Isolation and Characterisation of Bioactive Compound From the Root Bark of Ficus Sycomorus (LINN).

Isolation and Characterisation of Bioactive Compound From the Root Bark of Ficus Sycomorus (LINN).

CHAPTER ONE

Objectives of the Study

The aim will be achieved through the following objectives:

MAE and Target extraction on the plant
Preliminary Phytochemical screening of the
Separation, purification and isolation of the bioactive constituents using chromatographic techniques.
Characterization and structural elucidation of the isolated compound(s) using spectroscopic techniques such as:
- Infraredspectroscopy (IR);
- NuclearMagnetic Resonance (¹H NMR, ¹³C NMR and 2D NMR)
Determination of the antimicrobial activity of the plant extracts and the isolated compound.

CHAPTER TWO

LITERATURE REVIEW

Extraction

Medicinal plants are in considerable significance view due to their special attributes as a primary source of new medicine and lead compounds for the development of new drugs against various diseases (Parekh and Chanda, 2007) in addition to their ethno pharmacology.

Pre-extraction and the extraction procedures are the first and important steps of any medicinal plant study in the processing of the bioactive constituents from the plant materials (Azwanida, 2015). The extract thus obtained may be ready for use as a medicinal agent, it may be further processed to be incorporated in any dosage form such as tablets or capsules, or it may be fractionated to isolate individual chemical entities as modern drugs (Handa et al., 2008). Various solvents have been used to extract different phyto-constituents and alcohol among other solvents, in any case, remains a good all-purpose solvent for preliminary extraction (Harborne, 1998). El- Sayed and his co-workers reported that n-butanol (BuOH) fraction of Ficus sycomorus leaves has strong antioxidant activity and the compounds isolated from it were found as major components and principally responsible for the antioxidant activity of F. sycomorus (El-Sayed et al., 2010).

Medicinal plants are biosynthetic laboratories, not only for chemical compounds but for phytochemicals, which exert physiological and therapeutic effects (Barthel and Reuter, 1968). Due to the presence of many bioactive compounds in natural product extracts, synergistic effects or antagonistic interactions are likely to occur and as such; natural product extracts are claimed to have better pharmacological activity compared to single drug component. However, such claims are difficult to prove experimentally.

Synergistic effects, can result in the following: (i) the constituents of a natural product extract affects different targets (ii) they can interact with one another to improve the solubility and thereby enhancing the bioavailability of one or several substances of a natural product extract and (iii) compounds may also have their efficacy enhanced with agents that antagonize mechanisms of resistance (Wagner and Ulrich-Merzenich, 2009).

The analysis of the discrimination between antagonistic interactions or real synergism, of different classes of phytochemicals, can be carried out by investigating the complete phytochemical profile of a given plant species and fractionating the crude extract to obtain different classes of phytochemicals. This is known as target extraction, prior to chromatographic analysis. Target extraction, on an alkaloid-containing plant might be employed, that is based on varying polarity and basicity. However, modification is possible when investigating labile substances (Harborne, 1998).

CHAPTER THREE

MATERIALS AND METHODS

Materials

Solvents/Reagentsand Chromatographic Materials

Solvents used, which include methanol, ethanol, n-butanol, ethyl acetate, chloroform and n-hexane were of general purpose grade and were distilled before use. Chromatographic materials include: aluminium TLC plate pre-coated with silica gel 60 PF254, Shandon chromatographic tank (developing jar), Glass columns, Ultraviolet lamp (254 and 366 nm), Silica gel (Qualikens 60-120 mesh), 10 % H2SO4, conc. H2SO4 and conc. NaOH.

Equipment

Conventional microwave oven (MATSUI M180TC), digital pH meter (Hanna 4221), and the melting points apparatus (Suart SMP40 automatic melting point apparatus) were obtained from the Department of Chemistry, Ahmadu Bello University Zaria. Infra-red Spectroscopy (Shimadzu FTIR 400 Fourier Transform Infra-Red Spectroscopy) from the Multi user Laboratory, Department of Chemistry, Ahmadu Bello University Zaria. NMR analyses were run on a 600 MHz Bruker AVANCE spectrometer at the Department of Pure and Applied Chemistry, University of Strathclyde, Scotland-UK.

CHAPTER FOUR

RESULT

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Table 4.1 shows the percentage recovery of n-butano extact (3.67 %), chloroform fraction (0.15 %), chloroform-ethanol fraction (0.28 %) and ethanol fraction (0.20 %) from the root bark of Ficus sycomorus.

Table 4.2 shows the Phytochemical constituents present in the extract/fractions from the root bark of Ficus sycomorus.

Table 4.3 shows the solvent ratio of the eluting solvent, number of spots and their Rf value for each of the collections from first column chromatography of n-butanol extract.

Plate III shows the Thin-Layer Chromatography profile of the isolated compounds.

Table 4.4 shows the Rf value of each of the isolated compound at solvent system 9:1 (n- hexane : ethyl acetate)

Table 4.5 shows the result of the chemical test carried out on the isolated compounds.

Table 4.6 shows the melting point, physical state and the weight of the isolated compounds

CHAPTER FIVE

DISCUSSION

Plant Extraction

The result of the percentage yield of the extract/fractions from the root bark of Ficus sycomorus reported in Table 4.1 shows that the n-butanol extract from MAE had the highest recovery of 3.66 % followed by the fractions from Microwave-Assisted polarity based extraction; moderately polar chloroform-ethanol (0.28 %), more polar ethanol (0.20 %) and less polar chloroform (0.15 %).

Phytochemical Profiling

The result of the phytochemical analysis of n-butanol revealed the presence of flavonoids, alkaloids, steroids/triterpenes and tannins. The analysis also revealed the presence of flavonoids, steroids/triterpenes and tannins in the less polar CHCl3 fraction (MBC1), the moderately polar CHCl3-EtOH fraction (MBC2) had alkaloids, steroids/triterpenes and tannins and lastly the polar EtOH (MBC3) fractions had steroids/triterpenes (Table 4.2). These plant constituents were also reported from the parts of the plant extracts (El-Sayed et al., 2010 and Garba et al., 2007).

Isolation, Purification, Characterization and Biological Activity of the Isolated Compounds

Compound MB01 was isolated as a white crystalline solid with melting point of 213 – 215 °C. The ¹H NMR spectrum showed seven methyl signals at δH 1.53, 1.04, 0.98, 0.94, 0.83, 0.78 and 0.76 ppm. A doublet of doublets at δH 3.21 ppm characteristic of an α-oriented proton at C-3. Doublets for geminal protons at δH 4.69 and 4.57 ppm at C-29, along with the methyl signal at δH 1.53 ppm for C30, suggested that the

compound MB01 was a lupane-type triterpenoid. The ¹³C NMR spectrum further suggested compound MB01 as a lupane-type triterpene derivative. A total of 30 carbon signals were observed from the spectrum. The characteristic pair of sp2 hybridized carbon atoms comprising the double bond of lupeol was observed at δ 151.14 and 109.47 ppm. Oxygenated carbon shift was observed at δ 79.15 ppm for C3.

Consequently, after comparing these NMR data with data in the literature (Jamal et al., 2008), the compound was identified to be (3β)-Lup-20(29)-en-3-ol, more commonly known as lupeol (C30H50O). The FTIR spectrum complemented the assignment; a very intensely broad band at 3421 cm^-1 and moderately intense band at 1192 cm^-1 indicates the characteristic hydroxyl group (O-H). The corresponding C=C vibrations was shown around 1662 cm^-1 as a weak band. The stretching and bending vibrations of sp² C-H part were noticed by the intense band 2929 cm^-1 and medium intensity band at 1461 cm^-1, respectively. The stretching vibration of the sp³ C-H part was shown by the band at 2855 cm^-1. The IR absorbance values are in concordance with Silverstein et al. (2014).

CHAPTER SIX

CONCLUSION AND RECOMMENDATIONS

Conclusion

Microwave-assisted extraction (MAE) and Microwave-assisted polarity based extraction (target extraction) were carried out on the pulverized root bark of Ficus sycomorus. The fractions from the Microwave-assisted polarity based extraction were concentrated under reduced pressure while the n-butanol extract from MAE was evaporated and dried at room temperature. The highest percentage recovery was recorded on the n-butanol extract. The results of the phytochemical analysis of the extract/fractions revealed the presence of alkaloids, flavonoids, steroids/triterpenes and tannins, which are in agreement with the previous work (Garba et al, 2007) except for saponins, which were absent in all the extract/fractions of this work.

Silica gel column purifications followed by preparative thin layer chromatography led to the successful isolation of two compounds which were identified to be lupeol (30 mg) and lupeol acetate (940 mg) using ¹H NMR and ¹³C NMR and by comparing with literature data. The isolated compounds demonstrated good activity against the tested microorganism, which implies that the compounds are potential sources of antimicrobial agents against various ailments.

Recommendations

MAE with more polar solvent such as water or methanol should be carried outon the root bark of the plant with a view to isolating more potent bioactive
While in vitro assays can besensitive, quick and inexpensive, the results that are obtained may not necessarily predict in vivo activity as observed by Wright (2010), therefore, there will be a need to further screen the extracts and the isolated compounds using suitable in vivo assays.
The isolated compounds are potent bioactive compounds with characteristic sides of reaction, therefor, they can be synthetically modified with a view to improving their antibacterial activity.

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