Guiera senegalensis, Scoparia dulcis and Pulicaria crispa are plants known in Nigeria for the treatment of various infectious diseases. These plants were subjected to phytochemical, antimicrobial and anti-TB screening. Phytochemical screening of the plants revealed the presence of saponin in all the hexane fractions, ethyl acetate fraction of Scoparia dulcis and methanolic fraction of Guiera senegalensis. Cardenolides, phlobatanins, volatile oil and resins were absent in all the three solvent extracts. Flavonoids and volatile oils were present in only the ethyl acetate extract and methanolic extract of Pulicaria crispa . Cardiac glycosides, glycosides, balsams and phenols were only present in the methanolic extract of Scoparia dulcis. Steroids were found in the ethyl acetate extracts. Flavonoids, cardiac glycosides and glycosides were also found in the methanolic extract of Guiera senegalensis and Scoparia dulcis. The antimicrobial activity of the plant fractions tested against Gram positive bacteria (Staphylococus aureus, Bacillus subtilis, Streptococcus pyogenes) and Gram negative bacteria (Shigella dysenteriae, Salmonella typhii, Escherichia coli) and fungus (Candida albicans, Candida krusei) using agar in-well diffusion method have their activities ranging between 16 mm to 35 mm respectively as against the control (ciproflaxin and fluconazole ranging between 31mm to 41mm respectively) while the zone of inhibition of the test microbes against the three plant extracts were found to have high zone of inhibition in both ethyl acetate fraction and hexane fraction of Guiera senegalensis, at a concentration of 3.12 μg/ml as against S. aureus, also in ethyl acetate fraction of Pulicaria crispa at a concentration of 12.5 μg/ml as against P. mirabilis, also in all fractions o G. Senegalensis at a concentration 3.12 μg/ml as against C.albican, followed by the fractions of Pulicaria crispa at concentration of 3.12 μg/ml as against S. pyogenes and N.gonorrhea, the inhibitory effect of various extracts were compared with standard drug ciprofloxacin and fluconazole. Minimum inhibitory concentration and Minimum bactericidal and fungicidal concentration for both extracts were also determined and ranged between 12.25 to 25 μg/ml using broth dilution method. The anti-mycobacteria study showed that activity were found in methanol extract of Pulicaria crispa at concentration of 8.01±1.70 μg/ml, extracts of ethyl acetate of Scoparia dulcis at concentration of 12.03±0.86 μg/ml and methanol extract of Guiera senegalensis at concentration of 2.80±1.40 μg/ml, however low activity was observed for the hexane extract of Scoparia dulcis at concentration of 20.40±0.24 μg/ml and ethyl acetate extract of Guiera senegalensis at concentration of 40.01±1.20 μg/ml, hence when compared with the control drug rifampicin at concentration of 0.38±1.40 μg/ml, methanol fraction for
Guiera senegalensis showed the highest anti-mycobaterial activity. Extensive chromatographic separation of the methanol extract of Guiera senegalensis led to the isolation of compound Dy1 (oleanolic acid). The structure of the compound were established by spectral analysis including 1D NMR (1H, 13C and DEPT), 2D NMR (COSY, HSQC, HMBC and NOESY) hence showing 8 quatenary carbons, 5 methine carbons, 10 methylene carbon and 7 methyl carbons, hence the total of thirty carbons. The high anti-TB activity on the methanolic fraction of Guiera sengalensis is been reported for the first time from this plant. The use of the plants for the treatment of infectious disease is therefore justified
TABLE OF CONTENTS
Title page i
Dedication iv Acknowledgement v Abstract vi
Table of Contents viii List of Tables xiii List of Figures xiv List of Structures xv List of Abbreviation xvi
1.0 INTRODUCTION 1
1.1 Historical Background and Importance of Medicinal Plants 1
1.2 Ethnobotany and Pharmacology of Guiera senegalensis (J.F. Gmel),
Scoparia dulcis(Linn) and Pulicaria crispa (Forssk) 2
1.2.1 Guiera senegalensis (J.F. Gmel) 2 1.2.2 Scoparia dulcis (Linn) 3 1.2.3. Pulicaria crispa (Forssk) 5 1.3 Aim and Objective of Study 5 1.3.1 Aim 5 1.3.2 Objective 5 1.4 Justification of Study 6
CHAPTER TWO: 8 2.0 LITERATURE REVIEW 8
2.1 Natural Products in Drug Discovery and Development 8
2.2 The Global Burden of Tuberculosis 9
2.3 Etiology of Tuberculosis 11
2.4 Tuberculosis in Nigeria 12
2.5 Treatment of Tuberculosis 14
2.6 Problems with Controlling TB 17
2.7 In Vitro Assays for Evaluation of Anti-tubercular Activity 17
2.8 Drug Discovery from Natural Products 18
CHAPTER THREE 3.0 MATERIALS AND METHODS 25 3.1 Materials 25 3.1.1 Reagents 25 3.1.2 Equipment 25 3.1.3 Collection and identification of plant materials 25 3.2 Methods 26 3.2.1 Extraction 26 3.2.2 Phytochemical Screening 26 220.127.116.11 Test for Carbohydrates 26 18.104.22.168 Test for Tannins 27 22.214.171.124 Tests for Flavonoids 27 126.96.36.199 Test for Cardiac glycoside 28 188.8.131.52 Test for Steroids and Triterpenes 28
184.108.40.206 Test for Alkaloids 28
220.127.116.11 Test for Saponins 29 18.104.22.168 Test for Glycosides 29 22.214.171.124 Test for Phlobatannins 29 126.96.36.199Test for Phenols 29 188.8.131.52Test for Terpenoid 29 184.108.40.206Test for Resins 29 3.2.3 Antimicrobial Screening 30 220.127.116.11 Collection and Preparation of Microbial Culture 30 18.104.22.168 Antimicrobial Screening Test 30 22.214.171.124 Determination of Minimum Inhibitory Concentrations
(MIC) of the Extracts 31
126.96.36.199 Minimum Bactericidal Concentration/ Minimum
Fungicidal Concentration 32
3.2.4. Anti-Mycobacteria Screening 32
188.8.131.52 Microplate Alamar Blue Assay (MABA) 32
3.2.5 Chromatographic Purification of Extracts 33
184.108.40.206 Thin Layer chromatography (TLC) 33
220.127.116.11. Purification and Isolation of Compounds 33
18.104.22.168. Preparative Thin Layer Chromatography 33
3.2.6. Spectral Analysis 34 22.214.171.124. UV- Visible Spectrometer 34 126.96.36.199. Infra-Red (IR) 34 188.8.131.52. Nuclear Magnetic Resonance (NMR) 34
4.0 RESULTS 35
4.1 Result of Extraction of G.senegalensis, S. dulcis and P. crispa 35
4.2 Result of Phytochemical Screening 36
4.3 Result of Anti-Microbial Screening of Extracts 36
4.4 Result of Anti-TB Assay of the Extracts 43
4.5 Physical and Chemical Test on Dy1 44
4.6 Colum Chromatography 45
4.7 Thin layer Chromatography of the Methanol Extract of G.senegalensis 45 4.8 Spectral Analysis 46
4.8.1 Result of the 1HNMR Spectra of Dy1 46
4.8.2 Result of the 13CNMR Spectra of Dy1 47
4.8.3. DEPT 46
4.8.4 2D Spectrum of Dy1 56 4.8.5 Summary of Assignment (spectral data) Comparism with Literature. 56
5.0 DISCUSSION 58
5.1 Phytochemical Screening. 58
5.2 Antimicrobial Activity 59
5.3 Anti-mycobacterial Activity 60
5.4 Chromatograghic and Spectral Analysis. 61 CHAPTER SIX
6.0 Conclusion 63
6.1 Recommendation 63
1.1 Historical background and importance of medicinal plants.
Plants have been used for thousands of years to flavour and conserve food, to treat health disorders and to prevent diseases including epidemics. The knowledge of their healing properties has been transmitted over the centuries within and among human communities. The use of plants for treating diseases is as old as the humans (Balunas and Kinghorn, 2005). Plant materials have been used for the treatment of various diseases throughout the world before the advent of modern clinical drugs. The use of medicinal plants still plays an important role to cover the basic health needs in the developing countries and the industrialized societies. The extraction and development of several drugs have been traced from these plants as well as from traditionally used folk medicines (Shrikumar and Ravi, 2007). Popular observations on the use and efficacy of medicinal plants significantly contribute to the disclosure of their therapeutic properties, so that they are frequently prescribed, even if their chemical constituents are not always completely known. All over the globe, the use of medicinal plants has significantly supported primary health care (Maciel et al., 2002). From 250 to 500 thousand plant species are estimated to exist on the planet, and only between 1 and 10 % are used as food by humans and other animals (Cowan, 1999). About 80 % of the world population uses only 37 % of the commercially available drugs and depend exclusively on medicines of natural origin (Funari and Ferro, 2005). Thus, phytotherapeutics entered the market promising a shorter and cheaper production, since basic requirements to use medicinal plants do not involve strict quality control regarding safety and efficacy compared to the other types of drugs (Niero, 2010).
Infectious diseases represent an important cause of morbidity and mortality among the general population, particularly in developing countries. Therefore, pharmaceutical companies have been motivated to develop new antimicrobial drugs in recent years, especially due to the constant emergence of microorganisms resistant to conventional antimicrobials. Apparently, bacterial species present the genetic ability to acquire and transmit resistance against currently available antibacterial since there are frequent reports on the isolation of bacteria that are known to be sensitive to routinely used drugs and became multi-resistant to other medications available on the market (Nascimento et al., 2000; Sakagami and Kajimura, 2002). Consequently, common strategies adopted by pharmaceutical companies to supply the market with new antimicrobial drugs include changing the molecular structure of the existing medicines in order to make them more efficient or recover the activity lost due to bacterial resistance mechanisms (Chartone-Souza, 1998).
1.2 Ethnobotany and pharmacology of Guiera senegalensis (J.F. Gmel), Scoparia dulcis (Linn) and Pulicaria crispa (Forssk)
The plant selected for study in this project were root part of Guiera senegalensis (J.F. Gmel), whole plant part of Scoparia dulcis (Linn) and whole plant part of Pulicaria crispa (Forssk).These species were mentioned by traditional healers in Nigeria to be used in the treatment of tuberculosis symptoms and chest related infections according to an ethno-botanical survey that was done (Gautam, et al., 2007)
1.2.1. Guiera senegalensis (J.F. Gmel)
Guiera senegalensis is a flowering plant in the genus Guiera of the family of combretaceae and it is a native of tropical regions of Africa. The plant produces the Tannin 3, 4, 5- tri-o-galloylguinic acid and several alkaloids (Gmel et al., 2006). It is popularly named in Hausa as kululu or saabaraa in the northern parts of Nigeria. It is used to treat a variety of microbial infections, can be used as dyes. It is also used for the following purposes; as an essential oil,
exudates, fibre meals, fuel plant, medicinal plant, timber and vegetables. They are soft on both surfaces, with scattered black glands underneath (Hutchison and Dalziel, 1965). The leaves are bitter-tasting and have widespread acknowledgement in African medicine as a “cure-all” in herbal concoctions (Hiermann and Bucar, 1994). The usual form of preparation for internal use is in decoctions or mixed with food preparations G. senegalensis leaves are widely administered for pulmonary and respiratory complaints, for coughs, as a febrifuge, colic and diarrhea, syphilis, beriberi, leprosy, impotence, rheumatism, diuresis and expurgation (Hutchinson and Dalziel, 1965; Zeljan et al., 1998). In Northern Nigeria powdered leaves are mixed with food as a general tonic and blood restoratives (Koumare et al., 1968). In Nigeria and other West African Countries, the leaves are used to treat dysentry and fever due to malaria (Abbiw, 1990). Earlier findings by Etkin (1997) indicated Guiera leaf extracts markedly oxidized glutathione and also generated high levels of methaemoglobin in vitro, both conditions being unfavourable for the survival of Plasmodium in red cells. These reported activity and the application of the plant in herbal medicine for analgesic and antimalarial properties necessitated in vivo screening with parasitized animals. Leaf extracts of the plant were tested for antiplasmodial, analgesic and anti-inflammatory effects in vivo by Jigam et. al., 2009, his results indicated the safe dose of extracts as 600 mg/kg body weight of mice with LD50 of 1100 mg/kg body weight. Only the methanol fraction had antiplasmodial effect while ethyl acetate and hexane fractions were ineffective. Furthermore the methanol extract produced a significant (p<0.05) suppression of up to 67.52% levels. The extracts were found to have no prophylactic effect or high parasitaemia, rather it gave 44.83% analgesic effect which was devoid of anti-inflammatory activity.
1.2.2 Scoparia dulcis (Linn) Scoparia dulcis (Linn) belongs to the family Scrophulariaceae. It is an herbal plant found almost around the globe: In European, African, American and Asian countries. Synonyms are Scoparia grandiflora, Scoparia ternata, Capraria dulcis and Gratiola micrantha. In Nigeria the plant is known by such names as roma fada (Hausa), aiya (Igbo), bibiimbelemo (Ijaw), mesenmesèn gogoro (Yoruba), ndiyang (Efik), and ungungbuhi (Gwari).
The plant is an erect, shrubby herb that grows up to about 50 cm high (Orhue et al, 2006). It usually has many auxiliary shoots and reproduces from seeds. The stem is more or less woody, ribbed, mainly branched and glabrous. The leaves are opposite or three at a node, oval or narrowly oblanceolate; about 2.5 cm to 5.0 cm long and 1.5 cm wide. The Brazilian folkloric use it to treat bronchitis, gastric disorders, hemorrhoids, insect bites and skin wounds. Asian medicine uses the herb to treat hypertension Latha et. al., 2004. The herb also has antiulcer properties Cragg et. al., 2008. In tropical and subtropical regions, the fresh or dried plant of Scoparia dulcis has traditionally been used as one of remedies for stomach troubles (Satyanarayana, 1969), hypertension (Chow et al., 1974), diabetes (Perry, 1980), bronchitis (Freire et al., 1996), and as an analgesic and antipyretic (Gonzales, 1986). Three antidiabetic compounds have been isolated from Scoparia dulcis; amellin, scoparic acid D and diasulin. Amellin, an antidiabetic compound is a glycoside which has been reported in the leaf and stem of fresh green plants. An infusion of the leaf is used in fever, cough and bronchitis and as gargle for toothache. A hot infusion is a diuretic. An infusion of roots leaves and tops are useful in diarrhea and dysentery. The diterpenoid, scoparic acid A, isolated from the plant has been reported to be a potent α-glucoronidase inhibitor (Hayashi et al, 1992). The constituents, scopadulciol, scopadulcic acid-B and diacetylscopadiol, have been shown to be responsible for the inhibitory activity of the plant on gastric H+-K+ ATPase enzyme (Asano et al., 1990). All parts of the plant are useful as emetic. However, very limited work has been
carried out on this specie toward documenting its ethno medicinal uses and establishing its phytochemical and anti-TB finger prints. 1.2.3. Pulicaria crispa(Forssk) Pulicaria crispa (forssk) is an annual herb or sometimes a perennial sub shrub belonging to the family Asteraceae producing small bright yellow flowers and Genus Pulicaria is represented in the flora of Iran by five species (Nematollahi et al., 2006). P. undulata is an example of the species which is used as a medicinal plant by people of Southern Egypt to treat inflammation. This species are distributed in Iran, Saudi Arabia, Kuwait, Iraq, Egypt, Afghanistan, Pakistan, India and parts of North and West tropical Africa (Al-Rawi, 1987; Boulos, 2002).
Pulicaria crispa is used as an insect repellent (Stavri et al., 2007) and also as an herbal tea (Ross et al., 1997). These plants have been reported in the past to have medicinal values just as reviewed above, they possess potentials for inhibiting TB in the human body and hence justifying the research. 1.3 Aim and Objectives of the study
The aim of this work is to justify or otherwise the ethnomedicinal claims on the root of Guiera senegalensis, whole plant of Scoparia dulcis, and Pulicaria crispa as medicinal plants in the treatment of tuberculosis and any other microbial infectious diseases.
The objectives of this study are:
Collection, proper botanical identification and drying of the plants.
Extraction of the ground plant material using different solvents, from non-polar to polar ones.
Phytochemical screening of the crude extracts.
Antimicrobial screening of the extracts
To determine the anti-mycobacterial activity of the plant extractsChromatographic purification of the extracts
Spectroscopic Structural elucidation and characterization of the isolated compounds using available spectral techniques
To determine the anti-mycobacterial activity of the isolated compound.
1.4 Justification of the study
Trends in the incidence of tuberculosis together with the development of multi-drug and extensively drug resistant strains of tuberculosis raises the need to intensify the search for more efficient drugs to combat this disease (Corbett et al., 2003; CDC Report, 2005). There are widespread claims by some traditional healers that TB can be treated using herbs. However these claims have no scientific justification mainly because, Nigeria has one of the least published literatures on plants screened for anti-Tubercular activity (Mann et al., 2007). The results of this study will go a long way to authenticate the claims by 5 traditional healers and will as well enrich the databases on plants with anti-mycobacterial activity that can be used in drug discovery.
Among infectious diseases, tuberculosis is one of the leading killers of adults in the world today. The incidence of tuberculosis is exacerbated by the emergence of drug-resistant strains multi-drug resistant and extensive drug resistant (MDR and XDR) and HIV co-infection (Furin, 2007). Available treatment regimens are lengthy and complex, leading to problems of
non-adherence and inadequate response. In the case of MDRTB, second line drugs used are more toxic and expensive while XDRTB is virtually untreatable. HIV/AIDS patients presenting with tuberculosis stand a risk of drug adverse reactions as a result of possible drug-drug interactions. On the other hand a number of traditional medicinal plants have been reported to treat tuberculosis, however their efficacy and safety remains unknown. This study was conducted to determine the efficacy and safety of the plants (Guiera senegalensis, Scoparia dulcis, and Pulicaria crispa) that have been suggested in treatment of TB symptoms by traditional healers.
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