.2    Tannins determination                                                                        84

3.6.3    Total phenols determination                                                                            84

3.6.4    Saponins determination                                                                                  84-85

3.6.5    Flavonoids determination                                                                               85

3.7       proximate composition                                                                                  85

3.7.1    Moisture content determination                                                                      85-86

3.7.2    Ash content determination                                                                             86

3.7.3    Crude protein                                                                                                  86

3.7.4    Fat content determination                                                                               86-87

3.7.5    Crude fiber content determination                                                                 87

3.7.6    carbohydrate content determination                                                              87

3.8       Antimicrobial Screening                                                                                 87

3.8.1     Test microorganisms                                                                                      87-88

3.8.2     Antimicrobial assay                                                                                        88-89

3.8.3    Minimum inhibition concentration                                                                  89

chapter 4: RESULTS AND DISCUSSION               

4.1       Minerals Composition of Justicia spicigera                                                    90-91

4.2       Vitamins Composition of Justicia spicigera                                                   92-93

4.3       Phytochemicals Content of Justicia spicigera                                                94-98

4.4       Results of Proximate Composition                                                                 98-99

4.5       Characterization and Identification of Natural Products Isolated from

the leaves of Justicia spicigera                                                                       100-144

4.5       Results of Antimicrobial Assay                                                                      145-151

chapter 5: CONCLUSION AND RECOMMENDATIONS

5.1       Conclusion                                                                                                      152-154

5.2       recommendations                                                                                          155

References                                                                                                      156-174

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATE (S)

2.1:      Justicia spicigera Plant Structure.

LIST OF ACRONYMS

CHAPTER 1

INTRODUCTION

1.1       BACKGROUND OF THE STUDY

Bioactive natural products have enormous economic importance as special chemicals and they can be used as drugs, lead compounds, biological or pharmaceutical tools, feed stock products, excipients and nutraceuticals (Farnsworth, 1986; Pieters and Vlietinck, 2005). The exploitation of this potential source of medicine requires ethno-botanical, ethno-pharmacological, chemical, biological, pharmacological and toxicological studies (Gilani et al., 2005; Gurib-Fakim, 2006). About 75 % of useful bioactive natural products are derived from medicinal plants. Bioactive natural products used globally are discovered by systematic investigation of traditional medicines. The past decade has witnessed a tremendous resurgence in the interest and use of medicinal plants. The beneficial medicinal effects of plant materials typically result from the combination of secondary metabolites present in them. The abundance of scientific evidence indicates that bioactive natural products have biological properties such as antioxidant, antimicrobial, modulation of detoxification enzymes, stimulation of the immune system, decrease of platelet aggregation, modulation of hormone metabolism and anticancer activities (Tomoke et al., 2002).  

In recent years, herbal preparations have received great attention as an alternative way to compensate for perceived deficiencies in conventional pharmacotherapy worldwide. Despite the inadequacy of medical evidence to support their therapeutic efficacy and toxicological effects, the use of herbal medicine has increased considerably. According to World Health Organization (WHO)(2002), up to 80 % of the world population in underdeveloped and developing countries depend on traditional medicine practice for their primary healthcare needs. Traditional medicines have been given greater acceptance in Africa because the conventional medicines are unavailable, have unwanted side effects, have high cost, the healthcare facilities and the healthcare professionals are inadequate and some of the healthcare workers do not have adequate training (Hostellmann et al., 2002). The use of medicinal plants to treat diseases is often influenced by religious practices (Wambebe, 1999).

Medicinal activities of plants have long been associated with the production of plant chemicals called secondary metabolites which includes phenolic compounds (derived from carbohydrates), essential oils,  alkaloids (derived from amino acids), tannins, terpenoids (a group of lipids), steroids, saponins,  volatile oils and flavonoids (Yang et al., 2007; Timo et al., 2013). These secondary metabolites are responsible for plants therapeutic activities (Cowan, 1999; Rabe et al., 2000). Secondary metabolites are produced from the primary metabolites (the common sugars, amino acids, proteins, nucleic acids, chlorophyll, purines and pyrimidines). The bioactive non-nutrient secondary metabolites produced from plants are also known as phytochemicals. All plants produce phytochemicals as part of their normal metabolic activities. Phytochemicals are often confused with phytonutrients. All phytonutrients are phytochemicals but not all phytochemicals are phytonutrients. Phytochemicals are not essential nutrients and are not required by the body for sustaining life but they have important properties that help to prevent and fight common diseases. Phytochemicals promote health, slow the aging process and reduce the risk to certain diseases like cancer, heart disease, high blood pressure, stroke and other chronic diseases. It has been estimated that more than 5,000 phytochemicals have been identified but a large percentage still remain unknown. Phytochemicals protect the plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack. Plant secondary metabolites give humans health benefits because they act as synergistic agents that allow nutrients to be used more efficiently by the body. The health benefits of phytochemicals are low cost, low toxicity, easy availability and high biological activity. Justicia spicigera a medicinal plant from the rain forest biodiversity of Nigeria in particular contains biologically active secondary metabolites (Dominiguez et al., 1990; Euler et al., 1982). 

1.2       SECONDARY METABOLITES

1.2.1    Phenolic compounds

Phenolic compounds are one of the most important constituents of plant secondary metabolites and exist in plants as flavonoids, phenolic acids, tocopherols, stilbenes, coumarins and tannins (Ali et al., 2008). Other phenolic compounds are benzoquinones containing six carbon atoms, naphthoquinones with ten carbon atoms, norlignans with seventeen carbon atoms, lignans contain eighteen carbon atoms and condensed tannins which contain indefinite number of carbon atoms (n).  Phenolic compounds have enormous biological activities including inhibitory effects on enzymes, modulatory effects on some cell types, protection against allergies, antiviral, anti-malarial, anti-oxidant, anti-inflammatory and health promoting properties (Veitch et al., 2007). Phenolic compounds are a group of naturally occurring and widespread compounds which provide essential functions in the reproduction and growth of the plants in which they are found. They are characterized by the presence of an aromatic ring with one or more hydroxyl groups. They are responsible for the therapeutic, anti-fungal, antimicrobial and insecticidal properties in the plants. Phenolic compounds that contain carbon-3 side chain at a lower level and no oxygenare classified as essential oils and have anti-fungal properties. Polyphenols are the largest group of phytochemicals with over 8,000 identified compounds. Like carotenoids, polyphenols are powerful anti-oxidants (Hollman, 2001). Phenolic acids are hydroxybenzoic acids (P-hydroxybenzoic acid, protocatehuic acid, vannilic acid and syringic acid) and hydroxycinnamic acid derivatives (P-coumarin, caffeic acid, ferulic acid and sinapic acid). Phenolic acids have a wide range of therapeutic effect against degenerative diseases such as diabetes, cancer, cardiovascular, inflammatory and neurodegenerative diseases. The therapeutic effects of these phenolic acids are attributed to their antioxidant activity. Phenolic acids protect against polyunsaturated fatty acids (PUFA) and alcohol induced toxicity and they also enable the body to overcome deadly effects of alcohol and PUFA. Phenols in plant tissues are usually oxidized to co-quinones which subsequently form effective cross-links with the serum proteins of the skin to arrest bleeding and effect healing (Woodman et al., 2005).

1.2.2    Flavonoids

Flavonoids are a group of polyphenolic compounds present in fruits and vegetables. The flavonoids family includes monomeric flavonols, flavonones, anthocyanidins, flavones, flavanoids and isoflavonoids. The term flavonoid has been used to embrace compounds whose structure is based on flavone and they have low molecular weight. There are over 4,000 individual flavonoids (Woodman et al., 2009). Flavonoids have many biological functions such as anti-bacterial, anti-fungal, anti-cancer effects. Flavonoids are responsible for the bitter taste of leaves and fruits (El-kamali, 2009). Flavonoids anti-oxidant properties is due to mechanisms such as scavenging of free radicals, chelation of metals ions like iron and copper and the inhibition of the enzyme responsible for free radical generation (Akinmoladun et al., 2007). Plants containing flavonoids provide the natural anti-oxidant needed to enhance good living by scavenging free radicals that cause ill health in humans. Flavonoids are recognized by their hydrophilic nature and their common origin from Shikimic acid (Finar, 2006). Flavonoids have two phenolic nuclei that are connected to each other by three carbon units. Some flavonoids exhibit acidic character, some exhibit neutral character due to the presence of an acidic group or the absence of free phenolic group. Of the many types of flavonoid compounds, the coumarins (coumarin and warfarin) which are derivatives of α-pyrone are known (Leal et al., 2000). The largest group of naturally occurring phenols is about 500 in number and these are flavonoids that occur in the ‘free state’ as glycosides. The sugar in flavonoids can be either glucose or rhamnose. Most flavonoids are O-glycosides but a considerable number of C-glycosides are known. Dimeric compounds involving a 5′-8 carbon-carbon linkage are also known (that is the biflavonyls). Flavonoids are generally yellow in color and the intensity of the yellow color increases with the number of hydroxyl group and with the increase in the pH of the medium. Flavonoids contain a conjugated aromatic system which makes them show intense absorption bands in the ultra-violet and visible region of the spectrum. Flavonoids are present in all vascular plants but some classes are more widely distributed than others. Flavonoids occur in all parts of the plant such as the root, heartwood, sapwood, stem bark, leaves, fruits and flowers. The biological activity of flavonoids is attributed to their influence on arachidonic acid metabolism. Flavonoids have proven ability to improve cardiac function, decrease angina and lower cholesterol levels. Substances that contain high amount of flavonoids can restore cardiac function and improve blood circulation in patients with coronary heart disease and hypertension. Flavonoids of several classes can inhibit monoamine oxidase A and B thus working as anti-depressants which can improve the health conditions of Parkinson’s patients. Flavonoids common in diet are catechin, epicatechin, epigallocatechin, epigallocatechin gallate, theoflavins, thearubigins and proanthocyanidins (Nakanishi et al., 1977).

Flavonone glycosides are phenolic potent water soluble super anti-oxidants which prevent oxidative cell damage. They have anti-cancer and anti-inflammatory properties. Common examples of flavonones are hesperietin, Naringenin and Eriodictyol (Finar, 2006). Isoflavones are phytoestrogens, which means they are plant compounds that mimic the effect of estrogen in the human body. Isoflavones can cause hormonal disruptions in men and women, inhibit thyroid function and increase the risk of breast cancer (Morrison et al., 2006).

Flavones or anthoxanthins are yellow pigments that occur in the plant kingdom. Some flavones occur naturally in the uncombined state or as glycosides and their aglycon is anthoxanthidin or flavones can occur associated with tannins. Chemically, the flavones are very closely related to the anthocyanins. The flavones are hydroxylated derivatives of flavone (2- phenyl-4- chromone) which may be partially alkylated. The flavones naturally occur as ‘dust’ on flowers and leaves of plants. Flavones are widely distributed and occur mostly in higher plants and in the cell sap of young tissues.  Examples of flavones are apigenin, luteolin, orientin, chrysin, butin and fisetin. Flavones and the anthocyanins are sap soluble and have γ-pyrone ring. The anthocyanins are the most important and widely spread group of coloring matter in plants. The anthocyanins give colors such as pink, scarlet, red, mauve, violet and blue colors in leaves and fruits of higher plants. The anthocyanins are based on single aromatic chemical structure of cyanidin. All anthocyanins are derivatives of this cyanidin pigment by addition or subtraction of hydroxyl, methyl or glycosyl groups. Examples of anthocyanidins are delphinidin, malvidin, preonodin, cyanidin and petunidin. Anthocyanins, flavanols and flavanones have the ability to prevent inflammatory processes that lead to injury. Anthocyanins lower the risk of cancer, age related memory loss, control high blood pressure, and lower the risk of diabetes, heart attack and Alzheimer’s disease (Deanna, 2011).

Flavonols (3-hydroxy flavone) occur most frequently bound to sugar as glycoside that means aglycone or non-sugar portion is attached with a sugar portion. The sugar can either be glucose or rhamnose or a combination of the two. A flavonoid aglycone may occur in a single plant or in several glycosidic combinations. With over a hundred flavonol aglycones known, only four are of common occurrence; kaempferol, quercetine, isorhamnetin and myricetin. The other known flavonols are mostly simple structural variants of the common flavonols and are of limited natural occurrence. Flavonols are very widely distributed in plants, both as co-pigments to anthocyanins in petals and also in leaves of higher plants (Fang, et al., 2005). The three flavonols corresponding in hydroxylation pattern to the anthocyanidins are pelargonidin, cyanidin and delphinidin (Wang et al., 2004). There is a considerable range of flavonol glycosides present in plants. Over seventy different glycosides of quercetin alone have been described. Quercetin is a flavonoid widely distributed in plants, like many other phenolic heterocyclic compounds, glycosylated forms include rutin and quercetrin. Quercetin is a polyphenolic flavonoid with potential chemo preventive activity. The commonest flavonol glycoside is quercetin-3-rutinoside, known as rutin, which is of pharmaceutical interest in relation to the treatment of capillary fragility in man. Quercetine occurs as the glycoside quercetrine and quercetrine appears to be the most widely distributed natural plant pigment. The most common flavonoid in human diet is the flavonols. They help to lower the risk to cardiovascular diseases and lower blood pressure. Anthocyanins are powerful antioxidants which help to protect the liver, improve eyesight, reduce blood pressure and even reduce the risk to many diseases. Flavones differ from flavonols in lacking a 3-hydroxy substitution; this affects their UV absorption, chromatographic mobility and color reactions. There are only two common flavones -apigenin and luteolin, corresponding in hydroxylation pattern to kaempferol and quercetin. The flavone -tricetin corresponding to myricetin is known but it is of very rare occurrence. More common are 2- methyl ethers: Chrysoeriol (3'-methyl ether of luteolin), and tricin (3', 5'-dimethyl ether of tricetin). Flavones occur as glycosides but the range of different glycosides is less than that of the flavonols. A common type is the 7-glycoside, luteolin. Flavones may have sugar bound by a carbon - carbon bond. One example is the 8 -C- glycoside of luteolin called orientin. In the central nervous system, flavones bind to the benzodiazepine site on the GABA (A) receptor resulting in sedation, anxiolytic or anti-convulsive effect. The chalcones, aurones, flavanones and dihydrochalcones are isoflavones known as the "minor flavonoids", because these classes are of limited natural distribution. Their occurrence is either sporadic (example the flavanones) or limited to a very few plant groups (leguminosae and Iridaceae). Chalcones and aurones known as the "anthochlors" are yellow pigments which can be detected by a change to orange or red color when a yellow petal is fumed with the alkaline vapor of cigarette or with a vial of ammonia. These compounds occur characteristically in the compositae (especially in coreopopsis), but they have also been recorded in over ten other families (Salah et al., 1995). A typical chalcone is butein and a common aurone is aureusidin, both occur as glycosides. Okanin is a chalcone and sulphuretin is an aurone respectively. Dihydrochalcones have a different distribution pattern from chalcones, being mainly confirmed to the Rosaceae and Ericaceae. Isoflavones, of which over sixty are known, are isomeric with the flavones but of much less occurrence. They occur almost entirely in one sub-family (the lotoideae) of the leguminasae. Isoflavonoids can be divided into three classes depending on their physiolgical properties. Compounds such as 7, 4'-dihydroxyisoflavone (daizein), and 5, 7, 4- trihydroxyisoflavone are weak natural estrogens present in clover. Complex isoflavones such as rotenone are powerful natural insecticides. Isoflavones are difficult to characterize, since they do not respond specially to any one color reaction. Some isoflavones (example daizein) give a brilliant light blue color in UV light in the presence of ammonia, but most others (example genistein) appear as dull purple absorbing spots, changing to dull brown with ammonia. Other isoflavones are glycitein and orobol (Nakanishi et al., 1977; Salah et al., 1995).

Flavanones are isomeric with chalcones and the two classes are interconvertible in-vitro. Chalcones are frequently found in nature together with the flavanone analogues but the converse is not always true. Example flavanones accumulate in large quantity in citrus fruits without being accompanied by chalcones. Some flavanones have important taste properties example Naringin of the servile orange is very bitter. The difference between a flavone and a flavanone is that a flavanone structure has a hydroxyl group at carbon 3 and when there is no double bond between carbon 2 and carbon 3, it is a flavanone (Morrison et al., 2006).

1.2.3    Lactones

Lactones are cyclic esters of hydroxycarboxylic acids containing a 1-oxacycloalkan-2-one structure or analogues having unsaturation or heteroatoms replacing one or more carbon atoms. Lactones with 3 or 4 membered rings that are the α-lactones and β-lactones respectively are very reactive. The 5-membered ring lactones are the cardenolides example digitoxigenin, digoxigenin, gitaloxigenin and strophanthidin. The six membered ring lactones are the bufadicaoleates. Naturally occurring lactones are mainly saturated and unsaturated γ lactone and δ-lactones and the less common macrocyclic lactones. The γ- and δ-lactones are intra molecular esters of the corresponding hydroxyl fatty acids. Lactones contribute significantly to the flavor of fruits and of fermented and unfermented dairy products and are therefore used as flavors and fragrances. Lactone glycosides are isoflavonoids that are sometimes referred to as iridoid glycosides because they are present in glycosidic form (Morrison et al., 2006). Lactone rings occur widely as building blocks in nature, such as in ascorbic acid, kavain, nepeta-lactone, gluconolactone, hormones (spironololactone, mevalonolactone), enzymes (lactokinase), neurotransmitters (butyrolactone, avermectins), antibiotics (macrolides like erythromycin; amphotericin B) and anti-cancer drugs.

1.2.4    Xanthones

Xanthones are yellow phenolic pigments similar in color reactions and chromatographic mobility to flavonoids, but chemically they are different and are distinguished from them by their characteristic spectral properties (Morrison et al., 2006). Hydroxystilbenes are biogenetically related to the chalcones but have one less carbon atom in the basic skeleton. They are heartwood constituents in relatively few plants. A dihydrostilbene carboxylic acid is an important growth inhibitor in liverworts and algae. In these plants, lunularic acid replaces the sesquiterpene abscisic acid, which functions as the major dormancy hormone in all other plant groups (Morrison et al., 2006).

Sulphur containing compounds which are present in plants are the isothiocyanates, Indoles and allylic sulphur compounds. Isothiocyanates are produced when plants are injured. The natural forms of isothiocyanates are 2-phenylethyl-isothiocyanate, benzyl isothiocyanate and sulforaphanes. Isothiocyanates are usually accompanied by their glucosinolates. Isothiocyanates have marked chemo preventive capacity in animals and human cell cultures. Isothiocyanates are able to protect against tumor genesis in the lungs, breast, liver, stomach and esophagus. Indoles reduce the risk of cancer particularly breast and prostate cancers, and reduce the risk of tumor growth in cancer patients. Allylic sulphur compounds have hepatoprotective, immune-enhancing, anti-cancer and chemo-preventive activities. Isothiocyanates are recommended as a chemo preventive strategy to reduce lung cancer in smokers. Allylic compounds are formed when the allium is hydrolyzed by allinase. These compounds have very striking flavors and are generally hydrolyzed when the tissues of this allium present in cytoplasm of onions are disrupted. Organo-sulphur compounds possess hepato-protective, immuneenhancing, anticancer and chemo preventive activities. Some organo-sulphur compounds are anti-oxidants whereas others may stimulate oxidation (Ding et al., 2000; Wasshausen et al., 2004).

1.2.5    Glycosides

Glycosides are widespread in nature and many biologically important molecules contain glycosidic linkages example digoxin the biologically active component of digitalis is a glycoside containing a complex steroid alcohol linked to a disaccharide. Glycosides are natural sweeteners in many countries. Glycosides have therapeutic effect on humans and animals. Glycosides are used in modern and traditional medicine as cardio-tonic, purgative, analgesic, anti-rheumatism, demulcent and to restore adequate circulation in patients with congestive heart failure (Bradbury et al., 1991). Glycosides are classified based on the linkage between the aglycone and the glycone as either anthracene glycoside, saponin glycoside, flavonoidal glycoside, cyanogenetic glycoside, phenol glycoside, alcoholic glycoside, lactone glycoside, G-glycosides, C-glycoside (cardiac glycoside), O-glycoside, S-glycoside (isothiocyanate glycoside) or N- glycoside (nucleoside glycoside) or based on the type of sugar they contain as glucoside (those containing glucose) or rhamnoside (those containing rhamnose) or pentoside (those containing pentose) (Bradbury et al., 1991). Cardiac glycosides or sterol glycosides are found in higher plants. They are abundant in families such as Apocyanaceae, Asclepedaceae, Ranunculaceae, Sterculiaceae and Leguminosae among others. Steroidal glycosides are also found in monocotyledons like Liliaceae family. Anthocyanins are natural plant pigments. They are glycosides and their aglycone is anthocyanidin. They are responsible for the red, violet and blue color of plant flowers. Anthocyanins can inhibit the formation of free radicals thus protecting cardiomyocytes after ischemic episodes. The anti-inflammatory activity of anthocyanins is due to the fact that they inhibit cyclooxygenase enzyme.

1.2.6    Terpenoids

The terpenoids are a group of compounds with a large majority of them occurring in the plant kingdom. A few terpenoids are obtained from other sources other than plants. The mono- and sesqui-terpenoids are the major constituents of the essential oils (Takeda et al., 1993). The mono- and sesqui-terpenoids are the volatile oils obtained from the sap and tissues of certain plants and trees. Essential oils are used in perfume making from primordial times. The di-terpenoids and tri-terpenoids which are not steam volatile are obtained from plants, tree gums and resins (Tasdemir et al., 1998). Terpenoids have antioxidant properties and interact in most regulatory processes. Terpenoids also improve the skin tone, increase the concentration of antioxidant in wounds and restore inflammatory tissues by increasing blood supply. Triterpenoids are compounds in which the carbon skeleton is based on isoprene unit. Triterpenoids have cyclic structures that are relatively complex. Most triterpenoids are alcohols, aldehydes, ketones or carboxylic acids. Triterpenoids are mainly colorless crystalline, often high melting optically active substances which can be identified due to their color reactions. Most of them produce blue or green coloration and many plant terpenoids are toxins and feeding deterrent to herbivores or are attractants and many of the terpenoids possess pharmacological activity. The largest class of triterpenoids is the oleanane (β-amyrin) group and most oleananes exist in nature as saponins. The oleananes are pentacyclic triterpenoids and the other pentacyclic triterpenoids are ursane (α-amyrin) group and lupane (lupeol) group. Other classes of triterpenoids are the true terpenoids, sterols, saponins and cardiac glycosides. The latter two groups occur mainly as glycosides. Triterpenoids are found in the leaves and bark of plants. The anti-cancer properties of triterpenoids are wide including inhibition of proliferation, metastatic and angiogenic activities (Conolly et al., 1999). The tetra-terpenoids group of compounds is known as the carotenoids and it is usual to treat the carotenoids as a separate group. There are more than 750 known carotenoids and about 50 of them can be metabolized into vitamin A (having an effect on cellular differentiation and proliferation) and are antioxidants preventing free radical induced damage to cellular DNA and other molecules. Carotenoids broadly consist of α-carotene, β-carotene, γ-carotene, β-cryptoxanthin, lutein, zeaxanthin, astaxanthin and lycopene. The yellow plant pigments α-carotene, β-carotene, γ-carotene, and the red pigment of tomatoes lycopene can be converted to vitamin A or retinol in the liver. Carotenoids are strong antioxidants which help to reduce oxidative stress damage caused by free radicals. Lutein and zeaxanthin are compounds which help to filter light and protect the eyes like tiny internal sunglasses (Morrison et al., 2006). Lycopene has protective effect against colon, stomach, skin and lung cancers. Lycopene reduces the risk of arteriosclerosis by inhibiting aggregation and reducing inflammation. Lycopene helps to protect the skin from damage due to exposure to ultra-violet sun ray. Lycopene is most often used as an ingredient in anti-ageing creams and lotions. More than 10,000 sesquiterpenoids have been identified representing a wide variety of compounds having different skeletal types ranging from cyclic to tetracyclic systems (Conolly et al., 1999).

1.2.7    Alkaloids

Alkaloids are natural plant compounds which have basic character and contain at least one nitrogen atom in a heterocyclic ring system. The nitrogen atoms are usually in the tertiary state in a ring system. Alkaloids are present in the seeds, leaves, roots and stem bark of plants. Alkaloids generally occur as salts of various plant acids example acetic, oxalic, citric, maleic and tartaric acids. Different alkaloids can be found in different tissues of the same plant. There are more than 3,000 different alkaloids isolated from 4,000 plant species (Hadi et al., 2001). Certain plant species have an abundance of alkaloids like the poppy family (Papaveraceae), Ranunculaceae, Solanaceae and Amaryllidaceae are prominent alkaloids containing families. Alkaloids are colorless non-volatile solids which are insoluble in water but soluble in ethanol, ether, chloroform and other organic solvents. The alkaloids that contain oxygen in their molecular structure are usually colorless crystals at environmental conditions or the oxygen-free alkaloids such as nicotine or coniine are typically volatile, colorless, oily liquids. Alkaloids that are liquids are soluble in water example coniine and nicotine, some alkaloids are partially soluble in water like caffeine, codeine, cocaine, morphine, yohimbine (used as a stimulant and an aphrodisiac) and nicotine, and alkaloids that are colored like berberine having yellow color and sanguinarine having orange color. Most alkaloids have a bitter taste, are optically active and are weak bases but some alkaloids for example theobromine and theophylline are amphoteric in nature. The source of the alkaloid is usually considered as the most important characteristic of the compound. The alkaloids can be classified into different groups such as phenylethylamine, pyrrolidine, quinoline, isoquinoline, phenanthrene, indole, pyridine and piperidine. Alkaloids despite being poisonous show antitumor and bronchodilator activity which is why species of Justicia containing alkaloids are popularly used to treat cases of diarrhea, inflammations, renal disorders and HIV/AIDS (Konkwara, 1979). Alkaloids have a wide range of pharmacological activities including anti-arrhythmic activity example quinidine, anti-hypertensive and anti-cancer activity example homoharringtonine, cholinimimetic activity example galantamine, vasodilatory activity example vincamine, analgesic activity example morphine, anti-bacterial activity example chelerythrine, anti-hyperglycemic activity example piperine, antimalarial activity example quinine, anti-asthmatic activity example ephedrine (Hadi et al., 2001). Alkaloids also have neuro-protective, cholinergic and antioxidant activities in Alzheimer’s patients. Alkaloids activity is due to their being able to restrict oxidative stress and inflammatory reactions, enhance cholinergic transmission, elevate estrogen and other neurotropic agents and prevent β-amyloid toxicity. Majority of alkaloids are used in traditional or modern medicine or as starting material for drug discovery. Alkaloids can exhibit psychotropic activity example psilocin some exhibit stimulant activity example cocaine, caffeine, nicotine, theobromine used mainly in entheogenic rituals or as recreational drugs. Toxic alkaloids include atropine, acanitine and tubacurarine. Alkaloid related substances include serotonin, dopamine and histamine that act as important neurotransmitters in animals (Qiu, 2014).

1.2.8    Tannins

Tannins are found in plants and many types of tannin occur as glycosides. One of the best sources of tannin is nutgall. The tannins are colorless non-crystalline substances giving colloidal solutions in water. These solutions have an astringent taste and are responsible for the astringent taste of many plants in which tannins are present. Tannins precipitate proteins from solution and they form a bluish- black color with ferric salts, a basic requisite property for the production of ink. Tannins also precipitate many alkaloids from their solutions. Their ability to tan leather is not based on a class of compounds with a common basic structure. There are the hydrolysable tannins and the condensed tannins (proanthtocyanidins) which are derived from various forms of flavonoids (Boham et al., 1994). Condensed tannins refer to a mixture of polyflavonoids of different molecular weight ranging from 500-5,000 and are characterized by different linkages, functional groups and stereochemistry. All forms of tannins may participate in the control of glucose level in the blood. Tannins are responsible for the antiviral and antibacterial activities exhibited in the plants they are found (De-Ruiz et al., 2001; Ogunleye et al., 2003).  Tannins are traditionally used on swollen surfaces of mouth and in the treatment of catarrh, wounds, hemorrhoids, diarrhea and as antidote in heavy metal poisoning (Sodipo et al., 1991). Nguji (1988) reported that tannins are important in herbal medicines. Macerated bark or leaves or root of plants containing tannins is applied in arresting bleeding and as a healing dressing for wound healing. Some tissues of plants containing tannins are chewed as anti-scrobutin while the infusion of some bark and pods rich in tannin is used as tonic and sometimes provide remedy for dysentery, diarrhea, cough, fever and venereal diseases (Daziel, 1995).

1.2.9    Saponins

Saponins are plant compounds that occur either as steroid alkaloids, glycosides of triterpenoids or as steroids. Saponins are a special class of glycosides that possess the properties of precipitating and coagulating red blood cells and the plants that contain these saponins are used to treat fungal infections (Sodipo et al., 1991).  Saponins can reduce cholesterol, stimulate immunity, reduce glycemia and cancer cell proliferation. They are believed to act by enhancing anti-body production and in the stimulation of cell mediated immune system. Saponins are believed to have effect on disease causing cells and induce interferon and interleutin production thus mediating immune-stimulant effect. Saponins also exert anti-proliferative activity on prostate carcinoma cells by inducing apoptosis and cell cycle arrest. Saponins have anti-fungal and hypo-cholesterocemic properties and lower the risk of cancer and other chronic diseases.

1.2.10  Steroids

Steroids are a group of structurally related compounds widely distributed in animals and plants. Included in the steroids group are the steroids from which the name steroid is derived. Among these are Vitamin D, the bile acids, a number of sex hormones, the adrenal cortex hormones, some carcinogenic hydrocarbons and certain sapogenins. The structure of steroids is based on the 1, 2-cyclopentenophenanthrene skeleton. Sterols occur in animal and plant oils and fats. They are crystalline compounds and contain an alcoholic group. Sterols occur free or as esters of the higher fatty acids and are isolated from the unsaponifiable portion of oils and fats. Phytosterols occur either as sterols or stanols which are the saturated form of sterols. Phytosterols is comprised of sitosterol, campesterol, stigmasterol, campestanol, sitostanol and stigmastanol (Morrison et al., 2006). Cholesterol is an animal sterol notorious as the substance deposited on the walls of arteries. Cholesterol is an important part of all animal cell membranes and is needed to maintain their membrane structural wholeness and fluidity. Plants synthesize cholesterol in small amounts. Cholesterol is partially soluble in water. The concentrated form is crystalline cholesterol which is the major constituent of most gallstones. Lecithin and bilirubin also form gallstones but are less frequent in occurrence. Cholesterol is an extremely important part of all cells, and is an intermediate in the biosynthesis of other steroids. Ergosterol and stigmasterol are the principal plant sterols. Sterols obtained from plants are called phytosterols while sterols from animals are called zoosterols and sterols from fungi are called mycosterols. Ergosterol (ergost-5,7, 22-trien-3β-ol) is a sterol found in cell membranes of fungi and protozoa, serving many of the functions that cholesterol serves in animal cells. The enzymes that create ergosterol have become important targets for drug discoveries because many fungi and protozoa cannot survive without ergosterol. Ergosterol is a pro-vitamin form of vitamin D₂. Ergosterol is an important compound used in the preparation of antifungal drugs because it is present in the cell walls of fungi but absent in animal cell walls. This is the basis of some antifungals against West African sleeping sickness. Ergosterol is a smaller molecule than lanosterol. Ergosterol irritates the skin, the eyes and the respiratory tract. Ingestion of large amounts of ergosterol causes hypercalcemia.

Campsterol (ergos-5-en-3β-ol) is a phytosterols whose structure is similar to that of cholesterol. It has anti-inflammatory effects because it hinders several pro-inflammatory and matrix degradation mediators typically involved in osteoarthritis-induced cartilage degradation. It is the starting material for the synthesis of the anabolic steroid called boldenone which is commonly used in veterinary medicine to induce growth in cattle but is also one of the most commonly abused anabolic steroids in sports. Campsterol molecules usually compete with cholesterol thus reducing their absorption in the human intestine. When plant sterols are used in excess, they are implicated with the increased risk of cardiovascular diseases.  Campsterol, stigmasterol and β-sitosterol are the most abundant phytosterols in the human diet. Plant stanols that are most common in the human diet are sitostanol and campestanol. Stanols and sterols can equally reduce cholesterol levels. Steroid hormones are steroids that act as a hormone and are grouped into two classes - corticosteroids and sex hormones. Steroid hormones function in the control of metabolism, inflammation, immunity, salt and water balance, development of sexual characteristics and the ability to withstand illness and injury.     

Many plant steroids occur as glycosides, some have the property of stimulating the heart muscle and are referred to as cardiac glycosides. Other sterols have the property of forming foams in water (like soap solutions) and so are known as saponins. Steroids represents an important group of natural products found to be hormone regulators which possesses oxytocic, anti-inflammatory, anti -oxidant, anti-asthmatic, bronchodilator, anti-spasmodic, liver detoxifying actions and can normalize sticky blood (Xiong,1999; Morrison et al., 2006). Phytosterols structure and functions are similar to that of cholesterol. Beta-sitosterol is the main phytosterols in plants and it is also present in human serum and beta-sitosterol glycosides also present at lower concentrations. Beta-sitosterol and its glycosides can reduce incidences of inflammatory diseases and carcinogen induced cancer. Beta-sitosterol and its glycosides have immune moderating activities on conditions that are not infectious like rheumatoid arthritis and allergies, and chronic infectious conditions like tuberculosis and human papilloma virus. Beta-sitosterol influences the proliferation of T-lymphocytes. Beta-sitosterol and its glycosides have anti-inflammatory activity as they inhibit both tumor necrosis factor and interleukin-6 depending on their concentration. 

In nature a remarkable intermolecular alkyl coupling occurs in living organisms as part of the biosynthetic pathway to steroids including cholesterol in plants and the biologically active mammalian sex hormones estrogen and testosterone. Steroid biosynthesis occurs by enzyme catalyzed epoxidation of squalene to produce 2, 3-epoxy squalene. Enzymatic acid catalyzed ring opening occurs followed by sequential formation of four carbon bonds.  This is followed by acid catalyzed cyclization and then multiple carbocation rearrangement to yield lanosterol a biological precursor of cholesterol. Enzymatic degradation of lanosterol produces cholesterol which is itself converted by other enzymes to produce many other steroids (Morrison et al., 2006).

Stilbenes are a family of secondary metabolites derived from phenyl propanoid pathway that consist of trans-ethene double bond substituted with a phenyl on the two carbon atoms of the double bond. The major active component of stilbenes is trans-resveratrol synthesized from cinnamic acid derivatives and found in many plants. The stilbenes have anti-cancer and anti-inflammatory properties. Resveratrol synergistically inhibits the growth of human colon cancer cells. Resveratrol induces vasorelaxation and reduces thrombogenic potential in the body cells. Resveratrol has also shown anti-cancer activity (Finar, 1975). Another form of stilbene is pterostilbene. Stilbenes hinder DNA synthesis and replication also hinders the rapid increase of lymphocyte proliferation during immune suppressive therapies. Stilbene slows the gradual development of cancer thus inhibiting the development of neoplastic lesions (Morrison et al., 2006).

1.3       BIOACTIVE PHYTOCHEMICALS

Plant chemicals that are bioactive have potentials for use as therapeutic agents or in chemotherapy. Many compounds are also active in destroying bacteria but are harmful to tissues hence such compounds can only be used externally because they are disinfectants. Chemical compounds that can be used as therapeutic agents are active on the disease causing agents and not the host tissues. From 1907 organic dyes were used in chemotherapy but from then on, organic compounds of diverse chemical structures have been used in chemotherapy and many chemotherapeutic compounds are specific in their toxicity towards a particular micro-organism (Finar, 1975). The effectiveness of medicinal plants has been recorded since prehistoric times for the cure of different ailments (Sofowora, 1993; Hill, 1989). According to Bhat et al., (2013), more than 4.5 billion people in developing countries include medicinal plants as components of their healthcare. The high popularity of medicinal plants use in rural areas is because conventional drugs are costly and modern medicine has been unable to find an effective cure to diseases and many of the conventional drugs have side effect (Tolossa et al., 2013; Birhan et al., 2011).

All these factors resulted in the steady promotion worldwide of traditional medicinal plant use in primary healthcare in recent years (Samy et al., 2008). The bioactive compounds either act on different systems of animals including man, or act through interfering in the metabolism of microbes. Whether the microbes are symbiotic or pathogenic, the bioactive compounds present in medicinal plants plays a role in regulating host-microbe interaction to favor the host. An advantage of naturally bioactive molecules is that they have a milder side effect on the body in comparison to chemically synthesized drugs. With the increasing acceptance of herbal medicines as an alternative form of healthcare delivery, the screening of medicinal plants for bioactive compounds is imperative (Cowan, 1999). The important areas of application of natural products are in the treatment of human and veterinary ailments. Currently, at least 119 chemical compounds derived from 90 plants species are considered as important drugs made use of in many countries of the world (WHO, 1977). Although natural products use predates the first recorded history by the earliest humans, and humans have used various but specific plants to treat illnesses, the treatment of diseases with pure pharmaceutical agents is a relatively new phenomenon (McKenna et al., 2011). The role of traditional medicine in the discovery of potent chemicals is quite crucial. Among the earliest successes in developing drugs from natural products is the isolation of quinine a cinchona alkaloid used as an anti-malarial agent from Cinchona succirubra and pain reliever such as the morphine alkaloid from Papaver somniferum. Most recently, the vinca alkaloids vinblastine and vincristine have been isolated as anti-neoplastic agents from the Madagascar periwinkle Catharanthus roseus and the anti-cancer agent derivatives from them are vinorelbine and vindesine. Similarly, artemisinin was isolated from Artemisia annua as a remedy against the multi-resistant strains of plasmodium following the long use of the plant in native Chinese medicine. The semi-synthetic drug arthemeter has been synthesized from artemisinin. These accounts show not only the efficacy of natural products as a source of drug but also as the solid link between folk medicine, drug development and the necessity of natural products research.   Following this knowledge, chemists began to isolate compounds for use in various ailments (Morrison et al., 2006).  

There are about 2,500,000 higher plant species of which more than 80,000 are medicinal. India is one of the world’s 12 biodiversity centers with the presence of 45,000 different plant species (Chakravarty et al., 1982; Sharma, 1996; Surveswaran et al, 2007). Chemical substances obtained from higher plant origin are effective sources of chemotherapeutic agents without underlining side effects (Pinto et al., 2002; Iniaghe et al., 2008). 

In the developed countries, 25 % of the medical drugs are based on plants and their derivatives (Principe, 1991; Martins et al., 2005; WHO, 1977). Justicia spicigera is a native of Mexico and Central America. In Mexico there are about 23,400 vascular plants of which about 500 species are used for medicinal purposes (Newman, 2000).

In Nigeria, plants belonging to 325 species from 95 plant families, 13 of those species are from the Acanthaceae family are used by most Nigerians for the treatment of various common diseases. Some of these medicinal plants are cultivated in gardens while others grow wildly in bushes (Monier, 2016).

1.4       TRADITIONAL MEDICINE

WHO (2002; 2001) has described traditional medicine as the surest means to achieve total healthcare coverage of the world’s population. Nigeria has a long history of traditional healthcare based on largely rich, though unstandardized pharmacopoeia drawn mostly from plants used by women in the home for self-administration and by traditional health practitioners. Most of the knowledge of traditional medicine according to (WHO) (2002), used in the diagnosis, prevention and elimination of physical, mental or social imbalance whether it can be explained or not, depend exclusively on practical experience and observation handed down from generation to generation either through word of mouth or in writing. Traditional medicine is also considered to be a solid link of dynamic medical knowledge and ancestral experience. The medicinal use of plants is rapidly declining as a consequence of modernization and civilization and the younger generation show little or no interest in learning this valuable science of healing (Cox, 2005; Focho et al., 2009). A common practice in traditional medicine is to combine various medicinal plants or medicinal plants parts. The medicinal uses of plants are varied and the medicinal plants specie part used range from the leaves, roots, stem, bark and fruits only or two or more medicinal plants in a species can be combined with those of other medicinal plant species (Cano et al., 2004). Perusquia et al. (1995) reported that the use of Justicia spicigera and walnut leaves may purify and increase red blood cells. A variation for this infusion is the addition of Sarsaparilla for use in treating heart disorders. Some plant parts have been used as antimicrobial agents, especially their extracts either as decoctions, infusions or oral administration (Lai et al., 1976; Abayomi, 1984; Okemo et al., 2001). Extracts made from only plant leaves are mostly used, followed by extracts made from only plant roots. Some Justicia species are used as mixtures with the powdered roots of other substances to prevent pregnancy without any toxic effects. The abortifacient nature and other medicinal activities of the roots of plants has been studied (Badami et al., 2003; Wu et al., 1995; Schultes, 1972).

Several species of Justicia are popularly used in folk medicine for treating respiratory, inflammatory, gastro-intestinal as well as applications in rheumatism and arthritis. Justicia species also has effect on the central nervous system as hallucinogens, somniferous agents, sedatives and for treating epilepsy. Other Justicia species are popularly used for the treatment of headache, earache, nausea, teeth pain, stomach disorder and fever and this may be associated with their sedative and analgesic properties. Some species of Justicia are used for cancer treatment, diabetes, tuberculosis, asthma, cough, bronchitis, insanity (mental disorders) and HIV. The whole plant and the aerial part of Justicia spicigera are often used in folk medicine as a stimulant, against inflammations, scabies, gastrointestinal disorders, emotional disorders, kidney infection, menstrual disorders, dysentery, treatment of anemic condition, regulation and to reduce the level of glucose in the blood (Wang et al., 1983; MacRae et al., 1984; Lorenz et al., 1999; Sanmugapriya, et al., 2005; Lu et al., 2008; Lockleare et al., 2010).

1.5       OBJECTIVES OF THE STUDY

Natural products from medicinal plants either as pure compounds or as standardized extracts provide extensive opportunities for new drug discovery because of the unrivaled availability of various chemicals in plants. There is an increasing demand to screen plants seeking for therapeutic drugs from natural products with particular interest in edible plants. Herbal preparations for medicinal use contain various types of biologically active compounds. The objectives of this study are to:

1.         Extract biologically active compounds from Justicia spicigera using solvent extraction.

2.         Determine the biologically active phytochemicals present in Justicia spicigera.

3.         Apply chromatographic techniques such as High Performance Liquid Chromatography (HPLC) to isolate biologically active compounds and use Thin Layer Chromatography (TLC) to monitor the purity of the compounds isolated from the leaves of Justicia spicigera. 

4.         To characterize the bioactive constituents of Justicia spicigera through structure elucidation using Infra-red (IR) Spectroscopy, Proton (¹H) and DEPT-NMR Spectroscopy, Mass Spectroscopy (MS), Homonuclear ¹H,¹H-COSY (2D-¹H, ¹H COrrelated SpectroscopY) and   Heteronuclear Multiple Bond Correlation (HMBC) spectroscopy.

5.         To apply a non-chromatographic technique; antimicrobial immunoassay to determine the biological activity of the plant extracts on human pathogens.

1.6       SCOPE OF THE STUDY

This research work focuses attention on the medicinal plant Justicia spicigera from the Acanthaceae plant family.

1.7       justification of the study

Justicia spicigera, one of the eight species of Justicia found in Africa is popularly used in herbal medicine for treating many diseases. With about 7,000 species of medicinal plants in-bloom recorded in Africa more than 4,000 are medicinal plants, out of which 12 percent are found endemic due to environmental factors (Aderounmu, 2002; Elufioye et al., 2012). This is an alarming signal to the population which has for generations relied on plant resources to combat ailments of both humans and domestic animals (WHO, 1978; Obute et al., 2002; Ayodele, 2005).There is urgent need to preserve Justicia plant species by modern techniques like bio-technology, tissue culture and isolation and characterization of the bioactive natural products they contain (Genene et al., 2017; Akinyemi, 2000; Gbile et al. 1986). Over 25 percent of prescribed medicines in industrialized countries are derived directly or indirectly from medicinal plants while in developing countries the contribution is as much as 80 percent (Newmann et al., 2000). Medicinal plants used in traditional medicine has not been fully studied (Kirby, 1996). At present 32 % of drugs introduced into the international markets are of plant origin (Farnsworth, 1994). Ayodele (2005) has called on Nigerian taxonomists and conservation biologists to rise up to the task of properly identifying and conserving these important genetic resources.Several ethno-botanical studies relating to medicinal plants have been documented (Ekpendu et al., 1998; Balansard et al., 2000; Sing et al., 2001; Wang et al., 2002; Kumar, 2005; Pei, 2005; Surveswaran et al, 2007). In Nigeria, scanty information on ethno-botanical studies have been documented (Gill, 1992; Sofowora, 1993; Igoli et al., 1999). According to Gavamukulya et al. (2014), the absence of scientific investigation of medicinal plants to authenticate their use may cause serious negative effects. This study is an attempt to document information on the efficacy of the traditional medicinal plant Justicia spicigera based on scientific evaluation of its bioactive constituents.

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