Isolation and Characterization of Bioactive Compounds From Stembark Extract of Uapaca Pilosa Hutch
Chapter One
Aim of the Research
This research work aims to isolate and characterize bioactive components present in the plant.
Objectives of the Research
- phytochemical screening of crude plant extracts,
- antimicrobial screening of the crude extract of the plant,
- isolation and identification of phytochemicals present in the extracts and
- antimicrobial screening of isolated/identified compounds.
CHAPTER TWO
LITERATURE REVIEW
The EuphorbiaceaeFamily
Family Euphorbiaceae is one of the largest families of flowering plants, composed of over 300 genera and 8,000 species (Webster, 1994). According to researchers, the family is very diverse in range, composed of all sorts of plants ranging from large woody trees through climbing lianas to simple weeds that grow prostrate to the ground. Members are widely distributed all around the world constituting both old world and new world plants some of which are yet to be identified. Many family members are inhabitants of tropical climates surviving hot dry desert conditions while others are rainforest trees and herbs (Webster, 1994). The family is divided into five subfamilies which include Acalyphoideae, Crotonoideae, Euphorbioideae, Phyllanthoideae and Oldfieldoiideae (Webster, 1975). According to this classification, the first three subfamilies are characterized by one ovule per locule (uni-ovulate) while the last two have two ovules (bi-ovulate). Phytochemical and molecular phylogenetic studies eventually accumulated evidence pointing to non-monophyly of Euphorbiaceae (Mwine and Van Damme, 2011; Tokuoka and Tobe, 1995). This culminated into partitioning of the traditional Euphorbiaceae into five families, where only uni-ovulate subfamilies constituted family Euphorbiaceaesensu lato, others being upgraded with additions or subtractions into their own families (Webster, 1994) and was validated by the APG II group (Wurdack et al., 2005). The new classification left family Euphorbiaceaesensu lato with five subfamilies, 49 tribes, 317 genera and about 8,000 species (Webster, 1994).
The Uapacagenus
Uapaca is an evergreen, dioecious, small to medium-sized tree under Euphorbiaceae family and belongs to the order malpighiales. The plant is distributed in tropical Africa and Madagascar. Other member of the family include Uapaca togoensis, Uapaca guinessis, Uapaca bail, Uapaca paludosa, Uapaca le-testuana, Uapaca lebrunii, Uapaca littoralis, and Uapaca macrostipulata (Breteler, 2012). Uapaca species are part of ethno medicinal plants used by Africans in treating various diseases. Some of the species in the genus are represented in West Africa (Hutchinson and Dalziel, 1958; Heywood, 1978; Van Damme, 2001). Two of the species Uapaca stipularis and Uapaca kirkiana are commonly used in folkloric medicine, which is a practice by lay people in the West African sub region (Oliver, 1960). Uapaca heudelotii is ethnomedicinally reported to treat skin infections, female sterility, as an emetic, pile and gargle for tooth-troubles. It also serves as a source of highly-priced hard timber (Dalziel, 1937; Burkill, 1985). Uapaca togoensis is reported in the treatment of female infertility, as a restorative wash against fatigue and for making charcoal (Irvine, 1948). The wood of Uapaca staudtii is termite-proof, difficult to work upon because of its chemical composition and strength; it is used for making furniture, railway sleepers and barrel staves (Dalziel, 1937). Uapaca paludosa and Uapaca vanhouttei have also been reported for making charcoal and are used as firewood (Dalziel, 1937).
Uapaca pilosa
Uapaca pilosa (Hutch.) is an indigenous tropical African plant, which can be found in Madagascar, Congo, Nigeria and other Africa countries. It has well branched broad leaves, thick stem and edible fruit. To the best of our search of the literature, the phytochemistry and antimicrobial screening of any part Uapaca pilosa have not been studied.
Taxonomy of the Plant
Name: Uapaca pilosa (Hutch.)
Family: Euphorbiaceae
Genus: Uapaca
Species: pilosa
Idoma name: Obloblo
CHAPTER THREE
MATERIALS AND METHODS
- Materials
- Solvents
Solvents used were of general purpose grade and were distilled before used. The solvents used are:
n-hexane Chloroform Ethyl acetate Methanol Equipment
The equipment used includes the following: Analytical balance (AND Instrument, GR-200-EC)
Top loading balance (Denver Instrument, XP 600×0.02 g) Melting point apparatus (Ernst Leitzwetzlar)
Rotary evaporator Oven
Vacuum pump
FTIR Agilent Technologies Carey 630 FTIR spectrometer UV lamp 254-326 nm spectrophotometer (Hitachi U-3200) NMR Bruker AVANCE spectrometer III (600 MHz)
Reagents
1% Hydrochloric acid
10% Ammonium solution
10% Sodium hydroxide solution Ferric chloride solution
Fehling solution Conc. sulphuric acid Chloroform Dragendorff‟s reagent
Molisch‟s reagent
Mayer‟s reagent
Anhydrous acetic acid Glacial acetic acid 95% Ethanol
CHAPTER FOUR
RESULTS
Result of Extraction of the Stembark of Uapaca pilosa
The results of solvent extraction of stem bark from Uapaca pilosa showing composition of crude methanol extract, n-hexane, chloroform and ethyl acetate fractions are as presented in Table 4.1.
Result of Phytochemical screening
The results of the phytochemical screening of the stem bark extracts from Uapaca pilosa are as shown in Table 4.2.
CHAPTER FIVE
DISCUSSION
Extraction of the stem bark of Uapaca pilosa
The plant material was identified, dried, pulverised and extracted using cold maceration method. The plant have been used traditionally as medicine in the treatment of female sterility and other ailments. The percentage yield of the extraction result as presented in Table 4.1 showed methanol has the highest yield of 270 g about 18.01 % of the total weight of the plant while chloroform has the lowest yield with recovery weight of 8.62 g representing 0.58 % of the plant.
Phytochemical Screening of the Stem bark of Uapaca pilosa
The crude extracts from the stembark of Uapaca pilosa were subjected to phytochemical screening and the results (Table 4.2) revealed the presence of all the tested secondary metabolites in methanol and ethyl acetate fraction except saponnins which was absent in ethyl acetate fraction, while alkaloids, anthraquinones, flavonoids and saponnins were absent in chloroform fraction. The results also revealed the presence of alkaloids, anthraquinones, glycosides, steroids and triterpenes were observed hexane fraction. In general, the accumulation and concentration of secondary metabolites are responsible for antibacterial activity and this varies according to plant extracts depending on their polarity (Essawi and Srours, 2000). These metabolites are known to act by different mechanisms and exert antimicrobial, antioxidants, anticancer and antituberculosis actions (Aiyegoro and Okoh., 2009).
CHAPTER SIX
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The plant sample Uapaca pilosa was collected fresh from Otukpo, Benue state, Nigeria in February, 2015. It was identified by Mr Sanusi Namadi at the Herbarium unit of Department of Biological Sciences, Ahmadu Bello University Zaria with a specimen voucher number 1279. The stem bark was subsequently subjected to air drying, pulverization and cold maceration extraction method using methanol. The methanol extract was reconstituted in water and partitioned with hexane, chloroform and ethyl acetate respectively. Extract and fractions obtained were concentrated using rotary evaporator and dried at room temperature. Crude extract and fractions obtained after extraction were subjected to preliminary phytochemical screening and antimicrobial studies. Ethyl acetate fraction showed most promising antibacterial activity against the tested organisms. Silica gel column purification and preparative thin layer chromatography of the ethyl acetate fraction led to the isolation of two pure compounds coded GF1 and GF2. The isolated compounds were identified through spectroscopic analyses such as 1D NMR (1H, 13C and DEPT), 2D NMR (COSY, HSQC, HMBC and NOESY) and comparison with literature. The pure compounds were revealed to be betulin (GF1) and beta-sitosterol (GF2). The isolated compounds demonstrated good antimicrobial activity against tested microorganisms.
Conclusion
The result of this study revealed the antimicrobial activities of extracts and molecules isolated from Uapaca pilosa. This study established that the plant offer significant potential for the development of new antibacterial therapies and treatment of diseases associated with tested microorganisms. Results from this research validates the ethnomedicinal uses of Uapaca pilosa for the treatment of several ailments by traditional medicine practitioners. The compounds isolated from the plant have been isolated from different plants and their antibacterial, antitumor and anti-inflammatory activity have been reported, this is the first report on their isolation from Uapaca pilosa (Hutch.). Therefore, this research work is an addition to the world data on natural product.
Recommendation
This study was carried out on the stembark of Uapaca pilosa only since the aim of research is to justify the use of this plant for the treatment of infections. The following recommendations are therefore made:
- other parts of the plant should be screened for their antimicrobial activities so as to validate the whole plant`s antimicrobial properties,
- since the isolation work was made on the ethyl acetate extract only there is need to carry out work on the hexane, methanol and chloroform fractions of this plant, because they also showed some activities,
- structural modification of the isolated compounds for enhancement of their activities
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