ISOLATION, CHARACTERISATION AND BIOLOGICAL ACTIVITY OF SOME COMPOUNDS FROM RAPANEA MELANOPHLOEOS (L.) MEZ.
by
THABILE LUKHELE
Dissertation in fulfilment of the requirement for the degree
MASTER
in
CHEMISTRY
in the
FACULTY OF SCIENCE
of the
UNIVERSITY OF JOHANNESBURG
Supervisor : Prof. R.W.M. Krause Co-supervisor : Mrs D.K. Olivier
DECLARATION
I hereby declare that this dissertation, which I herewith submit for the research qualification
MASTERS DEGREE IN CHEMISTRY to the University of Johannesburg, Department of Chemical Technology, is, apart from the recognised assistance of my supervisors, my own work and has not previously been submitted by me to another institution to obtain a research diploma or degree.
______on this ____ day of ______
(Candidate)
______on this ____ day of ______
(Supervisor)
______on this ____ day of ______
(Co-supervisor)
i DEDICATION
This work is a true reflection of God‟s greatness, immeasurable love and faithfulness. It is dedicated to my family without whose support I would never have been able to complete it. Bo Mhlantiwendlunkhulu, thank you for your support, you mean the world to me. To my dearest dad; QomazithawaNgwane, Nabonyoni yasindvwa sisila kusuka esihlahleni, thank you for believing in me and always supporting me. You are my pillar of strength and my inspiration. To my lovely mom; Dlamini wekunene, Langeni lekutsenga eSwatini, a big thank you for your love, prayers and always reminding me that there is an end to every process. To my late big sister, thank you for your love. I know you would have been very proud of this work. Wherever you are, you will always be a bigger part of my life, I miss you dearly.
ii ACKNOWLEDGEMENTS
The following individuals and institutions are sincerely acknowledged for their invaluable contributions towards the success of this work:
My supervisors, Professor R.W.M Krause and Mrs D.K. Olivier for their supervision, advice and guidance throughout this work.
Professor Sandy van Vuuren for her assistance with the biological tests.
Dr Anna Moteete for helping with the deposition of herbarium specimens.
Mr Gugu Mavuso from the University of Swaziland and Ms I Johnson for their assistance with plant collection.
My friends, Bheki, Dumie, Innocent, Mfanaleni, Nikiwe, Njabu, Unathi, and Vinny for their support, love and for always offering a shoulder to lean on. From the bottom of my heart, thank you for being a part of my life, it‟s an honour and a blessing.
My colleagues, Dr Derek Tantoh Ndinteh, Mr Smart Mpofu and Xavier Siwe Noundou for being the best lab mates. I am grateful for your advice; help with some experiments and of course the crazy moments we shared. May God richly bless you and your families.
The University of Johannesburg through the Postgraduate Bursaries for financial support.
The Almighty God, for providing me with the opportunity and forever engulfing me with his love. Even when I pass through fire I know you are there and in the darkest hour you shine as bright as day because you alone are God.
iii ABSTRACT
The continued use and popularity of plant based traditional medicine necessitates scientific validation of the therapeutic potential of medicinal plants through phytochemical and pharmacological screening as well as the isolation and identification of bioactive compounds. Since the pharmacological effectiveness of medicinal plants is affected by several intrinsic and extrinsic factors, studies on the variations on chemical composition and biological activity are important as well. These provide a scientific rationale for using plants from different regions for the same medicinal purposes and allow traditional healers and consumers to make informed decisions with regard to the collection and use. Rapanea melanophloeos is a popular medicinal plant from the Myrsinaceae family widely distributed in southern Africa. It‟s bark, fruits and rarely the leaves are used traditionally for ailments ranging from stomach disorders, respiratory problems to disorders of the nervous system. Available chemical information reports on the accumulation of benzoquinones as major compounds, as well as some triterpenoid saponins and tannins. In view of the plant‟s wide distribution and medicinal use of different plant parts, this study comparatively evaluated the chemical composition of various crude extracts of the leaves, fruits and bark of plants collected from six localities. This was coupled with antibacterial tests to evaluate the therapeutic potential of different solvent extracts of the leaves, fruits and bark as well as the isolation of bioactive compounds from the fruits.
Plants were collected from six different localities between Swaziland and South Africa and sequentially extracted with petroleum ether, ethyl acetate, methanol and water as a series of increasingly polar solvents. Thin layer chromatography (TLC), Gas-Chromatography/Mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC) were used to obtain a semi-quantitative chemical composition profile of different extracts. The TLC fingerprints of petroleum ether, ethyl acetate and methanol extracts showed the accumulation of non-polar terpenes, benzoquinones, saponins, tannins and flavonoids in the three plant parts. Slight variations in the chemical composition of the leaves, bark, and fruits were noted. Some components occurred in specific plant parts and others
iv occurred in all three plants. The chemical profile of the leaves and bark were quite comparable in most instances with the fruits showing a generally different profile. This confirms previous literature reports on the comparability of leaves and the bark accounting for interchangeable use in traditional medicine.
From GC-MS analysis five compounds were observed to accumulate in the three plant parts with considerable quantitative variations and random plant to plant variations. Three of these were tentatively identified as (-)-spathulenol, caryophyllene oxide and α-cardinol. Caryophyllene oxide was identified as the major compound in the leaves and the fruits and as expected the concentrations of the five compounds were the highest in the fruits and lowest in the bark. Screening for amino acids using GC-MS provided some insight into the traditional uses of the plant for neuroactive disorders. About 29 free amino acids (standard and non- standard) were identified and quantified in all plant parts. These showed minor qualitative variations and remarkable quantitative variations between the leaves, fruits and the bark. The fruits accumulated higher amino acid concentrations as compared to the leaves and the bark. HPLC analysis as well showed minor differences in the chemical profile of the leaves, fruits and bark. Three major compounds were observed to occur in the leaves, fruits and bark with minor variations in their quantities. No distinct geographical trend was noted except for random plant to plant variations within and between populations.
Broad spectrum antibacterial activity of crude extracts of the bark, fruits, and leaves against B. cereus, E. faecalis, S. epidermidis, E. coli, K. pneumoniae and P. aeruginosa was noted. This provides an important basis for the justification of traditional medicinal uses of the plant against skin, stomach and respiratory infections. The highest activity was noted against gram positive strains with the fruit exhibiting superior activity than the bark and leaves. Medium polar compounds extractable with ethyl acetate were the most active, while the polar compounds (extractable in methanol) were somewhat surprisingly the least active. In all extracts compounds are likely to work synergistically to effect the observed activity, since no individual compound was active from the bio-autography assays.
v Although no single bioactive compound was localized from an auto-biography assay, an attempt was made to isolate some compounds from the fruits. About six compounds were isolated; two from the polar fraction and four from the non-polar fraction. Interestingly, preliminary spectroscopic analysis showed the lack of a benzoquinoid skeleton in five of the isolated compounds although benzoquinones, which are characteristic of Myrsinaceae plants were identified by TLC. From spectral analysis, one compound was successfully characterised as pentacos-4- ene-6,7-dione. Re-isolation of the remaining four compounds so as to fully resolve their structures is continuing in our lab.
Overall the study provided some justification into the traditional medicinal uses of R. melanophloeos. Although the leaves, fruits and bark showed some variations in their chemical profile, they don‟t have a pronounced effect on the antibacterial activity as all plant parts showed good antibacterial activity. Plants from different localities as well had similar chemical profiles only with minor quantitative variations and comparable antibacterial activity.
vi CONFERENCE PRESENTATIONS AND PUBLICATIONS
Some of the work in this dissertation has been presented in conferences and prepared for publication.
Conference presentations:
Lukhele T., Krause R.W.M., Olivier D.K., Isolation, Characterization and Biological Activity of Some Compounds from R. melanophloeos (L.) Mez. Oral Presentation. University of Johannesburg, Department of Botany and Plant Biotechnology, Annual Postgraduate Symposium. 22 October 2008.
Lukhele T., Krause R.W.M., Olivier D.K., Phytochemical and Biological Screening of Some Compounds from R. melanophloeos (L.) Mez. Poster Presentation. South African Chemical Institute (SACI) Annual Conference, Stellenbosch. 1-5 December 2008.
Lukhele T., Krause R.W.M., Olivier D.K., Van Vuuren S.F., A Comparative Phytochemical and Pharmacological Study of Rapanea melanophloeos (L.) Mez. Oral presentation. Indigenous Plant Use Forum (IPUF) Annual Conference Keimoes. 4-7 July 2010.
Lukhele T., Krause R.W.M., Olivier D.K., A Geographical Variation Study of Phytochemicals from Rapanea melanophloeos (L.) Mez. Oral Presentation. University of Johannesburg, Department of Botany and Plant Biotechnology, Annual Postgraduate Symposium. 26 October 2010.
Publications:
Lukhele T., Krause R.W.M., Olivier D.K., Van Vuuren S.F. (2011). Antibacterial activity of Rapanea melanophloeos (L.) Mez. (Myrsinaceae) extracts. Article currently being prepared for submission for publication in a peer reviewed journal.
vii TABLE OF CONTENTS
Section Page
Declaration ...... i
Dedication ...... ii
Acknowledgements ...... iii
Abstract ...... iv
Conferences and Publications ...... vii
Table of contents ...... viii
List of figures ...... xiii
List of tables ...... xvi
List of abbreviations ...... xviii
CHAPTER 1 ... INTRODUCTION ...... 1
1.0 The use of plants in traditional medicine ...... 1 1.1 A South African perspective ...... 2 1.2 Use of plants in drug development ...... 3 1.3 Justification of study ...... 4 1.4 Objectives of study ...... 5 1.5 Thesis outline ...... 6
CHAPTER 2 ... LITERATURE REVIEW ...... 10
2.1 Plant natural products ...... 10 2.1.1 Classes of secondary metabolites ...... 11 2.1.1.1 Phenols ...... 11 2.1.1.2 Terpenoids ...... 16 2.1.1.3 Alkaloids ...... 19 2.2 An overview of the family Myrsinaceae ...... 20 2.2.1 Rapanea melanophloeos (L.) Mez ...... 22 2.2.1.1 Botanical description ...... 22
viii 2.2.1.2 Plant Distribution ...... 23 2.2.1.3 Traditional medicinal uses ...... 25 2.2.1.4 Phytochemistry and pharmacological activity...... 25 2.3 An overview of techniques used in natural products chemistry ...... 26 2.3.1 Extraction techniques ...... 26 2.3.2 Purification techniques ...... 27 2.3.3 Chromatographic techniques ...... 27 2.3.3.1 Thin Layer Chromatography (TLC) ...... 27 2.3.3.2 Open Column Chromatography (CC)...... 29 2.3.3.3 High Pressure Liquid Chromatography (HPLC) ...... 29 2.3.3.4 Gas Chromatography-Mass Spectrometry (GC-MS) .... 30 2.3.4 Structure elucidation techniques ...... 31 2.3.4.1 Infrared Spectroscopy (IR) ...... 31 2.3.4.2 Nuclear Magnetic Resonance spectroscopy (NMR) ..... 31 2.3.4.3 Mass spectrometry...... 32
CHAPTER 3 MATERIALS AND METHODS ...... 40
3.1 Chemical variation study ...... 40 3.1.1 Plant collection ...... 40 3.1.2 Extraction of phytochemicals ...... 43 3.1.3 Extraction of Alcohol Precipitable Solids (APS) ...... 44 3.1.4 TLC screening of crude extracts ...... 44 3.1.4.1 Sample preparation...... 45 3.1.4.2 Development ...... 45 3.1.4.3 Visualisation ...... 46 3.1.5 GC-MS analysis...... 46 3.1.5.1 GC-MS analysis of PE extracts ...... 47
3.1.5.2 Screening of H2O extracts for free amino acids ...... 48
3.1.6 RP-HPLC analysis of MeOH and H2O extracts ...... 51 3.2 Antibacterial activity tests of crude extracts ...... 53 3.2.1 Determination of MIC values ...... 54 3.2.2 TLC bio-autography assay ...... 55 3.3 Bio-guided isolation of compounds from the fruits ...... 56
ix 3.3.1 Mass extraction ...... 56 3.3.2 Liquid–liquid fractionation of crude MeOH extract ...... 56 3.3.3 Fractionation of MeOH soluble fraction ...... 57 3.3.4 Antibacterial activity tests of fractions ...... 57 3.3.5 Fractionation of sub-fraction P4 ...... 58 3.3.6 CC fractionation of combined sub-fractions P5 and P6 ...... 59 3.3.6.1 Further purification of sub fractions P5 and P6...... 59 3.3.7 Fractionation of hexane soluble fraction (NP) ...... 60 3.3.7.1 Fractionation of sub-fraction NP 12-35 ...... 61 3.3.7.2 Fractionation of sub-fraction NP 62-69 ...... 62 3.4 Structure elucidation of isolated compounds ...... 63
CHAPTER 4 ... RESULTS AND DISCUSSIONS ...... 65
Introduction...... 65 4.1 Chemical variation study ...... 65 4.1.1 Extraction yields of the bark, fruit and leaf extracts ...... 66 4.1.1.1 Extraction yields of leaf samples ...... 66 4.1.1.2 Extraction yields of fruit samples ...... 68 4.1.1.3 Extraction yields of bark samples ...... 68 4.1.1.4 Geographical variation on extraction yields ...... 71 4.1.1.5 APS percentage yields ...... 72
4.1.2 TLC screening of PE, EtOAc, MeOH and H2O extracts ...... 73 4.1.2.1 TLC analysis of PE extracts ...... 74 4.1.2.2 TLC analysis of EtOAc extracts ...... 77 4.1.2.3 TLC analysis of MeOH extracts ...... 82
4.1.2.4 TLC analysis of H2O extracts ...... 85
4.1.2.5 TLC screening of H2O extracts for carbohydrates ...... 87 4.1.3 GC-MS analysis of PE extracts ...... 90
4.1.4 Screening of H2O extracts for free amino acids content ...... 94 4.1.4.1 Geographical variation on amino acid content ...... 97
4.1.5 RP-HPLC analysis of H2O and MeOH extracts ...... 98
4.1.5.1 RP-HPLC screening of H2O extracts ...... 98 4.1.5.2 RP-HPLC screening of methanol extracts ...... 100
x 4.1.6 Summary on the chemical variation study ...... 102 4.2 Antibacterial activity screening of crude extracts ...... 103 4.2.1 Determination of MIC values ...... 103 4.2.1.1 Antibacterial activity of the leaves ...... 104 4.2.1.2 Antibacterial activity of the bark ...... 104 4.2.1.3 Antibacterial activity the fruits ...... 105 4.2.1.4 Total biological activity ...... 106 4.2.1.5 Geographical variation on antibacterial activity ...... 107 4.2.2 Bio-autography assay ...... 108 4.2.3 Summary on the antibacterial activity of crude extracts ...... 109 4.3 Structure elucidation of isolated compounds ...... 111 4.3.1 Structure elucidation of compound TL 01 ...... 112 4.3.2 Structure elucidation of compound TL 02 ...... 113 4.3.3 Structure elucidation of compound TL 04 ...... 113 4.3.4 Structure elucidation of compound TL 06 ...... 114 4.3.5 Structure elucidation of compound TL 08 ...... 116
CHAPTER 5 … CONCLUSIONS AND RECOMMENDATIONS ...... 122
5.1 Conclusions ...... 122 5.2 Recommendations ...... 125
APPENDIX 1 ...... 126
APPENDIX 2...... 127
APPENDIX 3...... 131
APPENDIX 4...... 138
xi APPENDIX 5...... 141
xii LIST OF FIGURES
Figure Description Page
Figure 1.0: Examples of pharmaceutical drugs developed from plants...... 4
Figure 2.0: Phenol, parent compound of all phenolic compounds...... 12
Figure 2.1: Generic structure and examples of the different classes of flavonoids...... 13
Figure 2.3: Structure of the simplest condensed tannin...... 15
Figure 2.4: Structures of parent compounds for gallotannins and ellagitannins. .. 16
Figure 2.5: Structures of different classes of terpenoids...... 17
Figure 2.6: Structure of a steroidal saponin and a triterpenoid saponin ...... 19
Figure 2.7: Examples of alkaloids ...... 20
Figure 2.8: Examples of medicinally important Myrsinaceae plants ...... 21
Figure 2.9: Map of South Africa showing the natural distribution of R. melanophloeos...... 23
Figure 2.10: Rapanea melanophloeos leaves, fruits, flowers and bark...... 24
Figure 3.0: Map showing the KZN and SWD plant collection sites...... 41
Figure 3.1: Derivatisation reaction of amino acids...... 49
Figure 3.2: TLC plate of fractions collected from fractionation of P5 and P6...... 59
Figure 3.3: TLC plate showing compounds TL 01, TL 02 and TL◦03...... 60
Figure 3.4: TLC plate showing compounds TL 04,05,06 and 07 ...... 62
Figure 3.5: TLC plates showing the fractionation of sub-fraction NP-62-69...... 62
Figure 4.0: A comparison of mean % yields of leaf, fruit and bark extracts ...... 70
Figure 4.1a: TLC plate showing PE extracts of leaf samples from SWD plants. .. 75
Figure 4.1b: TLC plate showing PE extracts of leaf and bark samples from KZN plants ...... 75
Figure 4.1c: TLC plate showing PE extracts of bark samples from SWD plants. . 76
xiii Figure 4.1d: TLC plate showing PE extracts of fruit samples from SWD plants. .. 77
Figure 4.1e: TLC plate showing EtOAc extracts of leaf samples from SWD plants...... 78
Figure 4.1f: TLC plate showing EtOAc extracts of leaf and bark samples from KZN plants...... 79
Figure 4.1g: TLC plate showing EtOAc extracts of bark samples from SWD plants...... 80
Figure 4.1h: TLC plate showing EtOAc extracts of fruit samples from SWD plants...... 81
Figure 4.1i: TLC plate showing MeOH extracts of the leaves from SWD plants. .. 82
Figure 4.1j: TLC plate showing MeOH extracts of the leaves and bark from KZN plants...... 83
Figure 4.1k: TLC plate showing MeOH extracts of the bark from SWD plants. .... 83
Figure 4.1l: TLC plate showing MeOH extracts of fruit samples...... 84
Figure 4.1m: TLC plate showing H2O extracts of leaf samples from SWD plants. 85
Figure 4.1n: TLC plate showing H2O extracts of leaf and bark samples from KZN plants...... 86
Figure 4.1o: TLC plate showing H2O extracts of bark samples from SWD plants. 86
Figure 4.1q: TLC plate showing carbohydrates from H2O extracts of leaf samples from SWD plants...... 88
Figure 4.1r: TLC plate showing carbohydrates from H2O extracts of leaf and bark samples from KZN plants...... 88
Figure 4.1s: TLC plate showing carbohydrates from H2O extracts of bark samples from SWD plants...... 89
Figure 4.1t: TLC plate showing carbohydrates from H2O extracts of fruit samples from SD and KZN...... 89
Figure 4.2: Representative GC-MS chromatograms of the bark, fruits and leaves...... 93
xiv Figure 4.3: A comparison of the leaf mean concentrations of 14 amino acids of plants collected from four localities...... 97
Figure 4.4: Representative chromatograms of H2O extracts of the leaves, fruits and bark R. melanophloeos...... 99
Figure 4.5: UV spectra for the three major peaks from H2O extracts of the leaves, fruit and bark samples from R. melanophloeos...... 100
Figure 4.6: Representative HPLC chromatograms of MeOH extracts of the leaves, fruits and bark...... 101
Figure 4.7: UV spectra of the two major compounds observed in the MeOH extracts of the leaves, fruits and the bark...... 102
Figure 4.8: A 1,4-benzoquinone skeleton consistent with the spectral data for compound TL 01...... 113
Figure 4.9: Compound TL 06, pentacos-4-ene-6,7-dione...... 115
xv LIST OF TABLES
Table Description Page
Table 1.0: Examples of indigenous South African plants commonly used in traditional medicine...... 3
Table 2.0: Examples of different classes of phenolic compounds...... 12
Table 2.1: Examples of different classes of terpenoids., ...... 17
Table 2.2: Examples of some common medicinal Myrsinaceae plants ...... 22
Table 3.0: Approximate distance (km) between different SWD collection sites .... 41
Table 3.1: R. melanophloeos collection sites, sample names and herbarium voucher specimen numbers...... 42
Table 3.2: Mobile phases and visualizing reagents used in the TLC screening of R. melanophloeos bark, fruit and leaf crude extracts ...... 45
Table 3.3: GC-MS instrument settings for the analysis of R. melanophloeos bark, fruit and leaf samples extracted in PE...... 47
Table 3.4: GC-MS instrument settings for the screening of amino acids from H2O extracts of R. melanophloeos...... 50
Table 3.5: HPLC pump programme for the analysis of H2O and MeOH extracts . 52
Table 3.6: Representative samples screened for antibacterial activity ...... 53
Table 3.7: Human pathogenic bacterial strains used for the antibacterial activity tests for R. melanophloeos crude extracts ...... 54
Table 3.8: MIC values (mg/ cm3) of crude Hex and MeOH soluble fractions and column chromatography fractions ...... 58
Table 4.0a: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos leaf samples...... 67
Table 4.0b: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos fruit samples...... 68
xvi Table 4.0c: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos bark samples...... 69
Table 4.1: Mean percentage yields of extracts of the leaves and bark samples of plants collected from five different locations ...... 72
Table 4.2: Retention time and mass fragments of major compounds from GC-MS analysis of the PE extracts of R. melanophloeos ...... 91
Table 4.3: Occurrence of major compounds from GC-MS analysis of R. melanophloeos PE extracts of the leaves, bark and fruits...... 92
Table 4.4: Free amino acids detected in H2O extracts of the leaves, fruits and bark from R. melanophloeos...... 96
Table 4.5a: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of leaf samples...... 104
Table 4.5b: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of bark samples...... 105
Table 4.5c: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of fruit samples...... 106
Table 4.6: Total activity of R. melanophloeos bark, fruit and leaf extracts...... 107
Table 4.7: Mean MIC values (mg/ cm3) of leaf EtOAc extracts from 4 different localities...... 108
1 Table 4.8: H NMR (400 MHz, (CD3)2CO) data for TL 01 ...... 113
Table 4.9: 1H NMR (400 MHz), 13C NMR (100 MHz) and 2D NMR {1H-NMR (300 MHz) and 13C-NMR (75 MHz)} data for compound TL 06 ...... 115
xvii LIST OF ABBREVIATIONS
1D-NMR 1 Dimensional Nuclear Magnetic Resonance Spectroscopy
1H-NMR Hydrogen Nuclear Magnetic Resonance Spectroscopy
2D–NMR 2 Dimensional Nuclear Magnetic Resonance Spectroscopy
13C-NMR Carbon 13 Nuclear Magnetic Resonance Spectroscopy
AA Amino acid
ACN Acetonitrile
ACOOH Acetic acid
APS Alcohol Precipitable Solids
BuOH Butanol
CC Column Chromatography
CHCl3 Chloroform
DCM Dichloromethane
DE Diethyl ether
DMSO Dimethyl sulfoxide
ESI Electrospray Ionisation
EtOAc Ethyl acetate
EtOH Ethanol
FT-IR Fourier-Transform Infrared Spectroscopy
GC-MS Gas Chromatography Mass Spectrometry
Hex Hexane
HCOOH Formic acid
HPLC High Performance Liquid Chromatography
IS Internal Standard
KZN Kwa-Zulu Natal LC-MS Liquid Chromatography – Mass Spectrometry
xviii MeOH Methanol
MP Medium polar
MS Mass Spectrometry m/z Mass to charge ratio
NP Non polar
NMR Nuclear Magnetic Resonance Spectroscopy
PDA Photo Diode Array Detector
PE Petroleum Ether
PTLC Preparative Thin Layer Chromatography
Rf Retention factor
SD Standard
SPE Solid Phase Extraction
SWD Swaziland
TLC Thin Layer Chromatography
TMP Traditional Medical Practitioners
TOF-MS Time of Flight Mass Spectrometry
UV Ultraviolet
VS Vanillin in Sulfuric acid
(v/v) volume to volume ratio
WHO World Health Organization
xix
CHAPTER 1...
INTRODUCTION
1.0 The use of plants in traditional medicine
From time immemorial plants have been an important resource for the treatment of diseases from minor ailments to the more serious or acute diseases like cancer, malaria, tuberculosis and even HIV/AIDS. 1 Different societies from different regions of the world have systematically identified medicinal and poisonous plants forming part of their unique traditional medical systems over time. 2 Such indigenous knowledge is deeply rooted in the people‟s culture and forms part of their history being passed on orally from generation to generation. As such we can talk about Chinese traditional medicine, Arabic traditional medicine or African traditional medicine.1
Today the medicinal use of plants is still prominent in both developed and developing countries. The World Health Organization (WHO) estimates that 80 % of the world population relies (solely or partially) on traditional medicine for their primary health care.1 In developed countries such as Europe, North America and Australia, plant based medicines compliment “western” medicine which is accessible to the larger population. These are often self-administered as standardised complimentary or alternative medicines (CAM) available from retail pharmacy outlets. 3 , 4 In most developing countries however, a relatively larger portion of the population relies solely on traditional medicine for their primary health-care needs because of its easy accessibility, affordability and cultural relevance.5 In this case the traditional medicines may be self-administered but most often local traditional medical practitioners (TMP‟s) or herbalists are consulted. In those societies where traditional medicine occurs alongside modern medicine, the latter is used for acute conditions and the former for minor ailments, to reduce symptoms and for general maintenance of good health.2
1 Chapter 1: Introduction
1.1 A South African perspective
In South Africa, even though plant based traditional medicine occurs alongside conventional medicine it remains popular in both rural and urban societies. An estimated 27 million people (about 75 % of the population) rely on plant based traditional medicine for primary health-care.5 Apart from the easy accessibility and affordability of plant based medicines, the practice seems to be culturally relevant with some ailments identified as specifically requiring traditional medicine. The country boasts of a rich diversified flora of about 30 000 species which are mostly endemic and an estimated 3 000 of these are regularly used in traditional medicine.6 Table 1.0 shows some of the popular plant species commonly used in South African traditional medicine.
The continued use and escalating demand for plant based medicines has seen the emergence of a formal herbal sector and an informal plant trade industry.7,8 The formal herbal industry mainly services those with a better socio-economic standing through “over the counter” self-medication remedies or supplements processed from indigenous medicinal plants.8, 9 Stakeholders in the informal plant trade industry constitute mainly individuals who are less economically empowered. These include informal gatherers, hawkers, TMP‟s, owners of “muti” (herbal) shops and consumers of traditional medicines.7 This industry, often referred to as a „hidden economy‟, generates an annual income of about 270 million Rand through the trade of about 700 plant species. The escalating demand for plant based medicine has however, placed a challenge on plant sustainability.10 Some medicinal plants are reportedly becoming scarce due to over-exploitation and poor harvesting practices.7 Proposed initiatives to promote plant conservation include large-scale cultivation, the use of alternate plants and plant part substitution. Harvesting of the leaves, twigs and fruits in substitution of the bark and bulbs (where possible) is being promoted since their harvesting inflicts less damage. But these initiatives are met with reservations from stakeholders who always question or doubt the effectiveness of cultivated plants and alternative plants and plant parts.10
2 Chapter 1: Introduction
Table 1.0: Examples of indigenous South African plants commonly used in traditional medicine. 9
Plant species Common name Medicinal uses
Agathosoma betulina Buchu diuretic, stimulant tonic, kidney diseases
Aloe ferox Cape aloe laxative, bitter tonic, eczema, arthritis, conjunctivitis Artermisia affra African wormwood cough, cold, sore throat, influenza, headache, asthma Gunnera perpensa Wild rhubarb gynaecological problems
Siphonachilus aethiopicus African ginger malaria, thrush, fever, respiratory problems
1.2 Use of plants in drug development
Initially the medicinal use of plants was restricted to direct use as herbal mixtures, teas, concoctions, decoctions or functional foods taken for their medicinal value.3 The isolation of morphine as a pure natural product in 1826 from the opium poppy Papaver somniferum historically used as an analgesic was breaking ground to drug development from plants.11 Since then a number of pure compounds have been isolated from plants and used as prescription drugs or analogues for the development of pharmaceutical drugs. At the start of the 21st century it was estimated that 11 % of the 252 essential and basic drugs were of plant origin.12 Some examples of important plant derived pharmaceutical drugs in use today are shown in Figure 1.0 and they include morphine, codeine, atropine and quinine. The recent discovery of taxol, camptothecin, vincristine and vinblastine as anticancer drugs further demonstrates the great pharmaceutical potential of plants.13 The example on the isolation of morphine typically demonstrates the value of indigenous knowledge as a starting point for the discovery and isolation of pure bioactive compounds.1 Based on their use in folklore medicine some plants may be selected for the isolation of compounds for use as pharmaceutical drugs or analogues in their synthesis.14
3 Chapter 1: Introduction
Although drug development from plants tends to be a long, tedious and expensive process, its value surpasses other routes because plants possess an enormous potential. With an estimated 250 000 plant species worldwide and each plant producing hundreds or thousands of structurally diverse compounds, the potential of plants as sources of novel compounds cannot be matched.15 The advances in spectroscopic and chromatographic techniques used in the isolation of chemicals from plants further exuberates their pharmaceutical potential.15
OH O 3CH
O O
NH H 2 CH3 N
CH3 OH Morphine Codeine Figure 1.0: Examples of pharmaceutical drugs developed from plants.
1.3 Justification of study
The World Health Organization (WHO) estimates that 80 % of the world population relies (wholly or partially) on traditional medicine for primary health care. Consequently, it has acknowledged the significance and potential of plant based traditional medicine and considers the practice as one of the surest means to achieve total health care coverage of the world‟s population.16 However one major drawback of traditional medicine is the lack of authentication or scientific proof of effectiveness and safety. Medicinal plant research therefore, can be assumed to stem from the need to validate and improve traditional medicine as well as the discovery of new drugs and drug leads. 17 This is a multidisciplinary field encompassing different fields of knowledge such as taxonomy, botany, chemistry, pharmacology, microbiology and toxicology.12 In essence scientific validation of traditional medicine involves the identification of bioactive phytochemicals, determination of their mode of action, recommended dosages and toxicity levels.2 Since the pharmacological effectiveness of medicinal plants is affected by biochemical and external factors such as climate, geographical location, season
4 Chapter 1: Introduction
and growth conditions, studies on the variations on biological activity are also important. 18 These provide a scientific rationale for using plants from different regions for the same medicinal purposes and allow traditional healers and consumers to make informed decisions with regard to the collection and use.18
This study focuses on Rapanea melanophloeos (L.) Mez, an evergreen tree belonging to the Myrsinaceae family and used extensively in traditional medicine throughout Southern and Eastern Africa. The bark, fruits and rarely leaves are used for ailments ranging from stomach disorders, respiratory problems, fever, diabetes, cardiac disorders, muscular pain and wounds.19, 20, 21 Previous studies on the plant has resulted in the quantification of embelin (a benzoquinone) in the leaves, fruits and bark as well as the isolation of sakurasosaponin (a triterpenoid saponin) from leaf methanol extracts.22 The use of different plant parts and plants from such a wide distribution suggests the occurrence of different groups of bioactive compounds which are likely to vary considerably between populations and different plant parts.18 Thus this study aimed at investigating similarities and differences in the phytochemistry and antibacterial activity of the three different plant parts and plants from different localities. On top of that, it aimed at isolating more bioactive compounds especially from the fruits. Results from the study could add value to the limited information available with regards the phytochemistry and scientific validation of the traditional uses of R. melanophloeos and hopefully contribute to the continued search for more bioactive compounds from plants. Results from this investigation will also be used to evaluate possible substitution of the bark with the leaves or the fruits so as to promote the conservation of the plant which is prioritized for conservation in the Eastern Cape Province of South Africa.7
1.4 Objectives of study
The overall intention of the study is to provide a scientific basis for the use of different parts of R. melanophloeos collected from different geographical locations. in traditional medicine. Within this framework the following objectives were proposed:
5 Chapter 1: Introduction
To do a chemical variation study between the leaves, fruits and bark of plants collected in different localities between Swaziland and South Africa using TLC, GC-MS and HPLC.
To assess the antibacterial activity of different solvent extracts of the leaves, fruits and bark of plants collected from different locations using micro dilution methods.
To isolate some bioactive compounds from plant parts which have shown good antibacterial activity.
To characterise isolated compounds using chemical and spectroscopic techniques.
1.5 Thesis outline
In addition to the general introduction presented in this chapter, the thesis comprises of four more chapters. A brief description of what is discussed in each chapter is outlined below:
Chapter 2 (Literature Review)
This chapter presents an intensive literature survey discussing some major groups of plant natural products, some techniques used in medicinal plant research as well as a botanical description of the plant under study and related plants.
Chapter 3 (Materials and Methods)
This chapter outlines all the experimental methodology used to attain the objectives of the study. It outlines the analytical procedures, techniques and materials used for the chemical variation study of R. melanophloeos, the antibacterial activity tests and the isolation and characterisation of some major compounds from the fruits.
6 Chapter 1: Introduction
Chapter 4 (Results and Discussions)
The results on the chemical variation of R. melanophloeos, antimicrobial activity tests as well as the isolation and characterisation of some bioactive compounds are presented and discussed in detail in this chapter.
Chapter 5 (Conclusions and Recommendations)
This chapter is a summation of the conclusions and recommendations drawn from the outcomes of the study.
Appendix
Selected GC-MS and HPLC chromatograms of crude extracts, the MIC values of extracts as well as the FT-IR and NMR spectra of isolated compounds are illustrated in the appendix.
7 Chapter 1: Introduction
References
1 Gurib-Fakim A. (2006). Medicinal Plants: Traditions of Yesterday and Drugs of Tommorrow. Molecular aspects of Medicine 27: 1-93.
2 Van Wyk B–E. and Wink M. (2004). Medicinal Plants of the World. Briza Publications, Pretoria. Pg 8.
3 Heinrich M., Gibbons J.B.S., Williamson E.M., (2004). Fundamentals of Pharmacognosy and Phytotherapy. Churchill Livingstone, Spain. Pp 8-21.
4 Nyika A. (2009). The Ethics of Improving African Traditional Medical Practice: Scientific or African Traditional Research Methods? Acta Tropica 112: 32-36.
5 Fennell C.W., Lindsey K.L., McGaw L.J., Sparg S.G., Stafford G.I, Elgorashi E.E., Grace O.M., Van Staden J. (2004). Assessing African Medicinal Plants for Efficacy and Safety: Pharmacological Screening and Toxicology. Journal of Ethnopharmacology 94: 205-217.
6 Van Vuuren S.F. (2008). Antimicrobial Activity of South African Plants. Journal of Ethnopharmacology 119: 603-613.
7 Dold A.P. and Cocks M.L. (2002). The Trade in Medicinal Plants in the Eastern Cape Province South Africa. South African Journal of Science 98: 589-597.
8 Makunga N.P., Philander L.E., Smith M. (2008). Current Perspectives on an Emerging Formal Natural Products Sector in South Africa. Journal of Ethnopharmacology 119: 365-375.
9 Van Wyk B-E. (2008). A Broad Review of Commercially Important Southern African Medicinal Plants. Journal of Ethnopharmacology 119: 342-355.
10 Zschocke S., Rabe T., Taylor J.L.S., Jager A.K., Van Staden J. (2000). Plant Part Substitution – A Way to Conserve Endangered Medicinal Plants? Journal of Ethnopharmacology 71: 281-292.
8 Chapter 1: Introduction
11 Rishton G. M. (2008). Natural Products as a Robust Source of New Drugs and Drug Leads: Past Successes and Present Day Issues. American Journal of Cardiology 101: 43-49.
12 Rates S.M.K. (2001). Plants as Sources of Drugs. Toxicon 39: 603-613.
13 Raskin I., Ribnicky D.M, Slavko K. (2002). Plants and Human Health in the 21st Century. TRENDS in Biotechnology 20 (12): 522-531.
14 Jachack S.M. and Saklani A. (2007). Challenges and Opportunities in Drug Discovery from Plants. CURRENT SCIENCE 93 (9): 1251-1257.
15 Boris R.P. (1996). Natural Products Research: Perspectives from a Major Pharmaceutical Company. Journal of Ethnopharmacology 51: 29-31.
16 Rukangira E. (2001). Medicinal Plants and Traditional Medicine in Africa: Constraints and Challenges. Erboristeria Domani 8: 1-23.
17 Guza R.C. (2004). Isolation of Natural Products from Caesaria nigrescens. MSc Thesis. Virginia Polytechnic Institute and State University. Pg 1.
18 Burwa L.V. and Van Staden J. (2007). Effects of Collection Time on the Antimicrobial Activity of Harpephyllum caffrum Bark. South African Journal of Botany 73: 242-247
19 Van Wyk, B-E., Van Oudtshoorn, B., Gericke, N. (1997). Medicinal plants of South Africa. Briza Publications, Pretoria. Pg 208.
20 Watt J. (1962). The medicinal and poisonous plants of Southern and Eastern Africa. E and S Publishers, London. Pg 786.
21 Neuwinger H.D (2000). African Traditional Medicine: A Dictionary of Plant Use and Applications with Supplement Search System for Diseases. Medpharm Scientific, Germany. Pg 434.
22 Midiwo J., Yenesew A., Juma B., Derese S., Ayoo J., Aluoch A., Guchu S. (2002). Bioactive Compounds from Some Kenyan Ethnomedicinal Plants: Myrsinaceae, Polygonaceae and Psidia punculata. Phytochemistry Reviews 1: 311-323.
9
CHAPTER 2...
LITERATURE REVIEW
Introduction
This chapter presents an intensive literature survey discussing major groups of plant natural products, their biological and ecological significance in plants, their classification as well as occurrence in the family Myrsinaceae. The botanical description, medicinal uses and chemistry of R. melanophloeos and closely related plants is discussed as well. Some analytical techniques used in medicinal plant research with special emphasis on those techniques applied in this study are discussed.
2.1 Plant natural products
Plants are complex chemical factories continuously synthesizing natural products often referred to as phytochemicals. These structurally diverse compounds can be classified into two broad groups on basis of their role or functions in plants.1 Firstly there are primary metabolites, which are those compounds performing essential metabolic roles in a plant. These are directly involved in plant growth and development thus they occur in all plants in relatively large amounts.2 Examples include carbohydrates, amino acids, lipids and nucleotides. Then there are secondary metabolites, which are those compounds with no direct primary role in the growth and development of a plant, but which may play other roles such as protecting the plant from disease.3
Historically, secondary metabolites were considered as non-essential components and regarded as by-products of primary metabolism.3 Even though the exact reasons for their synthesis in plants is still being debated, there is now considerable evidence to the effect that many play a role as defence agents, as signals for pollinators and seed dispersers, and many other facets. Research has
10 Chapter 2: Literature Review
shown that secondary metabolites are often synthesized in response to attack by insects, microorganisms, herbivores and to suppress the growth of neighbouring competitor plants. Some are also produced in the form of aromas, flavours and colour to attract pollinators and seed dispersers.2,4 They are in essence important for the survival of a plant, and occur in all parts of higher plants in relatively small amounts.
Since secondary metabolites are synthesized or produced in response to some elicitors or stimulus, their occurrence in plants is considerably affected by external factors such as soil type, climate, ecology, stage of growth and taxonomy.5 The limited occurrence of some groups of secondary metabolites among taxonomic groups has been used to classify plants in a field referred to as chemotaxonomy.2 Most significantly the interest in secondary metabolites stems from their biological activity on other organisms especially animal cells.4 These are the major active ingredients of medicinal and poisonous plants. Thus their isolation, structure elucidation, chemistry, biological activity and synthesis are major focus areas for natural products chemistry and other fields like ethno-pharmacology and ethno- botany.
2.1.1 Classes of secondary metabolites
The wide structural diversity of secondary metabolites complicates their classification into distinct groups. Several classification approaches are used one of which groups them on basis of their biosynthetic pathway. Under this approach we find three major groups: the phenols, terpenoids and alkaloids.2 Examples of these major groups are discussed in the subsequent sections to give insight into the chemistry, distribution, ecological significance in plants and biological activity. Special attention is paid to those groups that occur in the family Myrsinaceae to which the plant under study belongs.
2.1.1.1 Phenols
This group of secondary metabolites is characterized by the presence of one or several hydroxyl (OH) groups attached to an aromatic ring.6 The parent compound
11 Chapter 2: Literature Review
can be assumed to be phenol, shown in Figure 2.0. Structures of phenolic compounds are very diverse and occur either as simple compounds with one aromatic ring or as complex polymers (polyphenols) with different functional groups attached. Their classification is based on the number of carbon atoms in the basic skeleton and major groups include the flavonoids, phenolic acids and tannins.6 Table 2.0 shows some examples of the different classes of phenols with their basic carbon skeleton and the number of carbons in the skeleton.
HO
phenol Figure 2.0: Phenol, parent compound of all phenolic compounds.
Table 2.0: Examples of different classes of phenolic compounds.6
No. of carbons Basic carbon skeleton Class of phenols
6 C6 simple phenols and benzoquinones
10 C6-C4 naphthaquinones
14 C6-C2-C6 anthraquinones
15 C6-C3-C6 flavonoids n (C6-C3-C6)n tannins
Phenolic compounds are widely distributed in the plant kingdom and accumulate in plants mainly as pigments responsible for the flavour and colour of different plant parts. For instance most floral pigments and the compounds responsible for the flavour of spices like ginger and cinnamon are phenolic. 7 The subsequent subsections look at the flavonoids, benzoquinones and tannins which are the major phenolic groups occurring in the Myrsinaceae family.
12 Chapter 2: Literature Review
a) Flavonoids
This is the largest and most diversified group of (poly)phenolic compounds structurally characterised by a C6-C3-C6 carbon skeleton referred to as the flavan nucleus (two benzene rings linked through a heterocyclic pyran ring). 8 These phenolic rings are referred to as A, B and C rings, respectively. Classification of flavonoids is commonly based on the oxidation or saturation of the intermediate C ring.9 Major groups include the flavonols, flavones, isoflavonoids, and cyanidins (Figure 2.1).
OH 3' 2' 4' B HO O 8 O 1' 5' 7 1 2 6' A C 6 3 5 4 OH O 4',5,7-trihydroxyflavone Generic flavonoid structure Flavone OH
OH HO O
HO O + O
OH O OH O OH quercetin daidzein Isoflavone Flavonol Anthocyanidin
Figure 2.1: Generic structure and examples of the different classes of flavonoids.
Naturally, flavonoids are ubiquitously distributed in the plant kingdom accumulating in different plant parts like the stem, fruits, seeds and flowers. Their principal function is protection against ultraviolet (UV) radiation and serving as signals to attract pollinators and seed dispersers. 10 For example anthocyanins give the attractive blue, pink, mauve, red and violet colours to flowers and fruits. Some species from Myrsinaceae family reportedly accumulate flavonols in their glycoside form but none have been reported specifically from R. melanophloeos. For instance about ten flavonol glycosides have been isolated from the leaves of
13 Chapter 2: Literature Review
Myrsine africana, a shrub native in South Africa and traditionally used for the expulsion of worms, blood purification, and skin diseases.11 The flavonoids include kaempferol, quercetin and several glycosides of myricetin.
The pharmacological properties of flavonoids include antibacterial, anti- inflammatory, antimicrobial, oestrogenic, anti-oxidant, cytotoxic and antitumor, activity.12,13 They are of also of dietary significance as most fruits, vegetables and plant based beverages like tea, coffee, and wine contain high amounts of flavonoids assumed to be responsible for their health benefits.10
b) Benzoquinones
Benzoquinones are naturally occurring quinoic compounds characterised by an aromatic ring containing two carbonyl groups (usually attached at carbon 1 and 4).1 Benzoquinones with long alkyl side chains (alkyl benzoquinones) occur in most Myrsinaceae plants as major compounds (Figure 2.2).14 These accumulate in almost all plant parts and are associated with insect anti-feedant properties.15
O
RO CH3
OH R= H (maesaquinone)
O R= -COCH3 (acetylmaesaquinone)
O O
MeO CH3 OH R
OH OH O maesanin O R= C11 H23 (embelin)
R= C13H27 (rapanone) Figure 2.2: Examples of major benzoquinones from Myrsinaceae plants.
A common benzoquinone according in most Myrsinaceae plants is embelin (2,5- dihydroxy–3-undecyl–2,5-cyclohxadiene-1,4-benzoquinone) which is known to accumulate in all plant parts often with higher quantities in the fruits. 15
14 Chapter 2: Literature Review
This compound is reported to have good medicinal properties including wound healing, antibacterial, anthelmintic and antioxidant activity. 16 , 17 , 18 , 19 Most significantly, the alkylbenzoquinones from Myrsinaceae have been used as taxonomic markers. Based on the occurrence of five alkylbenzoquinones: maesaquinone, acetylmaesaquinone, maesanin, embelin and rapanone, (Figure 2.2) commonly occurring in African Myrsinaceae plants the family has been split into two subfamilies.15 The subfamily Maesa to which the genus Maesa belongs is characterised by the occurrence of maesaquinone, acetyl maesaquinone and maesanin. While the sub family Myrsine to which three genera (Ardisia, Myrsine and Rapanea) belong is characterised by the occurrence of embelin and rapanone.
a) Tannins
These are naturally occurring polyphenolic compounds with high molecular weights and a characteristic ability to precipitate biological molecules like proteins, alkaloids, metal ions, and other macromolecules like polysaccharides.20 Based on chemical structure, they are grouped into two major groups; condensed (CT) and hydrolysable (HT) tannins. 21 Condensed tannins, often referred to as proanthocyanidins, are flavonoid polymers or oligomers consisting of flavonoid subunits.21 Figure 2.3 shows the chemical structure of a condensed tannin which is a dimer between epicatechin and catechin.
OH
OH O OH
OH OH OH O OH OH
OH OH Figure 2.3: Structure of the simplest condensed tannin.
15 Chapter 2: Literature Review
Hydrolysable tannins on the other hand have more complex structures that are derivatives of gallic acid (gallotannins) or ellagic acid (ellagitannins) esterified to glucose or a polyol residue (Figure 2.4).20
O
HO HO O O OH HO HO O OH OH
HO O gallic acid ellagic acid
Gallic acid Ellagic acid
Figure 2.4: Structures of parent compounds for gallotannins and ellagitannins.
Tannins are widespread in most plant families and occur mainly in the bark and foliage. 22 Most Myrsinaceae plants are tanniferous accumulating mostly condensed tannins and rarely hydrolysable tannins.14 In R. melanophloeos for instance, the bark contains 11 to 15 % tannins and the twigs about 4 %.23
Biologically tannins have anti-diarrheal, anti-oxidative, anticancer, wound healing and anti HIV properties.4 The ability of tannins to precipitate biological molecules is attributed to the multiple hydroxyl groups in their structures and accounts for most of their biological activities and uses in the leather tanning industry.24 However, the medicinal use of tannins has declined due to reported adverse effects like liver damage caused by high amounts of tannic acids.20
2.1.1.2 Terpenoids
The second class of secondary metabolites are the terpenoids, the largest and most diversified group of secondary metabolites structurally characterised by a basic skeleton constructed from repeating 5-carbon building units (isoprene), which are usually joined in a head to tail manner.25 Isoprene (2-methylbuta-1,3- diene) is shown in Figure 2.5, together with some examples of terpenoids.
16 Chapter 2: Literature Review
HO menthol
O
HO
OH
O
abscisic acid Abscisic acid (sesquiterpenoid) Menthol (monoterpenoid) Isoprene
Figure 2.5: Structures of different classes of terpenoids.
Even though most terpenoids have cyclic structures, some terpenoid skeletons often do not appear to be derived from isoprene on first inspection due to acid- catalysed re-arrangements during biosynthesis. For example steroids have a 27- carbon skeleton, typically violating the concept of having carbons in multiples of 5, but studies have shown their isoprene origin.5 Terpenoid classification is based on the number of isoprene units in the basic skeleton with C-5, C-10, C-15, C-20, C- 30 and C-40 skeletons being most common.26 Table 2.1 is a list of some of the major groups of terpenoids. The next subsections will be dedicated to the monoterpenoids and sesquiterpenoids as economically important groups as well as the saponins as compounds occurring in Myrsinaceae.
Table 2.1: Examples of different classes of terpenoids.6, 5
Class of terpenoids No. of carbons No. of isoprene units Example
Hemiterpenoid 5 1 isovalenic acid
Monoterpenoid 10 2 menthol, geraniol
Sesquiterpenoid 15 3 abscisic acid
Diterpenoid 20 4 taxol
Triterpenoid 30 6 oleanolic acid
Tetraterpenoid 40 8 ß-Carotene
17 Chapter 2: Literature Review
a) Monoterpenoids and sesquiterpenoids.
Sesquiterpenoids and monoterpenoids constitute plant oils commonly referred to as essential oils. Essential oils are the low molecular weight, volatile, aromatic, oily liquids synthesized by aromatic plants.27 Though they are complex mixtures with up to sixty components, they are characterised by two or three major components which occur at fairly high concentrations. These major components determine the biological activity and are usually the low molecular weight terpenes (monoterpenes and sesquiterpenes).28 Naturally in plants essential oils have a defensive role against herbivores as well as being anti-bacterial, anti-fungal, anti- viral and insecticidal activity.28 They are of economic importance in the food, pharmaceutical and cosmetic industries being explored for their fragrant and biological activity. They are widely used as additives, natural remedies, and in perfumes and make-up products. For example menthol with reported anaesthetic, and refreshing effects, is extracted from the oil of the field mint Mentha arvensis and has been used to flavour foodstuffs like sweets, tobacco and toothpaste.5 Examples of the different types of low molecular weight terpenoids of economic importance are shown in Figure 2.5.
b) Saponins
Saponins represent a distinct group of triterpenes (30 carbon compounds) with sugar moieties.29 They are characterised by their surfactant (soap like) properties and often have significant biological activity. Their structures consist of two parts: the aglycone (sugar free part) and the glycoside (the attached sugar residue). Based on the nature of the aglycone, saponins can be classified as steroidal or triterpenoid.30 Steroidal saponins have a typical tetracyclic 27-carbon skeleton and triterpenoid saponins have a pentacyclic 30-carbon skeleton. The structures of the aglycones may be complex due to the presence of a number of functional groups
(OH, -COOH, -CH3), the diversity of the sugar chain in composition, numbers, branching patterns and substitution, and stereogenic centres.31 Figure 2.6 shows examples of the two types of saponins.
18 Chapter 2: Literature Review
O
O
OH H O
H
AGlc Gal H Glc Rha O Rha 3-oxo-20,24-dammaradien-26-al 3- oxo-20, 24-dammaradiene-26-al (steroidal saponin) Sakurasosaponin (triterpenoid saponin)
Figure 2.6: Structure of a steroidal saponin and a triterpenoid saponin.
Saponins are widely distributed in the plant kingdom occurring in both wild and cultivated crops in different plant parts (flowers, leaves, roots, seeds, stems).30 From the family Myrsinaceae, some species contain relatively large amounts of triterpenoid saponins accumulating mostly in the leaves.14,23 For example sakurasosaponin (Figure 2.6), is a triterpenoid saponin with molluscidal and antifungal activity against C. cucumerinum isolated from the leaves of Rapanea melanophloeos. 32 Also plants from the genus Ardisia accumulate substantial amounts of triterpenoid saponins in their roots. 33 These have been found to possess a range of biological activity including immune-stimulatory, utero- contracting and anticancer properties with a possibility of development of pharmaceuticals from these compounds. Generally saponins exhibit a range of biological activities with haemolysis being the most general one shared by many structurally different saponins.34
2.1.1.3 Alkaloids
The third group of secondary metabolites are the alkaloids. This is a group of organic nitrogenous bases found principally in plants and to a lesser extent in microorganisms and animals.2,35 Alkaloids are distributed in about 20 % of all flowering plants and each plant species accumulates alkaloids in a unique defined pattern. Thus they are significant in chemotaxonomy as taxonomic markers.2
19 Chapter 2: Literature Review
Perhaps the most common alkaloids are the two stimulants caffeine from the coca plants and nicotine from the tobacco plant Nicotina tabacum.4 Alkaloids are known to have neuroactive properties and have been used for the treatment of ailments related to the central nervous system (CNS), malaria and cancer. But due to their toxic, narcotic and addictive nature, their use is regulated or restricted and most are rarely used in their pure form but rather semi-synthetic analogues are used.5 Examples of some of the most common alkaloids are shown in Figure 2.7. Alkaloids are generally not common in the Myrsinaceae family, and none have been reported in Rapanea.
O 3CH
CH3 3CH N N O
O N N N
CH3 CH3 Caffeine Codeine Figure 2.7: Examples of alkaloids.
2.2 An overview of the family Myrsinaceae
The plant under study belongs to the family Myrsinaceae commonly referred to as the Myrsine family. This is a relatively large family with about 1250 species spread in about 37 genera.36 In Southern Africa about seven species from four genera (Embelia, Mrysine, Maesa and Rapanea) are known. Other members of the family are distributed in temperate to tropical climates in areas such as China, Eastern Africa, Europe, India and Japan. Members of the family are mainly evergreen trees and shrubs and rarely herbaceous.36 Myrsinaceae plants generally have limited economic uses with some grown as ornamentals and a few others used in traditional medicine. Only five genera; Ardisia, Embelia, Maesa, Myrsine and Rapanea are of medicinal significance. The first four genera are the most important in African traditional medicine being used mostly as anthelmintics (for both livestock and humans) and antibacterials.14
20 Chapter 2: Literature Review
As mentioned earlier, the major compounds in Myrsinaceae plants are alkyl benzoquinones accumulating in almost all plant parts in varying concentrations. Other compounds known from members of the family are saponins, flavonoid glycosides and tannins. Typically some Myrsinaceae plants may contain crystals of calcium oxalate in their tissues. No iridoids (a group of highly oxygenated monoterpenoids with a cyclopentane skeleton) and cyanogenic glycosides (amino acid derived compounds that yield toxic hydrocyanic acids on hydrolysis) are reported from the family.14 The Myrsinaceae plants reportedly exhibit a range of biological activities including acaricidal, anthelmintic, antimicrobial, insecticidal, nematicidal and phototoxic activity.11 Figure 2.8 shows some of the medicinal plants from Myrsinaceae and Table 2.2 list some of the common Myrsinaceae plants with their traditional medicinal uses and known compounds.
Ardisia japonica Myrsine africana Maesa lanceolata Figure 2.8: Examples of medicinally important Myrsinaceae plants.
21 Chapter 2: Literature Review
Table 2.2: Examples of some common medicinal Myrsinaceae plants
Plant name Distribution Isolated/known Traditional medicinal compounds uses
Ardisia japonica China, Japan benzoquinones, respiratory problems, coumarins, saponins antidote, antioxidant, diuretic 33
Embelia ruminata Southern Africa embelin anthelmintic, general body (Ibhinini) tonic 23
Maesa lanceolata Central and benzoquinones, hepatitis, bacillary Eastern Africa triterpenoid saponins dysentery, cholera, sore throat, arthritis, tapeworm 15
Mrysine africana Southern and flavonol glycosides, gallic ringworm, skin diseases, (cape mrtyle) Eastern Africa acid, benzoquinones blood purification 11,17
2.2.1 Rapanea melanophloeos (L.) Mez
Rapanea melanophloeos is a popular medicinal plant often used for ornamental purposes perhaps appreciated for its evergreen foliage and persistent purple fruits. It is locally known as the Cape beech (English), Kaapse boukenhoute (Afrikaans), isiQwane-sehlati (Xhosa), uMaphipha (Zulu), Maphipha or Dzilidzili (siSwati), Tshididiri (Vhenda) and Mudonera (Shona).23,37,38 The English name is adapted from the European beeches commonly called „boukenhoute‟ which it resembles.39 It is one of only two species from the genus Rapanea to grow in Southern Africa. The other species is the threatened shrub Rapanea gilliana restricted in the Eastern Cape Province and having no records of traditional medicinal uses.23
2.2.1.1 Botanical description
This an evergreen tree with a variable height ranging between three and twenty meters.38,39 Morphologically the plant is characterised by mature leaves that are leathery and dull dark green. These appear darker above and paler below with distinct purplish petioles. When young, the leaves appear pale green with maroon
22 Chapter 2: Literature Review
petioles. These are about 100 mm long and occur clustered mainly at the end of branches. They are simple, oblong-lanceolate shaped.38
The flowers are very small, whitish or creamy yellow with a faint scent, and occur clustered along branchlets on knobs. Male and female flowers are borne on separate trees and appear from May to July.39 The fruits appear from August to November as a heavy cluster along the stem. They remain in the plant for many months and it is common to find flowers and fruits growing at the same time. These are small (about 8 mm in diameter) thinly fleshed, one seeded berries, green when young and purple when mature. They are edible and fed on by monkeys, baboons, birds and wild pigs. The bark is thick and grey often with small diamond shaped spots.23 Figure 2.10 shows the leaves, fruits, flowers and bark from the plant.
2.2.1.2 Plant Distribution
The plant is widely distributed throughout Southern Africa and grows in high rainfall areas on rocky cliffs and along coastal and mountain forests. It occurs from Table Mountain through the eastern parts of South Africa, Swaziland, Zimbabwe and Mozambique.23 Figure 2.9 shows the distribution of the plant in Southern Africa.
Figure 2.9: Map of South Africa showing the natural distribution of R. melanophloeos.23
23 Chapter 2: Literature Review
Young leaves, pale green with maroon Mature leaves, dull green, leathery, purple petioles petioles
Leaves and green unripe fruits clustered White flowers clustered along branchlet in along stems knobs, leaves typically clustered at end of
branchlet
Dry bark Grey thick bark with small diamond
spots Figure 2.10: Rapanea melanophloeos leaves, fruits, flowers and bark.23
24 Chapter 2: Literature Review
2.2.1.3 Traditional medicinal uses
R. melanophloeos is extensively used in traditional medicine all over Southern Africa and in some parts of Eastern Africa. The bark, fruits, and rarely roots or leaves have been used in mixtures with other plants or alone for a range of ailments associated with the stomach, respiratory tract, skin and the nervous system.14 In Southern Africa concoctions of the bark and rarely the roots have been used for fever, stomach disorders, respiratory problems, diabetes, nerve disorders, palpitations, wounds, blood purification, muscular pain, and as an anthelmintic, emetic and expectorant.23,40,41 The cleansing or purifying power of the bark makes it a very common ingredient in traditional herbal mixtures. For instance a mixture with either Pentanisia prunelloides (Rubiaceae) or Pterocelastrus echinatus (Celastraceae) is taken as a tonic, in which case R. melanophloeos is described as the “cleansing” component and the other plants are said to function as pain killers.42,43 The plant is also listed as one of the effective ethnoveterinary medicines in South Africa being used as an anthelmintic and in the treatment of “heart water” disease in a mixture with Curtisia dentata (Cornaceae).44 In Eastern Africa the edible fruits often prepared in milk or taken with thin porridge are used for the same ailments as the bark. The leaves are less popular throughout having been used as astringents in the Cape in South Africa.45
2.2.1.4 Phytochemistry and pharmacological activity.
As mentioned earlier the plant is reported to contain benzoquinones, saponins and tannins. Embelin has been identified as the major benzoquinone from R. melanophloeos, with the fruits having the highest concentrations and the leaves with lowest.15 The aqueous bark extracts have been tested against several microorganisms associated with the ailments for which the plant is used and there was no appreciable activity.46,47 Since different groups of secondary metabolites account for the therapeutic properties of plants, it could be interesting to evaluate the broad spectrum chemical profile of the different plant parts (non-polar and polar phytochemicals) and of plants from different localities to see if no other compounds can be isolated especially from the fruit and bark. The traditional
25 Chapter 2: Literature Review
medicinal uses of the plant suggest good antimicrobial activity, which could be ascertained by use of a different more sensitive assay to add to the limited pool of knowledge available on the phytochemistry and pharmacological activity of the plant.
2.3 An overview of techniques used in natural products chemistry
Plant natural products research stems from the traditional use of plants in medicine and the need to isolate small quantities of bioactive compounds from plants.48 Generally the processes involved are extraction, purification, isolation and characterisation. However, the specific aim of plant investigation determines the different types of techniques and the specific analytical pathways to be followed.48 The following sections present the different techniques used in natural product research giving insight into the different extraction techniques, chromatographic and spectroscopic techniques with special emphasis on those techniques used in this study.
2.3.1 Extraction techniques
Extraction of plant material serves to separate the compounds of interest from the insoluble cellular matrix as well as artefacts like chlorophylls, cellulose, starch, salts, and glycoproteins.49 With secondary metabolites constituting about 10 % of the total weight of plant material their extraction can be challenging and demand optimization. Ideally, a good extraction method should be simple, rapid, and give good extraction yields of the analytes of interest with minimum co extraction of impurities.48,49 While there are several methods of extraction, in this study solvent extraction was used to extract different groups of phytochemicals. This is one of the most traditional methods preferred for its simplicity, low cost and broad selectivity.50,51 In this method inorganic or organic solvents are used to dissolve the analytes from the insoluble cellular matrix. The analytes are then obtained through filtration and further concentration. To improve the efficiency, solvents of increasing polarity are used sequentially to afford the extraction of different groups of compounds.
26 Chapter 2: Literature Review
2.3.2 Purification techniques
These serve to remove the interferences (tannins, chlorophylls and waxes) from analytes as well as fractionate extracts into smaller fractions containing similar groups of compounds. 52 Liquid–liquid fractionation between two immiscible solvents is one of the most traditional purification techniques applicable for the purification of different groups of compounds.49 On an analytical scale liquid-liquid fractionation is being replaced by solid phase extraction (SPE) which is a liquid- solid extraction technique using pre packed cartridges of solid adsorbents.53 The sample is loaded onto the cartridge and either the analyte or interferences are adsorbed onto adsorbent and later eluted with a solvent selective to them. In addition to purification SPE serves as an analyte concentration step.52 In this study SPE was used in the purification of crude extracts for amino acid content analysis.
2.3.3 Chromatographic techniques
Chromatography is a separation technique in which analytes in a mixture are separated based on how they interact with two phases, the mobile and stationery phases.54 Separation is effected by differences in physical properties of analytes like molecular size, charge and solubility. Based on the mobile phase, chromatography is divided into gas and liquid chromatography. Chromatography was first discovered by Tswett in 1906 when he separated chlorophylls on paper.55 His pioneering work links the origins of chromatography to the need to study plants. Since then different types of chromatography have evolved and advanced overtime with the need to separate and purify phytochemicals. In the next section some chromatographic techniques that were used in this study are discussed.
2.3.3.1 Thin Layer Chromatography (TLC)
This is a form of chromatography in which the stationery phase is a thin layer of solid adsorbent encrusted on a solid support (glass, aluminium sheet or plastic) and the mobile phase a liquid.56 The sample mixture is spotted on the TLC plate along a straight line and developed by placing the plate in an upright position in a closed developing tank with the mobile phase (solvent). As capillary forces draw
27 Chapter 2: Literature Review
the solvent up the plate analytes separate at different rates depending on their solubility and retention by the stationary phase.56 Different spots equivalent to different analytes are characterised by their retention factor (Rf) values. This is defined as a measure of the fraction of the distance moved by an analyte in reference to the distance moved by the solvent, and is calculated as follows:
Rf = distance moved by analyte / distance moved by solvent front
TLC is one of the first chromatographic methods to be used in plant analysis with a range of applications. In this study it was used in the preliminary screening of crude plant extracts, monitoring columns during isolations, isolations and bio autographic assays. Coupled with UV detection and spray reagents, TLC gives valuable information about the types of compounds in an extract.48 It is advantageous because it‟s a simple, rapid and cost effective benchtop technique allowing the analysis of several samples in one run.55
a) Preparative Thin Layer Chromatography (PTLC)
This is a modified version of analytical TLC used in the separation and isolation of analytes from small quantities of sample. Often it is used in conjunction with column chromatography as a final purification step of relatively less complex mixtures.52 The major difference from analytical TLC is in the thickness of the plates. It uses preparative plates of about 1-2 mm in thickness allowing the application of larger volumes of sample. After development the plates are visualized under UV, and the band with the analyte of interest is scraped off together with the adsorbent using a spatula. The analyte is separated from the adsorbent by filtration and concentrate.56
b) TLC bio-autography
When TLC is combined with a biological detection method it is known as TLC bio- autography.57 This technique dates back to 1946 and has been used in bioactive guided isolation of antifungal, antibacterial, antitumor, antiprotozoal and antioxidant compounds from plants.58 It is a simple insitu method that permits the
28 Chapter 2: Literature Review
simultaneous separation of complex mixtures and the localization of active constituents on the same TLC plate.57 In direct bioautography, used in this study, the developed TLC plate is dipped in a suspension of the microorganisms grown in a proper broth and incubated in a humid environment. The microorganisms grow on the TLC plate, but in places with antimicrobial agents (compounds) inhibition zones are formed. These appear as white clear zones which can further be visualised by use of dyes or visualizing reagents. 59 TLC bioautography is advantageous especially in bio-guided isolations as it is simple, rapid and sensitive. However reproducibility is compromised by several variables such as sample solubility, medium composition, pH, and test microorganisms, making the quantitative interpretation of results difficult.59
2.3.3.2 Open Column Chromatography (CC)
This is a form of solid-liquid chromatography used for the isolation of phytochemicals.48 The sample mixture is loaded at the top of a glass column packed with an adsorbent (stationary phase) and eluted with a solvent (mobile phase). Due to differences in chemistry, analytes separate as they move down the column forming bands which are collected in small fractions. 60 These are continuously monitored with TLC and similar fractions are combined and concentrated. Depending on the complexity of the sample mixture, the column can be eluted in one of two ways. In isocratic elution a single solvent or solvent mixture is used whereas in gradient elution, a series of solvents with increasing polarity/ elution strength are used to gradually elute the column. Advantages of open column chromatography include its simplicity and low cost. However, it lacks automation, and is very tedious, labour intensive and time consuming. As analytes spend longer periods of time in the column they sometimes decompose and form artefacts which compromise separations.48
2.3.3.3 High Pressure Liquid Chromatography (HPLC)
This is an advanced form of column chromatography characterised by high pressure, narrow columns and adsorbents with small particle size accounting for
29 Chapter 2: Literature Review
improved resolution and shortened analysis time. The HPLC instrument is an automated closed system consisting of a pump system to generate pressure, narrow columns packed with small particle size adsorbents and a detector.61,49 The ability of HPLC to separate non-volatile and thermally labile compounds is a great advantage over gas chromatography allowing the analysis of a wide range of compounds of different sizes.61
In this study HPLC was used for a comparative preliminary screening of crude extracts. Coupled with UV photodiode array (PDA-UV) detection; HPLC gives a fingerprint of the chemical composition of a crude extract. The HPLC chromatogram provides information on the groups of phytochemicals present and the oxidation pattern in the case of compounds such as polyphenols.48 PDA-UV has an added advantage of detecting compounds with poor UV characteristics like terpenoids and polyketides which lack unsaturation and chromophores that give rise to characteristic UV absorption.1
2.3.3.4 Gas Chromatography-Mass Spectrometry (GC-MS)
Gas chromatography is a form of chromatography in which the stationary phase is a liquid bonded on a solid support and the mobile phase is a gas. It is used for the separation of volatile compounds.62 Analytes separate as they move along the column on basis of their boiling points and interaction with the stationary phase and the separated analyte signals are monitored by a detector.54 A GC-MS system is a Gas chromatograph system coupled to a mass spectrometer as a detector. This is a powerful tool for the simultaneous separation and identification of analytes. It is applied mainly for the quantification of plant constituents and identification by comparison of retention times and mass spectra of separated compounds with those of known compounds in spectral libraries.48 Though the application of gas chromatography is limited to volatile samples, it has an advantage of short analysis time, high sensitivity and a capability to separate closely related compounds.62
30 Chapter 2: Literature Review
2.3.4 Structure elucidation techniques
Structure elucidation involves the determination of factors such as atom number, number of bonds, configuration, and conformation of pure compounds.48 Most often the starting point is information from literature, the chemistry, and physical properties of compounds to be identified. Classical spectroscopic techniques such as UV-visible, infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy as well as mass spectrometry (MS), have always been and continue to be used for structure elucidation. Advances in technology with time have allowed the use of milligram quantities of sample, high resolution and minimised analysis times.63 The subsequent sections look at some of the structure elucidation techniques.
2.3.4.1 Infrared Spectroscopy (IR)
Infrared spectroscopy is useful in the identification of functional groups present in a compound.63 This technique is based on the absorption of electromagnetic radiation at wavelengths ranging between 4000 and 400 cm-1. At this range of wavelengths specific functional groups give characteristic vibration, bending and stretching vibrations at characteristic wavelengths. This is recorded in a spectrum by the IR instrument and gives basic information on the structure.64
2.3.4.2 Nuclear Magnetic Resonance spectroscopy (NMR)
This is the most comprehensive spectroscopic technique in organic chemistry giving detailed structural information useful for the complete identification of both simple and complex compounds.64,48 Under radiofrequency energy, nuclei with non-zero spin values (1H, 13C, 31P, 15N) in a static magnetic, field absorb energy with frequencies characteristic of the nuclei. Consequently the NMR spectrum is a plot of frequencies of absorbed energy against the peak intensities. The following are some of the most useful experiments in NMR.64
1H-NMR (proton NMR) spectra shows the chemical shift values of protons against their intensities. The chemical shift values reveal the nature of different protons in a molecule and the splitting patterns (multiplicities) give information on the number
31 Chapter 2: Literature Review
of neighbouring protons.64 Such information gives the basic framework on the structure and purity of a compound and can be useful to assign a compound into a specific class of compounds. A13C-NMR (carbon NMR) spectrum is a plot of the chemical shift of different carbon environments against their intensities. The signals normally appear as singlets due to decoupling of attached protons. It is possible to classify the carbons into primary, secondary and tertiary through an experiment called Distortionless Enhancement by Polarization Transfer (DEPT).65
Detailed structural information useful for the complete characterisation of a compound, can be obtained from two dimensional NMR experiments (2D-NMR). These experiments correlate the proton and carbon spectra giving information on which proton is attached to which carbon.65 Correlated Spectroscopy (COSY) is the most widely used 2D experiment showing the correlation of mutually coupled protons. The plot shows the 1H-NMR spectra of a compound on both axis. Correlated protons are reflected by dark spots and if one draws a diagonal line across the plot, correlations are observed which reveal mutually coupled protons and the attachment of protons to neighbouring carbons. Heteronuclear Correlation Spectroscopy (HECTOR) experiments show the correlation between carbons and protons that are directly bonded to each other. 66 Heteronuclear Multiple Bond Correlation (HMBC) experiments show the correlation of carbons and protons as far as three bonds away. A combination of the different types of 2D-NMR enables the determination of the molecular skeleton of a compound.
2.3.4.3 Mass spectrometry
In mass spectrometry a compound is ionized under high energy of a specific voltage. The resultant ions are then separated on basis of their mass to charge ratio (m/z) in a magnetic or electric field. A spectra of the separated ions is recorded which is a plot of the detected m/z ratios against their relative abundance.64 An analysis of the mass spectrum reveals the molecular weight of the compound and the fragmentation patterns of the parent compound. The fragmentation patterns put together give a molecular skeleton of the compound.66
32 Chapter 2: Literature Review
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2 Croteau R., Kutchan T.M., Lewis N.G. (2000). Natural Products (Secondary Metabolites). In Buchanan B., Gruissem W., Jones R., (Eds.) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists. Pp 1250-1318.
3 Prachersky E. and Gang D.R. (2000). Genetics and Biochemistry of Secondary Metabolites in Plants: an Evolutionary Perspective. Trends in Plant Science 5 (10): 439-445.
4 Van Wyk B-E. and Wink M. (2004). Medicinal Plants of the World, 1st Ed. Briza Publications, Pretoria. Pp 8, 371-394.
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6 Mann J., Davidson B.S., Hobbs J.B., Banthorpe D.V., Harbone J.B. (1994). Natural Products, Their Chemistry and Biological Significance. Longman group UK limited. Pp 3-54.
7 King A. and Young G. (1999). Characteristics and Occurance of Phenolic Phytochemicals. Journal of American Dietetic Association 99: 212-218.
8 Cook N.C. and Samman S. (1996). Flavonoids: Chemistry, Metabolism, Cardio protective Effects and Dietary Sources. Journal of Nutritional Biochemistry 7: 66-76.
9 De Souza L.M., Cipriani T.R., Lacomini M., Gorin P. A.J, Sassaki G.L. (2008). HPLC/ESI–MS and NMR Analysis of Flavonoids and Tannins in Bioactive Extracts from Leaves of Maytenus ilicifoli. Journal of Pharmaceutical and Biomedical Analysis 47: 59-64.
10 Peterson J. and Dwyer J. (1998). Flavonoids: Dietary Occurrence and Biochemical Activity. Nutritional Research 18 (12): 1995-2018.
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11 Arot M.L.O., Midiwo J.O., Kraus W. (1996). A Flavonol Glycoside from Myrsine africana. Phytochemistry 43: 1107-1109.
12 Dweck A.C. (2009). The Internal and External Use of Medicinal Plants. Clinics in Dermatology 27: 148-158.
13 Havsteen B.H. (2002). The Biochemistry and Medical Significance of the Flavonoids. Pharmacology and therapeutics 96: 67-202.
14 Hutchings A., Scott A.H., Lewis G., Cunningham A. (1996). Zulu Medicinal Plants: An Inventory. University of Natal Press, Scottsville 3209. Pp 227- 228.
15 Midiwo J., Yenesew A., Juma B., Derese S., Ayoo J., Aluoch A., Guchu S. (2002). Bioactive Compounds from Some Kenyan Ethnomedicinal Plants: Myrsinaceae, Polygonaceae and Psidia punculata. Phytochemistry Reviews 1: 311-323
16 Joshi R., Kamat J.P., Mukherjee T. (2007). Free Radical Scavenging Reactions and Antioxidant Activity of Embelin: Biochemical and Radiolytic Studies. Chemico-Biological Interactions 167: 125-134.
17 Choudhury P.R., Ibrahim A., Bharati H.N., Venkatasubramanian P. (2007). Quantitative Analysis of Embelin in Mrysine africana Using HPLC and HPTLC. Electronic Journal of Food and Plant Chemistry 2 (1): 20-24.
18 Krishra V., Shankamurthy K., Abdul R.B., Mankani K.L., Harish B.G., Raja Naika H. (2007). Wound Healing Activity of Embelin Isolated from the Ethanol Extract of Leaves of Embelia ribes Burm. Journal of Ethnopharmacology 109: 529-534.
19 Githiori J.B., Höglund J., Waller P.J., Baker L.R. (2003). Evaluation of Anthelmintic Properties of Extracts from Some Plants Used as Livestock Dewormers by Pastoralists and Small Holder Farmers in Kenya Against Heligmosomoides polygyrus Infections in Mice. Veterinary parasitology 118: 215- 226.
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20 Hagerman A.E. (2002). The Tannin Handbook. Downloaded from http//www.users.muohio.edu/hagermae/tannin.pdf.
21 Salminen J.P, Ossipov V., Haukioja E.P.K. (2001). Seasonal Variation in the Content of Hydrolysable Tannins in Leaves of Betula pubescens. Phytochemistry 57: 15-22.
22 Chavan U.D., Shahidi F., Naczk M. (2001). Extraction of Condensed Tannins from Beach Pea (Lathyrus maritimus L.) as Affected by Different Solvents. Food Chemistry 75: 509-512.
23 Van Wyk B-E, Van Oudtshoorn B., Gericke N. (1997). Medicinal Plants of South Africa. Briza Publications Pretoria. Pp 120, 208.
24 Schofield P., Mbugua D.M., Pell A.M. (2001). Analysis of Condensed Tannins– A Review. Animal Feed Science and Technology 91: 21-40.
25 Zwenger S. and Basu C. (2008). Plant Terpernoids: Applications and Future Potentials. Biotechnology and Biology Reviews 3 (1): 1-7.
26 Des las H.B., Rodriguez B., Bosca L., Villar A.A. (2005). Terpenoids: Sources, Structure Elucidation and Therapeutic Application in Inflammation. Current Topics in Medicinal Chemistry 3: 171-185.
27 Burt S. (2004). Essential Oils: Their Antibacterial Properties and Potential Application in Foods - A Review. International Journal of Food and Microbiology 94: 223-253.
28 Bakkali F., Averbek S., Averbek D., Idaomar M. (2008). Biological Effects of Essential Oils – A Review. Food and Chemical Toxicology 46: 446-475.
29 Francis G., Kerem Z., Makkar H.P.P., Becaker K. (2002). The Biological Action of Saponins : A Review. British Journal of Nutrition 88: 587-605.
30 Vincken J-P., Heng L., De Groot A., Gruppen H. (2007). Saponins: Classification and Occurrence in the Plant Kingdom. Phytochemistry 68: 275- 297.
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31 Oleszek W. and Bialy Z. (2002). Chromatographic Determination of Plant Saponins - An Update (2002-2005). Journal of Chromatography A 112: 78- 91.
32 Hostettmann K., Marston A., Ndjoko K., Wolfender J–L. (2000). The Potential of African Plants as Sources of Drugs. Current Organic Chemistry 4: 973-1010.
33 Kobayashi H. and De Mejía E. (2005). The Genus Ardisia: A Novel Source of Health-Promoting Compounds and Phytochemicals. Journal of Ethnopharmacology 96: 347-354.
34 Dinan L., Harmatta J., Lafant R. (2001). Chromatographic Procedures for the Isolation of Plant Steroids. Journal of Chromatography A 935: 105-123.
35 Dewick P. M. (2002). Medicinal Natural products: A Biosynthetic Approach. John Wiley and Sons LTD, England. Pg 291.
36 Dyer R.A. (1975). The Genera of Southern African Flowering Plants Volume 1 - Dicotyledons. Department of Agricultural Technical Services Botanical Research Institute. Pg 439.
37 Compton R.H. (1976). The Flora of Swaziland. Journal of South African Botany Supplimentary volume 11: 414-415.
38 Moll E. (1981). Trees of Natal. ABC Press, Cape Town. Pg 291.
39 Palmer E. and Pitman N. (1961). Trees of South Africa. A.A. Balkerma Publishers, Capetown. Pp 262-263.
40 Dlamini Z., Taxonomist based at the Malkerns Research Station in Swaziland, Personal communication, (4 April 2008).
41 Msibi K., Herbalist based at Manzini Muti Market in Swaziland. Personal communication, (27 March 2008).
42 Mavuso G., Herbalist based at the Swaziland Institute for Research in Indigenous Medici.nal Plants (SIREMIP). Personal communication (28 March 2008).
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43 Amusan O.O.G., Dlamini P.S., Msonthi J.D., Makhubu L.P. (2002). Some Herbal Remedies from the Manzini Region of Swaziland. Journal of Ethnopharmacology 79: 109-112.
44 Dold A.P. and Cocks M.L. (2001). Traditional Veterinary Medicine in Alice Street of the Eastern Cape Province, South Africa. South African Journal of Science 97: 375-379.
45 Watt J. (1962). The Medicinal and Poisonous Plants of Eastern and Sourthern Africa. E and S Publishers, London. Pp 786-788.
46 Steenkamp V., Fernandes A.C., Van Rensburg C.E.J. (2007). Antibacterial Activity of Venda Medicinal Plants. Fitoterapia 78: 561-564.
47 Steenkamp V., Fernandes A.C., Van Rensburg C.E.J. (2007). Screening of Venda Medicinal Plants Against Candida albicans. South African Journal of Botany 73: 256-258.
48 Walton N.J. and Brown D.E. (1999). Chemicals from Plants: Perspectives on Plant Secondary Products. Imperial College Press, London. Pp 1-26,91- 186, 187.
49 Stitcher O. (2008). Natural Products Isolation. Natural products Reports 25: 517-554.
50 Lijun W. and Curtis L.W. (2006). Recent Advances in Extraction of Neutraceuticals from Plants. Trends in Food Science and Technology 17: 300-312.
51 Ramanik G., Gilgenast E., Przyjazny A., and Kaminski M. (2007). Techniques of Preparing Plant Material for Chromatographic Separation and Analysis. Journal of Biophysical Methods 70: 253-261.
52 Hostettmann K., Marston A., Hostettmann M. (1997). Preparative Chromatography Techniques: Applications in Natural Products Isolation. Springer Berlin, Heidelberg. Pg 5.
53 Mendonça-Filho R.R. (2002). Bioactive Phytocompounds: New Approaches in the Phytosciences. In Ahmad I, Aqil F., Owais M., (Eds) Modern
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Phytomedicine: Turning Medicinal Plants into Drugs. Wiley-VCH Verlag. Pp 1-24.
54 Christian G.D. (2004). Analytical Chemistry. John Wiley Inc. USA. Pp 556- 558, 574, 604, 627.
55 Heinrich M., Barnes J., Gibbons S., Williamson E.M. (2004) Fundamentals of Pharmacognosy and Phytotherapy. Churchill Livingstone Spain. Pp 3-24, 170-184.
56 Fried B. and Sherma J. (1986). Thin-Layer Chromatography, Techniques and Applications, 2nd ed. Marcel Dekker, INC, New York. Pp 1;4;116;186;136.
57 Marston A. (2010) Thin Layer Chromatography with Biological Detection. Journal of Chromatography A (Article in Press).
58 Die H.N. (2005). Bioactivities and Chemical Constituents of a Vietnamese Medicinal Plant, Jasminium subtriplinerve blume (Che Vang). MSc Thesis. Roskilde University, Denmark. Pg 24.
59 Choma I.M. and Grzelak E.M. (2010). Bioautography Detection in Thin layer chromatography. Journal of Chromatography A, (Article in Press).
60 Gilbert J.C. and Martin S.F. (1998). Experimental Organic Chemistry, A Miniscale and Microscale Approach. Saunders College Publishers, Fortworth. Pp 100-103.
61 Meyer V.R. (1994). Practical High Perfomance Liquid Chromatography. John Wiley and Sons. Pp 1-14, 45.
62 McMaster C. and McMaster M. (1998). GC-MS: A Practical Users Guide. John Wiley and Sons Inc. New York. Pp 3-31.
63 Cordell G.C. (1995). Changing Strategies in Natural Products Chemistry. Phytochemistry 40 (6):1585-1612.
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64 Silverstein R.M., Webster F.X., Kiemle D.J. (2005). Spectrometric Identification of Organic Compounds. John Wiley and Sons Inc. USA. Pp 1, 72,127, 204, 245.
65 Nadja B-W., Till K., Detlef M., Oliver Z. (2005). Strategies and Tools for Structure Determination of Natural Products Using Modern Methods of NMR Spectroscopy. Chemistry and Biodiversity 2: 147-175.
66 Field L. D., Ternhell S., Kalman J.R. (2002). Organic Structures from Spectra. 3rd Edition. John Wiley and Sons England. Pp 5-15.
39
CHAPTER 3
MATERIALS AND METHODS
Introduction
This chapter outlines all the experimental methods, procedures, techniques and materials used for chemical variation study on R. melanophloeos, antibacterial activity assays as well as the isolation and characterisation of major compounds from the fruits.
3.1 Chemical variation study
The chemical variation study aimed at comparing the chemical composition of the leaves fruits and bark from R. melanophloeos plants collected from six different localities. Crude extracts of varying polarities were phytochemically screened by TLC, GC-MS and HPLC. Procedures for plant collection, solvent extraction as well as TLC, GC-MS and HPLC analysis are outlined in the next sections.
3.1.1 Plant collection
Plant material was collected from naturally growing mature plants from Swaziland (SWD) and Kwa-Zulu Natal (KZN) Province in the Republic of South Africa. In Swaziland, plant material was collected from 19 plants distributed in five different sites along the western part of the country separated by an approximate distance ranging between 25 and 120 km. From each location, a minimum of three plants were harvested with the distance between plants ranging between 5 and 10 m. At the time of collection all plants were either flowering or in fruit and the leaves, bark and fruits (where available) were collected from all plants. Plants were identified by Mr Zachariah Dlamini, a taxonomist based at the Malkerns Research Station and herbarium specimens were prepared and deposited at the University of
40 Chapter 3: Experimental Methodology
Johannesburg Herbarium (JRAU). Table 3.0 shows the names of the five SWD collection sites and the approximate distances between them.
In KZN plants were harvested at Karkloof, and at the time of collection plants were at the vegetative stage of growth with no fruits or flowers. The leaves and bark were harvested from three plants and herbarium specimens were prepared and deposited at the Pietermaritzburg Botanical Gardens Herbarium. Figure 3.0 is a map showing the KZN and SWD collection sites in relation to each other and Table 3.1 shows the plant collection sites, assigned sample names and herbarium voucher specimen numbers for all samples.
Table 3.0: Approximate distance (km) between different SWD collection sites
Locality Approximate distance (km)
Nhlangano Bhunya Fonteyn Lundzi
Sicunusa (A) 90 50 100 70
Nhlangano (B) 100 130 110
Bhunya (C) 70 30
Fonteyn (D) 40
KZN
Figure 3.0: Map showing the KZN and SWD plant collection sites.
41 Chapter 3: Experimental Methodology
Table 3.1: R. melanophloeos collection sites, sample names and herbarium voucher specimen numbers.
Locality name GPS co-ordinates Plant Plant parts Herbarium voucher and region and elevation name collected* specimen number
A1 L, F ,B T Lukhele 01 26° 50.771 S Sicunusa (A) ° L, B T Lukhele 16 030 57.783 E A2 L, B T Lukhele 17 Manzini 1300 m A3
B1 L, B T Lukhele 02 26° 58.368 S Nhlangano (B) ° B2 L, B, F T Lukhele 03 030 17.290 E Shiselweni B3 L, B, F T Lukhele 14 1053 m B4 L, B T Lukhele 15
B5 L, B, F T Lukhele 18
B6 L, B, F T Lukhele 19
C1 L, B T Lukhele 04 26° 31.303 S Bhunya (C) ° C2 L, B T Lukhele 05 030 59.187 E Manzini C3 L, B T Lukhele 06 1069 m C4 L, B T Lukhele 07
D1 L, B, F T Lukhele 09 26° 18.499 S Fonteyn (D) ° D2 L, B T Lukhele 10 031 09.803 E Hhohho D3 L, B T Lukhele 11 1333 m D4 L, B T Lukhele 12
D5 L, B,F T Lukhele 13 26° 26.125 S Lundzi (E) 030° 57.324 E E1 L, B T Lukhele 08 Manzini 1505 m
F1 L,B I. Johnson 1214 29° 31.755 S Karkloof (F) F2 L, B I. Johnson 1215 030° 25.618 E KZN L, B I. Johnson 1216 1362 m F3 *Plant part collected: B=bark, F= fruit, L= leaves
42 Chapter 3: Experimental Methodology
3.1.2 Extraction of phytochemicals
After collection, all plant material was dried to constant weight at room temperature in open air in the laboratory away from direct sunlight. The dry plant material was then ground to a fine powder using a Warring commercial blender (Model 32BL79, Dynamics Corporation USA). The powdered plant material was stored in tightly closed glass bottles in the dark at room temperature. The drying of plant material makes handling, working on and storing of plant material much easier. It also improves extraction efficiency as some membranes of some organelles containing phytochemicals are destroyed during drying. However, labile or volatile compounds can be lost and some undesirable artefacts may be formed so caution is taken to dry plant material at ambient temperatures away from direct sunlight. To avoid the formation of mould plant material is preferably dried in open air and turned over periodically.1
The dry powdered plant material was sequentially extracted with solvents of increasing polarity: petroleum ether (PE), ethyl acetate (EtOAc), methanol (MeOH) and water (H2O). Ten grams (10 g) of each of the leaf, fruit and bark samples was extracted sequentially with 100 cm3 of each of the four solvents. The mixtures were shaken gently in a mechanical shaker for about one hour to increase extraction efficiency and left to stand at room temperature for 24 hours. Extraction solutions were filtered through filter paper onto pre-weighed flasks and organic solvents were removed through evaporation under a stream of air at room temperature in the fume hood. Water was removed through freeze drying in a WirTis freeze dryer. Dry extracts were weighed and the extraction yields (w/w) calculated as follows:
Percentage yield = (weight dry extract / weight dry material) x 100
The extraction afforded four different types of crude extracts (PE, EtOAc, MeOH and H2O) for each of the bark, fruits and leaf samples. Dry extracts were kept in the refrigerator in tightly closed vials and used for the TLC and HPLC analysis. The percentage yields of all samples are presented and discussed in Chapter 4.
43 Chapter 3: Experimental Methodology
3.1.3 Extraction of Alcohol Precipitable Solids (APS)
Alcohol precipitable solids are water soluble macromolecules that precipitate on treatment with an alcohol. These are mainly highly acidic polysaccharides and glycoproteins often associated with antidiabetic and wound healing properties.2 The APS‟s were extracted from all the leaf, fruit and bark samples as follows: One gram (1 g) powdered dry plant material was soaked in 10 cm3 boiling water and allowed to stand for 24 hours at room temperature. The solution was filtered through cotton wool in a syringe and the filtrate treated with 96 % rectified ethanol to make up a 50 % (v/v) ethanolic solution. This solution was left to stand at room temperature for one hour as the APS‟s precipitated, after which it was centrifuged at 4100 rpm for 20 minutes and decanted to separate the precipitate from the supernatant. The precipitate was collected and left under the fume-hood under a stream of air to dry. The supernatant was taken for a second extraction following the same procedure as outlined above. The two precipitates were combined, weighed and the % APS calculated using the formula below. Results for the APS extraction are presented and discussed in Chapter 4.
%APS = (weight dry APS extract / weight dry plant material) x 100
3.1.4 TLC screening of crude extracts
The PE, EtOAc, MeOH and H2O extracts were subjected to TLC analysis to screen for the presence of different groups of phytochemicals in the leaves, fruits and bark from all plant samples. PE extracts were used to screen for the presence of non polar compounds like monoterpenoids, sesquiterpenoids and fatty acids. EtOAc extracts were screened for the presence of medium polar compounds like benzoquinones, diterpenoids, saponin aglycones and sugar free flavonoids. MeOH extracts were used to screen for the presence of polar compounds and H2O extracts were used to screen for the very polar compounds, and carbohydrates.
44 Chapter 3: Experimental Methodology
3.1.4.1 Sample preparation
The dry extracts were dissolved/reconstituted in different organic solvents to make up a final concentration of 10 mg/ cm3. PE extracts were reconstituted in n- hexane, EtOAc extracts were reconstituted in 50 % (v/v) EtOAc in ethanol (EtOH),
MeOH extracts were reconstituted in MeOH and H2O extracts were reconstituted 50 % (v/v) aqueous MeOH. To ensure homogeneity all extracts were mixed gently in a vortex.
3.1.4.2 Development
Preliminary screening tests were performed to identify optimum solvent systems for use as mobile phases. Common solvent systems for specific groups of phytochemicals previously used in our lab were tried and modified accordingly to give optimum separation for PE, EtOAc, MeOH and H2O extracts. The different mobile phases and visualising reagents used for the different extracts are presented in Table 3.2.
Table 3.2: Mobile phases and visualizing reagents used in the TLC screening of R. melanophloeos bark, fruit and leaf crude extracts
Extract Groups of Mobile phase Visualizing compounds screened reagent
PE Non polar (NP) Hex–DE (3:2) Vanillin in H2SO4
EtOAc Medium polar (MP) Hex–EtOAc (2:3) Vanillin in H2SO4
MeOH Polar (P) BuOH–H2O–ACOOH (4:1:1) Vanillin in H2SO4
H2O Very polar (VP) EtOAc–HCOOH–ACOOH-H2O Vanillin in H2SO4 (100:11:11:26)
H2O Carbohydrates EtOAc–ACOOH-MeOH (60:15:15:10) Chromic acid
45 Chapter 3: Experimental Methodology
Samples were spotted on either 10 x 10 cm or 20 x 10 cm aluminium backed TLC plates (Machenery Nagel silica gel 60 F254). For all samples a 15 µL volume (in 5 µL aliquots) was spotted using a micropipette. For each of the different extracts
(PE, EtOAc, MeOH and H2O) leaf, fruit and bark samples were spotted on separate plates. In each plate samples were spotted 1 cm apart and developed for a distance of 8 cm in a closed glass development tank saturated with the relevant mobile phase.
3.1.4.3 Visualisation
The developed chromatograms were air dried at room temperature and visualized under UV light (Camac universal UV lamp) at 254 nm and 366 nm to detect UV visible compounds. These were later chemically visualized by dipping in the relevant visualising reagent (VR). The visualising reagent was poured into a rectangular glass container and the plates were dipped for several seconds and removed slowly to drain excess reagent. Dipping is preferred over spraying because it ensures uniform or even distribution of the reagent and is less dangerous especially when working with carcinogenic or corrosive reagents. It is also cost effective because the reagent can be used over and over again as long as it is filtered before use.3 Visualised plates were baked at 110 °C in an oven for about five minutes to enhance colour development. After visualization the different compounds depicted by different coloured spots were noted and their Rf values calculated. Visualized plates were documented by scanning as a coloured picture. The procedure for preparing visualising reagents is summarized in Appendix 1.
3.1.5 GC-MS analysis.
GC-MS is a powerful tool used for the concurrent separation, identification and quantification of volatile compounds.4 As part of the chemical variation study on R. melanophloeos, GC-MS was used to compare the chemical profile of the bark, fruit, and leaf samples of plants from the different localities. Petroleum ether extracts were screened for the presence of non-polar compounds and water
46 Chapter 3: Experimental Methodology
extracts were screened for the presence of free amino acids using the same technique.
3.1.5.1 GC-MS analysis of PE extracts
Because non polar compounds tend to be volatile, a fresh batch of PE extracts was prepared for the GC-MS analysis to ensure extract stability. These were prepared by soaking two grams (2 g) of each of the leaf, fruit and bark samples in 20 cm3 of PE. The solutions were shaken gently in a mechanical shaker for one hour, allowed to stand at room temperature for 24 hours, filtered through filter paper and allowed to dry overnight at room temperature. The dry extracts were kept in tightly closed vials and used within three days. The dry PE extracts of the three different plant parts were re-dissolved in 5 cm3 spectroscopic grade n- hexane, mixed gently with a vortex and filtered through a 0.45 µm nylon filter. A 1.5 cm3 volume of each sample solution was transferred into a GC sample vial for analysis in a Varian 3800 Capillary GC coupled to a Saturn 2000 MS. The instrument settings for analysis are shown in Table 3.3.
Table 3.3: GC-MS instrument settings for the analysis of R. melanophloeos bark, fruit and leaf samples extracted in PE.
Column type TRX-5MS (5% diphenyl-95 % dimethylpolysiloxane),
30 m x 0.25 mm
Injection temperature 60 °C
Injection volume 1.0 μL (2 injections per sample)
Carrier gas Helium
Column flow 1.0 cm3/ minute
Oven programme 50 °C for 1 minute increased to 210 °C at 4 °C/ minute
then increased to 300 °C at 45 °C/ minute
Scanning mode EI full mode (35-600 m/z)
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3.1.5.2 Screening of H2O extracts for free amino acids
For this analysis a fresh batch of water extracts of the leaves, fruits and bark were prepared as follows: Dry powdered plant material (0.3 g) was soaked in 3 cm3 boiling water, and the solutions shaken gently and allowed to stand at room temperature for 24 hrs. The solutions were very slimy and could not be filtered through filter paper so a piece of cotton wool stuffed in a syringe was used. The filtrates were freeze-dried in a Virtis freeze dryer to remove the water. The dry extracts were kept in the freezer in tightly closed vials until analysis time. Before analysis, amino acids were extracted by solid phase extraction (SPE) and derivatised through a procedure adapted from an EZ-faast amino acid analysis and derivatisation kit from Phenomenex. A brief discussion of SPE and derivatisation precedes the overall sample preparation procedure.
a) Solid phase extraction (SPE)
SPE is a purification technique or analyte enrichment step which serves to simultaneously increase the concentrations of analytes above detectable limits and remove interferences. 5 SPE uses pre-packed adsorbent cartridges onto which the sample is loaded. Either the analyte or interferences are adsorbed onto the adsorbent and later eluted with a selective solvent. The water extracts, reconstituted in 50 % (v/v) aqueous methanol were loaded into an SPE cartridge with an amino acid selective adsorbent. The amino acids were adsorbed onto the adsorbent and interferences like sugars, acids and some of the very polar phytochemicals remained un adsorbed. The non-retained interferences were then washed with n-propanol and the amino acids were eluted together with the adsorbent. These were later extracted from the adsorbent with iso-octane.
b) Derivatisation
Derivatisation is defined as the chemical conversion of a compound into its derivatives that differ in character and chemical properties.6 The basic requirement for a compound to be analysed on GC is its volatility and amino acids being polar and quite large molecules they are not volatile.4 So, before analysis they are
48 Chapter 3: Experimental Methodology
converted to volatile derivatives through chemical derivatisation. Several derivatisation agents for amino acids are available and for this work an alkyl chloroformate agent was used. The derivatisation reaction is illustrated in Figure 3.1 below.
R O
3CH O Cl O OR' catalyst + 2HCl + CO2 + NH OR' NH OH 2 OR'
Amino acid O Derivatisation reagent Amino acid derivative
Figure 3.1: Derivatisation reaction of amino acids.
c) Sample preparation
The whole sample preparation procedure (extraction, derivatisation and concentration) as adapted from the EZ-faast amino acid analysis and derivatization kit from Phenomenex is outlined below:
Dry aqueous extracts were reconstituted in 1 cm3 of 50 % (v/v) aqueous methanol and mixed gently in a vortex. A 100 µL volume of 0.2 Mm norvaline (internal standard) was added to the sample mixture and mixed in a vortex. The sample solution was loaded onto an SPE sorbent tip to extract amino acids. The amino acids adsorbed on the adsorbent and interferences were washed with 200 µL of n- propanol. The amino acids together with the sorbent tip adsorbent were eluted with 200 µL of a mixture of sodium hydroxide and n-propanol (3:2 v/v). The amino acid solution was then derivatized by adding a 50 µL chloroform solution containing the derivatizing agent (alkyl chloroformate). The mixture was vortexed gently and allowed to stand for one minute to allow the derivatisation reaction to continue to completion. A 100 µL of iso-octane was added to the amino acid solution to extract the derivatized amino acids. A two layer solution formed from the extraction step. The upper organic layer containing the amino acids was transferred into a labelled vial using a micropipette and dried under a gently
49 Chapter 3: Experimental Methodology
stream of nitrogen. The dry amino acid mixture was re-dissolved in a mixture of chloroform and iso-octane (2:4 v/v) taken for analysis. Analysis were performed in a Varian GC system with auto sampler and coupled to a Time of Flight Mass Spectrometer (HT TOF MS) and a ChromaTOF workstation for data capturing and processing. The GC-MS instrument settings are presented in Table 3.4.
d) Internal standard
A 100 µL of Norvaline was added to all samples and calibration standards as an internal standard (IS). Often in GC-MS analysis, an equal volume of internal standard is added to all samples and standards to compensate for variations resulting from human error during sample preparation. Ideally, an internal standard is a solute with retention time close to that of the analyte.7
Table 3.4: GC-MS instrument settings for the screening of amino acids from
H2O extracts of R. melanophloeos.
Column type ZB-AAA (10 m X 0.25 mm) Amino Acid Analysis GC Column from Phenomenex ®
Injection temperature Split 1:15 @ 250 °C
Injection volume 2 µL (2 injections per sample)
Carrier gas Helium
Column flow rate 1.1 cm3/ min
Oven program 30 °C/ min from 110 to 320 °C
MS source temperature 240 °C
Scan range 45-450 m/z
Sampling rate 22 (3.5 scans/ s)
50 Chapter 3: Experimental Methodology
e) Calibration standards
The analysis kit included three amino acid standard mixtures: SD1 containing 23 amino acids each 200 µL in volume, SD2 containing complementary amino acids un stable in acidic conditions and SD3 containing complimentary urine amino acids. These were mixed in different proportions to make calibration standards for quantification following the same preparation procedure used for the aqueous extract samples. Three calibration levels of standards ranging from 50 nmole/ cm3 to 200 nmole/ cm3 were prepared as follows:
Calibration level I (50 nmole/ cm3) – mix 25 µL SD1, plus 25 µL of SD2 and 25 µL of SD3 solution and 100 µL of internal standard.
Calibration level II (100 nmole/ cm3) – Mix 50 µL of each of the three SD solutions and add 100 µL of the internal standard.
Calibration level III (150 nmole/ cm3) – Mix 100 µL of each of the three SD solutions and add 100 µL of the internal standard.
e) Quantification
Compound identification was achieved by spectral matching using an EZ-Agilent amino acid library. A processing method was developed in the GC-MS system and used for the quantification of data. Calibration curves were obtained by a plot of the peak area and concentration of the identified compounds. Results for the amino acid analysis are presented and discussed in Chapter 4.
3.1.6 RP-HPLC analysis of MeOH and H2O extracts
Methanol and water extracts of the bark, fruits and leaves of plants from the six different localities were analysed in HPLC to investigate any similarities and variations between plant parts and between plants from different localities. RP- HPLC is a type of liquid chromatography in which the stationary phase is less polar than the mobile phase. 8 Coupled with UV photodiode array (PDA-UV)
51 Chapter 3: Experimental Methodology
detection, it gives information on the groups of phytochemicals present in extracts and in the case of compounds such as polyphenols, the oxidation pattern.9
Before analysis, methanol extracts were redissolved in 5 cm3 chromatographic grade methanol from Chromasolv and water extracts were redissolved in 3 cm3 of 50 % (v/v) aqueous methanol. All sample solutions were mixed gently in a vortex and filtered through a 0.45 μm nylon syringe filter membrane and transferred to auto sampler vials. HPLC analysis of all the samples was carried out on a Waters 600E HPLC system (Millipore) equipped with a Photodiode Array Detector (PDA). A Luna C18 column (150 mm x 4.60 mm and a 5 μm stationary phase) from Phenomenex was used. The flow rate was kept constant at 1 cm3/ min and the injection volume was 20 μL. Chromatographic grade solvents were used to prepare two mobile phase systems used for gradient elution. Mobile phase A was 100 % acetonitrile (ACN) and mobile phase B was 10 % ACN in 1 % aqueous acetic acid. All mobile phase systems were filtered through a 0.22 µm nylon filter membrane and degassed by sonication before use. The pump programme identified to give better separation is shown in Table 3.5.
Table 3.5: HPLC pump programme for the analysis of H2O and MeOH extracts
Time (minutes) Proportion of solvent (%)
A ( 100 % ACN) B (10 % aq. ACN, 1 % ACOOH)
0 10 90
10 10 90
25 0 100
27 0 100
32 10 90
34 10 90
To monitor the system pressure and avoid sample carry over, acetone was run before and between samples. The run time was 35 minutes, and each sample was
52 Chapter 3: Experimental Methodology
injected twice. Results for the HPLC analysis are presented and discussed in Chapter 4.1.4.
3.2 Antibacterial activity tests of crude extracts
The crude PE, EtOAc and MeOH extracts of the leaf, fruit and bark were subjected to antibacterial activity assays. Minimum Inhibitory Concentrations (MIC) were determined as an evaluation of antibacterial activity and a TLC bio-autography assay was used to localize individual bioactive compounds. The water extracts were not screened because their chemical profile is similar to that of methanol extracts. Representative plants from the six localities were screened and these are shown in Table 3.6. On selecting plants for analysis priority was placed on plants with the leaves, bark and fruits collected.
Table 3.6: Representative samples screened for antibacterial activity
Locality No. of samples Sample Name Part screened* screened
Sicunusa (A) 1 A1 B, F, L
Nhlangano (B) 4 B3 B, F, L
B4 B, L
B5 F
B6 F
Bhunya (C) 2 C2, B, L
C3 B, L
Fonteyn (D) 2 D1 B, F, L,
D5 B, L
Lundzi (E) 1 E1 B, L
KZN (F) 2 F1 B, L
F2 B, L *Plant part tested, B = bark, F= fruits, L= leaves
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3.2.1 Determination of MIC values
Antibacterial activity tests were done in the microbiology lab of the University of Witwatersrand Medical School. A fresh batch of crude extracts was prepared as discussed in Section 3.1.2 and extracts were analyzed within a week of extraction to maintain extract stability. The samples were tested against six bacterial strains (gram positive and gram negative) associated with gastrointestinal, skin and respiratory problems. These are shown in Table 3.7 with their reference strain numbers and the ailments they are commonly associated with.
Table 3.7: Human pathogenic bacterial strains used for the antibacterial activity tests for R. melanophloeos crude extracts
Test microorganism Reference strain Gram +ve Pathogen primarily number* associated with infections or –ve related to:
Klebsiella pneumoniae ATCC 13883 Gram –ve Respiratory tract
Escherichia coli ATCC 8739 Gram –ve Gastro intestinal tract
Pseudomonas ATCC 9027 Gram –ve Wounds aeruginosa
Enterococcus faecalis ATCC 29212 Gram +ve Respiratory tract
Bacillus cereus ATCC 11778 Gram +ve Gastro intestinal tract
Staphylococcus ATCC 2223 Gram +ve Skin epidermidis
*Reference strain number, ATCC = American Type Culture Collection
The minimum inhibitory concentration (MIC) values of extracts were determined using the serial dilutions microplate assay.10 First the dry extracts were weighed and reconstituted in a relevant solvent to make up a starting concentration of 32 mg/ cm3. The PE and EtOAc extracts were reconstituted in sterile acetone and MeOH extracts were dissolved in sterile water. Serial dilutions of extracts were carried out aseptically in 96-well microtitre plates on a laminar flow bench. First,
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100 µL of sterile water was added to all the wells using a multichannel micropipette. Then a 100 µL of plant extract was added to each of the wells in the first row and mixed with the water. An eight fold serial dilution was achieved by transferring a 100 µL of the extract solution from the first row into the second row, mixing thoroughly and taking a 100 µL into the next row and so on until the eighth row. The serial dilutions achieved a maximum concentration of 16 mg/ cm-3 and a 3 minimum concentration of 0.125 mg/ cm .
The test pathogens grown overnight were diluted in Tryptone Soya Broth (TSB) at a ratio of 1:100, yielding an approximate inoculum size of 1 x 108 colony forming units (CFU)/ cm3. A 100 µL aliquot of the culture was added to each of the wells under ambient laboratory conditions. The plates were tightly sealed with sterile seal plate films and incubated at 37 °C overnight to stimulate bacterial growth. A 40 µL p-iodonitrotetrazolium violet (INT) (0.2 mg/ cm3) solution was added to the plates after incubation and left to stand at room temperature for six hours. INT is reduced by living cells to a red coloured complex. Consequently, bacterial growth is indicated by the appearance of a red/pink colour in the wells.10 The MIC was taken to be the last clear well in each row. Acetone and DMSO in water were used as negative controls and ciprofloxacin (0.01 µg/ cm3) as the positive control. The controls were prepared the exact same way as the samples. MIC‟s were determined at least in duplicate for all samples and controls to ensure that a standard error of not more than one dilution factor is obtained. Extracts which showed activity at the lowest concentration were further diluted to an even lower starting concentration of 5, 3.2 or 0.5 mg/ cm3.
3.2.2 TLC bio-autography assay
To localize individual bioactive compounds, the crude extracts were subjected to bio autography against Bacillus cereus as this was one of the most susceptible pathogens. The crude PE, EtOAc and MeOH extracts of the leaves, fruits and bark were (15 μL) spotted separately onto aluminium backed TLC plates (Machenery
Nagel silica gel 60 F254) and developed in the optimum solvent systems identified in Section 3.1.4. The chromatograms were first sterilized under UV light for three
55 Chapter 3: Experimental Methodology
hours and then placed on square petri dishes with covers. Agar mixed with the bacterial suspension to a final inoculum size of 1 x 108 colony forming units (CFU)/ cm3 was distributed over the developed TLC plates. After solidification of the media, the plates were kept in the refrigerator for one hour to enable better diffusion of compounds, and later incubated at 37 °C and 100 % humidity for 24 hours. During incubation microorganisms grow on the TLC plate, and in places with antimicrobial agents (compounds) growth is inhibited. These appear as clear white zones. The results for the bio-autography assays are presented and discussed in Chapter 4.
3.3 Bio-guided isolation of compounds from the fruits
3.3.1 Mass extraction
Since the fruits had shown superior activity over the leaves and bark, an attempt was made to isolate compounds from them. Methanol was used to try and extract a wide range of different groups of compounds as all extracts had shown appreciable activity and no individual compound was localized as being active. Powdered plant material collected from six plants (125 g) was combined and extracted with 1000 cm3 methanol at room temperature for 72 hours. The filtrate collected from filter paper (Whatman no.1), was dried under reduced pressure in a rotary evaporator at 40 °C yielding 25 g (about 20 %) of a sticky red-brown extract.
3.3.2 Liquid–liquid fractionation of crude MeOH extract
This crude methanol extract was subjected to liquid–liquid fractionation between hexane and methanol in a separation funnel affording two fractions, a non-polar hexane soluble (NP) and polar methanol soluble fraction (P). From both fractions the solvent was removed in a rotary evaporator at 40 °C and extracts dried completely under the fumehood. Both fractions were then subjected to extensive TLC analysis and column chromatography as outlined below:
56 Chapter 3: Experimental Methodology
3.3.3 Fractionation of MeOH soluble fraction
After intensive TLC analysis to identify an optimum solvent system giving good separation of compounds, the methanol soluble fraction (18 g) was prepared for column chromatography. This was dissolved in a minimum amount of methanol and mixed with silica gel to make a paste, dried completely in the fume hood and loaded on an evenly packed silica gel column. The column was eluted gradually with a mixture of dichloromethane (DCM) and methanol in increasing polarity from 100 % DCM, to 100 % MeOH. A hundred and ninety-five (195) fractions (50 cm3) were collected and pooled to 8 major sub-fractions (P1-P8) on basis of their TLC profiles. These fractions were taken for antibacterial activity tests to identify the most active subfraction to be considered for further fractionation.
3.3.4 Antibacterial activity tests of fractions
The eight subfractions from column chromatography of the polar fraction together with the crude fractions NP and P were taken for antibacterial tests to identify bioactive fractions to be considered for further fractionation. A starting concentration of 16 mg/ cm3 was prepared in 50 % (v/v) aqueous dimethyl sulfoxide (DMSO) for all samples. These were tested against E. faecalis (ATCC 13883) and S. epidermidis (ATCC 2223). The minimum inhibitory concentrations were determined using a micro dilution assay as discussed in Section 3.2. The MIC values of fractions are shown in Table 3.8 below. All the fractions showed appreciable antibacterial activity against both pathogens. The hexane soluble fraction (NP) was more active as compared to the crude extract and the methanol soluble (P) fraction. So it was subjected to further fractionation. From the methanol soluble sub-fractions activity decreased with the polarity and sub-fractions P3 and P4 were the most active. However these two fractions were rather complex and small in quantity so they were not considered for further fractionation. Instead two fractions (P5 and P6) were combined and subjected to further fractionation as discussed in the next section.
57 Chapter 3: Experimental Methodology
Table 3.8: MIC values (mg/ cm3) of crude Hex and MeOH soluble fractions and column chromatography fractions
Fraction MIC mg/ cm3
E. faecalis S. epidermidis
Crude methanol extract 2.00 1.00
Hexane soluble fraction (NP) 0.06 0.03
0.50 1.00 Methanol soluble fraction (P)
0.06 1.00 Sub-fraction P1
0.25 0.50 Sub-fraction P2
0.03 0.25 Sub-fraction P3
0.08 0.31 Sub-fraction P4
0.50 1.00 Sub-fraction P5
0.50 1.00 Sub-fraction P6
2.00 2.00 Sub-fraction P7
2.00 2.00 Sub-fraction P8 Positive control (Ciprofloxacin 0.01mg/ ml) 6.25x10-4 3.13x10-4
3.3.5 Fractionation of sub-fraction P4
This fraction yielded compound 1(TL 01) which precipitated as it was being dried in the fume hood. This was collected by filtration through filter paper and dried in the fumehood. Solubility tests showed that it dissolves in DMSO and TLC analysis in acetone - chloroform - ethyl acetate - methanol - water (40:30:12:10:8) (TLC 3) revealed a single spot with an Rf value of 0.7. A representative TLC plate for this compound is shown in Figure 3.4.
58 Chapter 3: Experimental Methodology
3.3.6 CC fractionation of combined sub-fractions P5 and P6
The combined fractions (P5 and P6) were fractionated in a silica gel column and eluted with a gradient of DCM and MeOH from 100 % DCM to 10 % DCM in MeOH. About 362 small fractions in vials were collected and pooled on basis of TLC analysis to four major fractions (series): 1-60, 61-157, 166-228, 231-314, 315- 360 and 362 which were all crude and needed further purification. Figure 3.2 is a TLC plate showing the major fractions collected from the column chromatography fractionation of P5 and P6 combined fractions. Two of these (61-157 and 362) purified further.
Figure 3.2: TLC plate of fractions collected from fractionation of P5 and P6.
3.3.6.1 Further purification of sub fractions P5 and P6.
a) Series 61-157
Series 61-157 (72 mg) was further purified by liquid-liquid fractionation between dichlomethane and methanol. This afforded two fractions, a DCM soluble and MeOH soluble which were both subjected to TLC analysis. The methanol soluble fraction afforded compound 2 (TL 02). TLC analysis revealed that this compound as a single spot with Rf 0.7 in TLC 3, staining yellow in vanillin-sulfuric acid and not visible under UV. A representative TLC plate for this compound is shown in Figure 3.3.
59 Chapter 3: Experimental Methodology
b) Series 362 (18.6 mg)
This fraction (18.6 mg) was purified by liquid-liquid fractionation between DCM and MeOH. First it was dissolved in small volumes of DCM until a clear solution was obtained. The resultant insoluble part was dissolved in MeOH. Both the DCM and the MeOH soluble fractions were subjected to TLC analysis. The DCM soluble fraction appeared was still crude. From TLC analysis the MeOH soluble fraction (TL 03) appeared as two spots(yellow and blue) that are non-separable. Analysis in the LC-MS showed three peaks with different mass spectra. Because of relatively small quantities the fraction could not be taken for further purification. Figure 3.3 is a TLC plate showing the two compounds.
TL 01 TL 02 TL 03 Figure 3.3: TLC plate showing compounds TL 01, TL 02 and TL◦03.
From the polar fraction (methanol soluble (P)) of the fruits two compounds were isolated and purified. These were later subjected structure elucidation techniques.
3.3.7 Fractionation of hexane soluble fraction (NP)
The hexane soluble fraction (4 g) was chromatographed on silica gel and eluted with an increasing (5 % increments of polar solvent) gradient of hexane and ethyl acetate from 100 % hexane to 100 % ethyl acetate. Eighty small fractions (50 cm3) were collected and pooled to 8 major subfractions based on their TLC fingerprints (1-10, 12-35, 36-39, 41-43, 44, 45-61, 62-69, 70-80). These were all crude and
60 Chapter 3: Experimental Methodology
needed further purification. Two of these sub-fractions: (12-35) and (62-69), were purified further as discussed in the next sections.
3.3.7.1 Fractionation of sub-fraction NP 12-35
Fraction (12-35) (184 mg) was chromatographed in a column packed with silica gel F60 and eluted gradually with hexane and ethyl acetate in increasing polarity starting with 100% hexane to 1 %, 2 %, 3 % and 4 % ethyl acetate in hexane. About 180 fractions (25 cm3) were collected and pooled to 5 major sub fractions (1-26, 30-80, 89-141, 152-159 and 160-177)
From the series 89-141, compound 04 (TL 04) (2.6 mg), Rf 0.2 in Hex - CHCl3 (10:1) was collected. It is a white amorphous solid soluble in chloroform appearing as white fluorescence under UV254 nm and not visible on visualization with vanillin- sulfuric acid. This compound precipitated as the fractions were being concentrated and was collected by filtration. It appeared as a single spot on analysis with TLC (Figure 3.4)
The series 152–177 was subjected to preparative thin layer chromatography in
CHCl3 - MeOH (100:1) and three compounds were isolated. Compound 05 (TL
05), Rf = 0.4 (CHCl3 – MeOH, 100:1) appearing as a purple spot under UV 254 nm and not visible on visualization with vanillin-sulfuric acid. Compound 06 (TL 06) with Rf = 0.2 in CHCl3-MeOH (100:1), a white amorphous solid not visible under UV and staining blue on visualization with vanillin-sulfuric acid. Compound 07 (TL
07), Rf = 0.9, a white solid appearing as white fluorescence under UV 366 nm and staining blue in vanillin-sulfuric acid. Figure 3.4 shows a TLC plate of the compounds isolated from series 152-177.
61 Chapter 3: Experimental Methodology
TL 07
TL 05 TL 04 TL 06
Figure 3.4: TLC plate showing compounds TL 04, 05, 06 and 07
3.3.7.2 Fractionation of sub-fraction NP 62-69
This fraction was subjected to Silica gel F60 column chromatography and eluted isocratically with a mixture of Hex-EtOAc (3:2). A total of 28 fractions (25 cm3 each) were collected. Fractions 1-5 were pooled (13 mg) and separated on PTLC plates developed in Hex-EtOAc (2:3). Compound 8 (TL 08), a sticky yellow solid with Rf 0.8 in Hex-EtOAc (2:3) and staining red with vanillin-sulfuric acid was isolated. This compound is shown in Figure 3.5 below.
TL 08
Figure 3.5: TLC plates showing the fractionation of sub-fraction NP-62-69.
62 Chapter 3: Experimental Methodology
3.4 Structure elucidation of isolated compounds
The purity of compounds was checked with TLC and confirmed with Liquid chromatography. Liquid chromatography experiments were performed in a Waters e 2695 Separations module coupled to a 3100 Mass detector. Analyses were done in positive and negative Electrospray Ionization (ESI) mode. FT-IR spectra were recorded on a Perkin Elmer 100 Series FT-IR spectrometer. Proton and carbon nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance II 400 MHz instrument. This structural elucidation of the isolated compounds is presented and discussed in Chapter 4.
63 Chapter 3: Experimental Methodology
References
1 Eloff J.N. and McGaw L.J. (2002). Plant Extracts Used to Manage Bacterial, Fungal and Parasitic Infections in Southern Africa. In Ahmad I, Aqil F., Owais M., (Eds) Modern Phytomedicine: Turning Medicinal Plants into Drugs. Wiley-VCH Verlag. Pp 97 to 121.
2 Arapitsas P. (2008). Identification and Quantification of Polyphenolic Compounds from Okra Seeds and Skins. Food chemistry 110: 1041-1045.
3 Fried B., Sherma J. (1986). Thin-Layer Chromatography, Techniques and Applications, 2nd ed. Marcel Dekker INC, New York. Pp 1;4;116;186;136.
4 McMaster C., McMaster M. (1998). GC-MS A Practical Users Guide. John Wiley and Sons Inc. New York. Pp 3-5.
5 Ramanik G., Gilgenast E., Przyjazny A., Kaminski M. (2007). Techniques of Preparing Plant Material for Chromatographic Separation and Analysis. Journal of Biophysical Methods 70: 253 -261.
6 Drozd J. (1981). Chemical Derivatisation in Gas Chromatography. Elsevier Scientific Publishing Company, New York. Pp 1,66.
7 Christian G. D. (2004). Analytical Chemistry. John Wiley Inc. USA. pg 589.
8 Meyer V.R. (1994) Practical High Performance Liquid Chromatography, John Wiley and Sons. Pp 1-14.
9 Hostettmann K., Marston A., Ndjoko K. and Wolfender J–L. (2000). The Potential of African Plants as Sources of Drugs. Current Organic Chemistry 4: 973-1010.
10 Eloff J.N. (1998). A Sensitive and Quick Microplate Method to Determine the Minimal Inhibitory Concentration of Plant Extracts for Bacteria. Planta Med 64: 711-713.
64
CHAPTER 4 ...
RESULTS AND DISCUSSIONS
Introduction
Despite the fact that medicinal plants have been used for centuries as medicines, the practice is still engulfed in a cloud of controversy in as far as efficacy and safety is concerned. 1 Consequently, WHO advocates for intensified efforts on medicinal plant research with emphasis on providing scientific explanations to the use of plant based traditional medicine and isolating new bioactive compounds that can be used as drugs or analogues in drug development.2 This study focused on R. melanophloeos, a medicinal plant widely distributed in Southern Africa and used extensively for ailments ranging from stomach disorders, respiratory problems and skin infections but a plant about which little chemical information is known.3 This study qualitatively and quantitatively evaluated and assessed the phytochemical profile of the leaves, fruits and bark of plants collected from six localities. This was coupled with an assessment of their antibacterial activity and isolation of some compounds from the fruits. This chapter is a detailed presentation and discussion of the results obtained in the overall study.
4.1 Chemical variation study
Phytochemicals typically show quantitative and qualitative differences between plants or different plant parts. These variations which could either be geographical or seasonal are often controlled by hereditary factors, environmental factors and developmental stage. Genetic factors account for both qualitative and quantitative variations whilst the other two account mostly for the quantitative variations. 4 Chemical variation studies reveal any differences or similarities between different plant parts and plants from different localities. These provide a scientific justification for using different plant parts and plants from different regions for the same medicinal purposes and allow traditional healers and consumers to make
65 Chapter4: Results and Discussions
informed decisions with regard to their collection and use. 5 A chemical variation study on R. melanophloeos leaves, fruits and bark of plants collected from six localities was carried out as mentioned in Section 3.1.1. For the chemical variation studies a coding system indicating the location, plant number in specific location and plant part, has been adopted. For example the code AB1: A = locality A, B = bark, 1 = plant 1 in specific locality (A). In some instances this is shortened to show only the location and plant number in that location. For example, A1= plant 1 from locality A. The results on the extraction yields as well as TLC, GC-MS and HPLC analysis are presented and discussed in the next section.
4.1.1 Extraction yields of the bark, fruit and leaf extracts
Four solvents of increasing polarity (petroleum ether, ethyl acetate, methanol, and water) were used sequentially to extract phytochemicals from the leaves, bark and fruit samples of all plants from the six localities (A to F) as discussed in Section 3.1.2 and the percentage yields (w/w) calculated. The percentage (extraction) yields reflect the amount or yield of extractable components relative to the weight of dry plant material. It is a factor of several variables including the extraction solvent and extraction method. Different solvents, depending on their selectivity and polarity, extract varying amounts of different groups of phytochemicals.6 The percentage yields of the leaves, fruit and bark samples are presented separately below.
4.1.1.1 Extraction yields of leaf samples
The percentage yields of leaf samples from the six locations extracted in the four different solvents are shown in Table 4.0a. The results show that methanol extracts the highest percentage of phytochemicals ranging between 8 % (A2) and 20 % (C4). The mean percentage yield stands at 15.6 % while that of water, petroleum ether, and ethyl acetate are 5.3 %, 1.8 % and 1.2 %, respectively. Generally plant to plant variation (within and between populations) exists in the percentage yields. For instance the percentage yields of methanol extracts of plants collected in Nhlangano (B) are, 13.3 %, 14.4 %, 19.0 %, 20.1 %, 13.2 %,
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and 16.5 % for B1, B2, B3, B4, B5 and B6 respectively, and these show some intrapopulational variation. These are more pronounced in the methanol and water extracts as depicted by the relatively high standard deviations (3.33 for methanol and 1.82 for water)
Table 4.0a: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos leaf samples.
Locality Sample % extractable material
name PE EtOAc MeOH H2O APS Sicunusa A1 1.4 1.1 16.2 3.0 7.2 (A) A2 1.6 0.9 8.3 6.6 5.8 A3 2.2 1.3 10.5 7.5 5.0 Nhlangano B1 1.5 1.3 13.3 4.5 1.2 (B) B2 1.5 1.0 14.4 4.5 0.8 B3 1.4 1.1 19.0 4.3 0.1 B4 1.3 1.1 20.1 3.6 0.4 B5 1.7 1.4 13.2 6.7 5.6 B6 1.7 1.6 16.5 3.3 5.0 Bhunya C1 1.9 1.1 15.1 4.2 0.3 (C) C2 1.7 1.1 19.0 5.3 3.5 C3 1.6 1.2 12.1 4.4 3.1 C4 1.9 1.1 20.4 5.4 6.1 Fonteyn D1 1.5 1.5 12.9 4.2 5.5 (D) D2 2.0 1.7 17.7 2.7 4.0 D3 1.5 1.3 13.6 5.2 0.2 D4 1.8 1.3 18.8 6.5 1.4 D5 2.0 1.4 20.1 5.2 4.2 Lundzi (E) E1 1.7 1.3 12.8 4.4 0.8 KZN F 1 2.3 1.0 18.3 9.3 0.6 (F) F 2 2.6 0.9 14.7 7.9 0.7 F 3 2.4 0.8 15.4 8.9 0.8 Average 1.8 1.2 15.6 5.3 3.2 Std dev.(absolute) 0.35 0.24 3.33 1.82 2.54
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4.1.1.2 Extraction yields of fruit samples
The percentage yields of all crude extracts of the fruits are shown in Table 4.0b. Methanol extracted the highest percentage of phytochemicals ranging from 10.6 % (A1) to 15.4 % (B5) and EtOAc extracted the least percentages with values ranging between 1.7 % and 4.7 %. The mean percentage yield of methanol extracts stands at 12.4 % while those of water, petroleum ether and ethyl acetate are 4.3 %, 3.6 %, and 2.7 % respectively. Relatively high standard deviations are observed which could be an indication of random plant to plant variation.
Table 4.0b: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos fruit samples.
Location Sample name % extractable material
PE EtOAc MeOH H2O APS Sicunusa (A) A1 2.9 2.3 10.6 5.9 4.4 Nhlangano (B) B2 3.0 2.8 13.8 3.4 1.8 B3 4.0 1.7 10.7 3.1 4.8 B5 3.8 2.3 15.4 3.0 5.9 B6 4.5 2.8 10.9 3.0 5.8 Fonteyn (D) D1 3.3 4.7 12.9 6.4 6.1 Average 3.6 2.7 12.4 4.3 4.8 Std dev. (absolute) 0.63 1.03 1.99 1.58 1.81
4.1.1.3 Extraction yields of bark samples
The percentage yields of the crude extracts of the bark are shown in Table 4.0c. As observed with the leaves and fruits, methanol extracts the highest percentage of phytochemicals with percentage yields ranging from 8.5 % (F3) to 23.1 % (C1). The mean percentage yield of methanol extracts stands at 15.5 % whilst those of water, ethyl acetate, and petroleum ether extracts are 3.2 %, 0.76 % and 0.73 % respectively. Unlike in the leaves and fruits where EtOAc extracted the lowest yields, with the bark petroleum ether recorded the lowest yields. Generally random plant to plant variation within and between populations could exist. For example the percentage yields of water extracts range between 0.6 % (B2) and 4.6 % (A1)
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with an absolute standard deviation of 3.2. On the other hand the percentage yields of water extracts of plants from Sicunusa (A) are 4.6 %, 1.3 % and 3.6 % for A1, A2, and A3 respectively showing slight variations between plants from the same location collected at the same time.
Table 4.0c: Percentage yields of PE, EtOAc, MeOH, H2O and APS extracts of R. melanophloeos bark samples.
Location Sample % extractable material
name PE EtOAc MeOH H2O APS
Sicunusa A1 0.6 1.4 14.4 4.6 0.8 (A) A2 0.5 0.6 10.8 1.3 4.5 A3 0.4 0.8 12.1 3.6 3.8 Nhlangano B1 0.7 0.8 18.3 3.8 0.6 (B) B2 0.5 0.7 14.7 0.6 1.4 B3 0.3 0.8 16.1 1.3 0.6 B4 0.5 0.6 17.0 3.1 1.2 B5 0.4 0.8 17.5 7.7 4.3 B6 0.3 0.5 15.1 4.7 4.1 Bhunya C1 0.4 0.4 16.0 6.6 0.7 (C) C2 0.4 0.3 23.1 1.8 0.4 C3 0.6 0.9 18.8 4.0 1.1 C4 0.5 1.2 13.9 2.8 2.9 Fonteyn D1 0.6 0.9 17.6 1.8 4.2 (D) D2 0.6 1.6 18.4 3.0 2.2 D3 0.7 0.6 15.0 3.1 2.0 D4 0.5 0.8 23.4 4.9 4.4 D5 0.4 0.5 12.5 1.9 4.2 Lundzi (E) E1 0.5 0.8 15.4 1.7 0.8 KZN F1 2.6 0.6 13.1 3.7 0.6 (F) F2 2.7 0.7 9.8 3.3 0.7 F3 1.3 0.4 8.5 1.7 0.8 Average 0.73 0.76 15.5 3.2 2.1 Std dev.(absolute) 0.65 0.30 3.73 1.75 1.59
Considering that secondary metabolites, normally account for about 10 % of dry plant material, the yields recorded with R. melanophloeos (bark, leaves and fruits) are relatively high.6 Accounting for this could be the use of a sequential extraction
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procedure which allows the selective and efficient extraction of different groups of phytochemicals.6 In all three plant parts, methanol extracted the highest percentage of phytochemicals, followed by water. For the leaves and fruits the least amounts were extracted by ethyl acetate, whereas in the bark the least amounts were extracted by petroleum ether. The high methanol yields could suggest that the plant is rich in polar compounds than the non-polar ones or it can be explained on basis of methanol being a polar solvent capable of extracting a wide range of compounds from non-polar to polar.
Typically, depending on their role in plants, secondary metabolites are stored in different parts accounting for qualitative and quantitative variations between plant parts.5 For example non polar phytochemicals accumulate in high amounts stored in the fruits, reproductive organs and the foliage.7 A comparison of the leaves, fruits and bark percentage yields reveal slight variations with the leaves recording the highest percentage yields of polar phytochemicals (water and methanol) while the fruits recorded the highest yields of non-polar phytochemicals (petroleum ether and ethyl acetate). Figure 4.0 shows a comparison of the mean percentage yields of the bark, fruits and leaves.
Average percentage yields of R. melanophloeos leaf, fruit and bark extracts
24 23 20 Leaves
15.6 15.5 Fruits 12.4 Bark
5.3 3.6 4.3 Mean % yield Mean% yield 2.7 3.2 1.8 0.7 1.2 0.8
PE EtOAc MeOH H2O Total extracted
Figure 4.0: A comparison of mean % yields of leaf, fruit and bark extracts
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On average the leaf percentage yields (24 %) are the highest followed by the fruits (23 %) and the bark‟s are the least (20 %). Since plants often contain several bioactive compounds which could be stored in any plant part, all extracts are considered potentially bioactive until proven otherwise. 8 Often the percentage yields of plant extracts are correlated to bioactivity through the calculation of total activity (cm3/ g).9 This is defined as a measure of the degree to which one gram plant material can be diluted with bio-active compounds still exhibiting activity. It is equivalent to efficacy in pharmacological terms and the higher the percentage yields the higher the total activity. On that regard the differences in percentage yields of the different plants somehow questions the traditional medicinal uses of the plant, where the bark and fruits are the most widely used and the leaves are less popular.10 To correlate the percentage yields with biological the total activity of all extracts were calculated presented in Section 4.3.
4.1.1.4 Geographical variation on extraction yields
The percentage yields of the leaves and bark from five localities (A, B, C, D, F) were compared to investigate geographical variations. Locality E was omitted because only one plant was harvested there. For each extract (e.g. PE extracts of the leaves) the mean percentage yield of plants from each locality was calculated and data were subjected to analysis of variance (ANOVA) to assess significant differences between the means. Differences between means at (p ≤ 0.05) were considered statistically significant. Table 4.1 shows the mean percentage yields of the different localities. Depending on the plant part and extraction solvent, percentage yields either showed a significant variation between the five locations or no significant variation. For the leaves, PE, MeOH and H2O extracts there is a significant difference (p ˂ 0.05) in the percentage yields of plants collected from the five localities and only the EtOAC extracts showed no significant variation(p ˃ 0.05). For the bark extracts on other hand there is no significant variation in the percentage yields of the PE, EtOAC and H2O extracts. Only the MeOH extracts showed a significant variation.
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A significant variation in the percentage yields implies that the amounts of extractable phytochemicals differ between plants collected from different localities. Whereas the lack of significant variation means that any observed variation is just random and cannot be accounted for by the differences in plant collection site.
Table 4.1: Mean percentage yields of extracts of the leaves and bark samples of plants collected from five different locations
Locality Mean % yield
Leaves Bark
PE EtOAc MeOH H2O P.E EtOAc MeOH H2O
Sicunusa 1.7 1.1 11.7 5.7 0.5 0.9 12.4 3.2
Nhlangano 1.8 1.3 15.3 4.4 0.5 0.7 16.4 3.5
Bhunya 1.8 1.2 16.1 4.8 0.5 0.7 17.9 3.8
Fonteyn 1.8 1.4 14.9 4.8 0.6 0.9 17.4 2.9
KZN 2.4 0.9 16.1 8.7 2.2 0.6 10.5 2.9 1.80 1.2 15.6 5.3 0.73 0.76 16.0 3.2 Overall mean 0.35 0.24 3.33 1.82 0.65 0.30 3.73 1.75 Std deviation 0.00006 0.0064 0.33 0.003 1.35 0.58 0.01 0.98 P-value * *p-value calculated from ANOVA, (p ≤ 0.05) implies a significant statistical difference between means
4.1.1.5 APS percentage yields
Alcohol Precipitable Solids (APS) consist of the high molecular weight phytochemicals like polysaccharides and glycoproteins. 11 These have been identified as the major compounds responsible for the therapeutic properties of aloes and the % APS is often used for quality assurance. They reportedly have important medicinal properties like antidiabetic, gastroprotective, immunostimulatory and wound healing properties.12 From the slimy appearance of plant material on treatment with water, and from the medicinal uses of R. melanophloeos we suspected the accumulation of APS. Subsequently, these were extracted as a white gelatinous solid from all samples as discussed in
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Section 3.1.3. The percentage yields were calculated and are presented in Tables 4.0a to 4.0c.
The percentage APS‟s extracted from R. melanophloeos are higher than to those previously recorded from other plants (ranging between 0.31 and 3.97 %).13 The relatively high amounts of APS‟s observed in the plant could give a justification for its traditional medicinal uses for wound healing and diabetes. In traditional medicine the bark is reportedly chewed or pulverized and applied as a topping for healing wounds while decoctions are taken to lower blood sugar.14 Comparing the percentage APS of the three plant parts (leaves, fruits and bark), higher percentages were extracted from the fruits, with a mean percentage of 4.8 % exceeding the leaves (3.2 %) and the bark (2.1 %). Though, contradicting the traditional uses of the plant where the bark is most popular, higher percentages in the fruits compared to the leaves and the bark can be justified on basis of their nutritional value. Random variation exists between individual plants. But this cannot be accounted for by the differences in collection site as comparison of the percentage yields of plants from different localities (through ANOVA) showed no significant variation (p ˃ 0.05). Since APS‟s are mostly complex mixtures of compounds with large molecular weights, an attempt was not made to isolate them any further.
4.1.2 TLC screening of PE, EtOAc, MeOH and H2O extracts
The leaf, bark and fruit extracts of plants collected from all six localities were subjected to TLC analysis to screen for the presence of non-polar (NP), medium polar (MP) and polar (P) phytochemicals as discussed in Section 3.1.4. A comparison of the chemical profiles of the different plant parts and plants from different localities was necessitated by the extensive medicinal use and wide distribution of R. melanophloeos. The Rf values of different spots, behavior under UV light and color formation on chemical visualization were considered in the analysis of results. In essence since TLC‟s were spotted in a consistent manner, they give a semi quantitative picture of the crude extracts. The number of spots with different Rf values is indicative of the number of compounds and the
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behaviour of different spots on visualization with different agents gives comprehensive information on the types or groups of compounds.15 Results for the TLC screening of different extracts are presented and discussed separately in the sections that follow.
4.1.2.1 TLC analysis of PE extracts
PE extracts were screened for the presence of non-polar phytochemicals as outlined in Section 3.1.4. All samples were developed in Hex-DE (3:2) and visualized in acidic vanillin. The non-polar compounds are mainly components of essential oils. They generally appear as purple, blue, red and yellow spots on treatment with vanillin in sulfuric acid (VS).16 The representative TLC plates for the leaves, fruits and bark samples are shown in Fig 4.1a to 4.1d. In all TLC plates compounds occurring universally in all plant parts are marked with a star.
a) Leaf samples
TLC plates for the screening of PE extracts of leaf samples from SWD and KZN are shown separately in Fig 4.1a and 4.1b. Qualitatively there is no significant variation between plants. Both plates are identical showing several blue staining components with different Rf values. The most prominent component appears at Rf = 0.78 and appears to be a mixture of compounds as it moves with the solvent front. Other minor components appear at Rf = 0.61, 0.48, and 0.15. A great quantitative variation as depicted by the difference in intensities of spots is observed between plants. For instance the two samples, DL3 and DL5 both from locality D differ considerably in their chemical profile, DL3 has all the major compounds and DL5 does not show any of the prominent compounds.
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0.78
0.61
0.48
0.15
Figure 4.1a: TLC plate showing PE extracts of leaf samples from SWD plants.
0.9 0.81
0.63
0.61 0.41
0.48
0.15 0.05
Leaves Bark Figure 4.1b: TLC plate showing PE extracts of leaf and bark samples from KZN plants.
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b) Bark samples
TLC plates for the screening of PE extracts of bark samples from KZN and SWD are shown separately in Fig 4.1b and 4.1c.
0.81
0.63
0.41
0.0.0505 Yellow streak
Figure 4.1c: TLC plate showing PE extracts of bark samples from SWD plants.
Both plates show an identical chemical profile, with prominent components at
Rf◦=◦0.81, 0.63, 0.41 and 0.05 all staining blue-violet. The occurrence of these components shows quantitative variations between individual plants depicted by the differences in spot intensity. Compared to the leaves, the bark samples accumulate fewer compounds and differ mainly with the occurrence of a yellow streak at the bottom of the plate which is missing in the leaves.
c) Fruit samples
A representative TLC plate for the screening of fruit samples (Fig 4.1d) shows a number of blue, purple and red staining compounds typical of components of essential oils. Two purple staining components appear at Rf = 0.78 and 0.69. These are poorly resolved and appear to be a mixture since they move with the solvent front. At Rf = 0.53 a blue staining compound appearing as a streak occurs in all samples. This appears under UV254 nm as a purple streak and at 366 nm as a mixture of at least three compounds with red and white fluorescence. A red
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staining component appears in all samples at Rf = 0.25 just below the blue streak. The intensity of different components vary considerably between individual plants implying some quantitative variations.
0.78
0.780.69
0.53
0.780.25
Yellow streak
AF1 BF2 BF3 BF5 DF1
Figure 4.1d: TLC plate showing PE extracts of fruit samples from SWD plants.
A comparison of PE extracts of the leaves, fruits and bark reveal some similarities and slight differences in their chemical composition. At least one purple staining component moving with the solvent front seems to accumulate in all three plant parts. The leaves and bark samples are more comparable with most of the components observed in the bark also appearing in the leaves at almost the same
Rf values. The fruit samples however are quite different with the occurrence of extra red straining compounds (Rf = 0.25).
4.1.2.2 TLC analysis of EtOAc extracts
The ethyl acetate extracts were used to screen for the presence of medium polar compounds such as diterpenoids, saponin aglycones, flavonoids and benzoquinones.16 These were developed in Hex-EtOAc (2:3) and visualised in
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acidic vanillin as discussed in Section 3.1.4. The EtOAC extracts physically had a crystalline nature and a bright orange colour which is common with benzoquinones.17 Representative TLC plates for the leaves, bark and fruits are shown in Fig 4.1e to 4.1h.
a) Leaf samples
TLC plates for the screening of EtOAC extracts of leaf samples from SWD and KZN are shown separately in Fig 4.1e and 4.1f. The SWD and KZN plants show a comparable chemical profile with slight qualitative variations.
0.96 Chl
0.77
0.48
0.10
AL1 AL2 BL1 BL2 BL4 BL5 CL1 CL2 CL3 CL4 DL1 DL3 DL4 DL5 EL1
Figure 4.1e: TLC plate showing EtOAc extracts of leaf samples from SWD plants.
From both plates about four major components staining blue appear at Rf = 0.96, 0.77, 0.48 and 0.1. In addition to the blue staining components some green staining components which are possibly chlorophylls (Chl) are observed between
Rf = 0.8 and 0.9. These components show intraspecific and interspecific quantitative variation between plants. For instance the components at Rf = 0.48 and Rf = 0.77 both show great variation in their intensities from plant to plant.
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0.96 0.85
0.63
0.50
0.40
0.16 0.10
Leaves Bark Figure 4.1f: TLC plate showing EtOAc extracts of leaf and bark samples from KZN plants.
a) Bark samples
TLC plates showing the chemical profile of EtOAC extracts of bark samples from KZN and SWD are shown separately in Fig 4.1f and 4.1g. Both TLC plates shows the occurrence of four major components in all plants at Rf = 0.85, 0.63, 0.16 and 0.10. This components show quantitative variations between plants from same population and plants from different populations. The component at Rf = 0.85 is the most prominent and appears to be same component occurring in the leaves
(Rf = 0.96). In addition to the four components, a yellow streak appears at the bottom of the plate. This is typical of phenolic compounds and shows great quantitative variation between individual plants.16
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0.85
0.63
0.160.85
0.10 Yellow streak AB1 AB2 AB3 BB1 BB2..BB3..BB4..BB5 BB6 CB1 CB2 CB3 CB4 DB1 DB2 DB3 DB4 DB5
Figure 4.1g: TLC plate showing EtOAc extracts of bark samples from SWD plants.
b) Fruit samples
The TLC plate of EtOAC extracts of fruit samples (Fig 4.1h) shows the occurrence of a variety of purple, blue and red staining compounds. About 3 major components are observed. A purple staining compound appears at Rf = 0.96 and seems to be the same compound observed in the leaves (Rf = 0.96) and the bark
(Rf = 0.85). At Rf = 0.79 a red staining compound appears and shows some quantitative variation between individual plants. It appears in all plants except for plant A1 and B3. Another major component staining blue appears at Rf = 0.53. It is the most prominent component and also shows some variation between plants with A1 and B3 showing the low intensities. A yellow streak, also observed in the bark and leaf samples, occurs at the bottom of the plate with some quantitative variations between individual plants.
The variation observed with the two compounds (Rf = 0.79 and Rf = 0.53) could mean the occurrence of two chemotypes. One consisting of plants with significant amounts of both compounds and the other consisting of those plants lacking the red compound and having small quantities of the blue compound. These are however not geographical chemotypes since even plants from the same location collected at the same time show this variation.
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0.96*
0.79
0.53
Yellow streak
Figure 4.1h: TLC plate showing EtOAc extracts of fruit samples from SWD plants.
Generally all plant parts (leaves, bark, fruits) show several compounds of medium polarity. There is some variation however between the three plant parts with some compounds occurring universally and others being specific in their occurrence. A similarity in the chemical composition is evident with a blue staining compound moving with the solvent front in all three plant parts. Also a yellow streak typical of phenolic compounds appears at the bottom of the TLC plates in all plant parts. It is of significance as it shows considerable quantitative variation between the different plant parts and among individual plants. The fruits show relatively larger quantities of this compound compared to the bark and leaves. The leaves differ from the bark and fruits with the appearance of green staining compounds (Chl) which could be chlorophylls. The fruits also differ from the bark and leaves with the appearance of a red staining compound at Rf = 0.79 not observed in the bark and leaves.
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4.1.2.3 TLC analysis of MeOH extracts
The methanol extracts were used to screen for the presence of polar compounds such as flavonoids, tannins, saponins, and triterpenoids which mainly occur as glycosides. These extracts formed a stable soap-like foam (froth) when shaken with water which is indicative of the presence of saponins.18 The extracts were developed in BuOH-H2O-ACOOH (4:1:1) and visualized in acidic vanillin as discussed in Section 3.1. Representative TLC plates for the leaf, and bark and fruit samples are shown in Fig 4.1i to 4.1l.
0.91
0.45 0.35
Figure 4.1i: TLC plate showing MeOH extracts of the leaves from SWD plants.
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0.8
0.4
0.3
Tannins
FL1 FL2 FL3 FB1 FB2 FB3 Leaves Bark Figure 4.1j: TLC plate showing MeOH extracts of the leaves and bark from KZN plants.
0.9
0.3
0.2
Figure 4.1k: TLC plate showing MeOH extracts of the bark from SWD plants.
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0.8
0.4
0.30.8
Tannins
AF1 BF2 BF3 BF5 BF6 DF1
Figure 4.1l: TLC plate showing MeOH extracts of fruit samples.
Compared to the petroleum ether and ethyl acetate, fewer components were extracted with methanol. All three plant parts (bark, leaves and fruits) show almost the same chemical profile with yellow, red and blue-black staining compounds characteristic of flavonoids, tannins and saponins respectively.16 In all samples the occurrence of different components shows some quantitative variation and no significant geographical variation. The TLC plates of the leaves, fruits and bark show the occurrence of a yellow staining (characteristic of flavonoids) component with an Rf value between 0.8 and 0.9. This appears to be a mixture of two components that are not separable under the TLC analysis conditions. Two other components staining blue-black and likely to be saponins, occur at Rf = 0.3 and 0.4. The occurrence of saponins in the leaves and tannins from the bark has previously been reported.19, 20 The intensities of the blue-black and yellow spots appear to be stronger in fruit samples compared to the bark and leaves implying higher concentrations. All samples appear to contain some tannins observed from the red streak at the bottom of plates. The concentrations seem to be higher in the bark and leaf samples such that they overshadow the other compounds.
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4.1.2.4 TLC analysis of H2O extracts
Water extracts were used to screen for the presence of very polar phytochemicals in all samples. The extracts were developed in EtOAc-HCOOH-ACOOH (100:11:11:26) and visualized in acidic vanillin as discussed in Section 3.1. Generally very polar compounds consist of amino acids, carbohydrates, flavonoid glycosides, and tannins.16 The water extracts contain the fewest number of compounds compared to the methanol, ethyl acetate, and petroleum ether extracts. Representative TLC plates for all three plant parts are shown in Fig 4.1m to 4.1p and they all show a comparable chemical profile. From the leaf samples two yellow staining components at Rf = 0.7 and 0.19 occur in all plants as shown in Fig 4.1m and 4.1n. Tannins (staining red) still appear at the bottom of the plate though in lower intensities as compared to MeOH extracts. Bark extracts (Fig 4.1n and 4.1o) shows only a streak of tannins at the bottom of the plate. The fruit samples (Fig 4.1p) show a blue-black staining compound and some tannins at the immersion line (bottom of the plate).
0.70
0.19
Figure 4.1m: TLC plate showing H2O extracts of leaf samples from SWD plants.
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0.86
0.68
0.25
0.14 0.12
Leaves Bark
Figure 4.1n: TLC plate showing H2O extracts of leaf and bark samples from KZN plants.
Tannins
Figure 4.1o: TLC plate showing H2O extracts of bark samples from SWD plants.
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Figure 4.1p: TLC plate showing H2O extracts of fruit samples.
4.1.2.5 TLC screening of H2O extracts for carbohydrates
Water samples were screened for the occurrence of carbohydrates in all samples. The extracts were developed in EtOAc-MeOH-ACOOH (100:15:15) and visualized in chromic acid. The carbohydrates appeared as white spots against a yellow background. Carbohydrates are generally classified as primary metabolites based on their synthetic pathway and universal occurrence in all plants. However they are important in medicinal plants because they possess some therapeutic properties and are used in pharmaceuticals as bulking agents.2 The TLC profiles of the leaves, bark and fruits (Fig 4.1q to 4.1t) are comparable showing one compound with Rf = 0.4 in all plants.
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0.4
AL1 AL2 AL3 BL1 BL2 BL3 BL4 BL5 BL6 CL1 CL2 CL3 CL4 DL1 DL2 DL3 DL4 DL5 EL1
Figure 4.1q: TLC plate showing carbohydrates from H2O extracts of leaf samples from SWD plants.
0.4
Figure 4.1r: TLC plate showing carbohydrates from H2O extracts of leaf and bark samples from KZN plants.
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0.4
Figure 4.1s: TLC plate showing carbohydrates from H2O extracts of bark samples from SWD plants.
0.4
Figure 4.1t: TLC plate showing carbohydrates from H2O extracts of fruit samples from SD and KZN.
Despite the availability of a whole range of sophisticated analytical techniques, and a drawback of non-reproducibility, TLC remains one of the fastest, cheapest
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and effective methods to obtain a characteristic analytical picture of plant extracts.6 Recently applications to compare the chemical composition of various plant parts of threatened South African medicinal plants revealed that for some plant species the chemical composition of leaf and bark samples are more comparable.21 In this study, TLC analysis of different solvent extracts of the bark, fruits and leaves revealed the accumulation of non-polar terpenes (components of essential oils) and confirmed the accumulation of tannins, saponins, benzoquinones and flavonoids. Slight qualitative and quantitative differences in the chemical composition of the bark, fruit, and leaves were noted. Some components occur universally in all three plant parts while others are selective in their occurrence.
With polar extracts (MeOH and H2O) the chemical profiles of all three plant parts are comparable and show the occurrence of the same groups of compounds in varying concentrations. With the non-polar extracts however, the fruit samples differ considerably from the leaves and bark which are quite comparable. Plant to plant variation between and within populations was observed especially in the concentrations of different components. However no distinct geographical chemotypes were observed. The different extracts were subjected to antibacterial activity tests to determine if the observed variation has any effect on their bioactivity. This is discussed in Section 4.2.
4.1.3 GC-MS analysis of PE extracts
GC-MS is a powerful analytical tool used extensively in the separation and identification (from mass fragments and in the presence of standards) of non-polar phytochemicals with applications ranging from geographical variation studies to the authentification of essential oils in industry.6 A comparison of the GC-MS profile of any two samples reveals any quantitative and qualitative variations. The PE extracts of the leaves, fruits and bark were subjected to GC-MS analysis to further screen for the presence of non-polar volatile phytochemicals (monoterpernoids, sesquiterpenoids) and to compare their occurrence in the different plant parts and plants from different localities as discussed in Section 3.1.5.1. Based on the retention time and mass spectra of peaks, five compounds occur at 14, 20, 25, 30 and 35 minutes retention time. Retention times and mass
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fragments of these 5 compounds are shown in Table 4.2. As there was no standard available, the results could not be quantified thus they are all qualitative. The mass spectra of these compounds were compared with those of known compounds in a library search (NIST 08) and three of them were identified as (-) spathulenol, caryophyllene oxide and α-cardinol. The mass fragments of the other two compounds suggest that they have relatively high molecular weights and could be diterpenoids or even triterpenoids. To the best of our knowledge there are no available reports on the occurrence of non-polar volatile compounds from R. melanophloeos. The occurrence of the five compounds in different samples of the leaves, fruits and bark is summarised in Table 4.3 and Fig 4.2 shows representative GC-MS chromatograms of the leaves, fruits and bark. Other chromatograms are shown in Appendix 2.
Table 4.2: Retention time and mass fragments of major compounds from GC-MS analysis of the PE extracts of R. melanophloeos
Compound Retention time Mass fragments (m/z) * Tentative identity from NIST (minutes) 08 Library 43, 119, 205 (-) spathulenol 1 14
43,109,188, 220 caryophyllene oxide 2 21
43, 121,204 α- cardinol 3 25
4 30 73, 268, 355 u
5 35 73, 356, 401 unknown * major mass fragments, underlined = base peak
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Table 4.3: Occurrence of major compounds from GC-MS analysis of R. melanophloeos PE extracts of the leaves, bark and fruits.
Compound (retention time ) Plant part Sample 1 2 3 4 5 (14 min ) (20 min) (25 min) (30 min) (35 min) A1 tr tr _ _ x Leaves A2 tr tr tr _ x B2 x xxx xx x x B3 tr tr tr tr x B4 tr xxx tr tr _ C1 xx xxx xx x x C3 x xxx xx x x C4 x xxx xx x x D1 x xxx xx x x D3 xx xxx xx x x D5 x xxx xx x x E1 x xxx xx x x F1 xx xxx xx x _ F2 x xxx xx x _ F3 x xxx xx x x A1 _ tr _ _ _ Bark B2 _ _ _ _ _ C1 _ _ _ _ _ D3 tr tr tr tr tr E1 _ tr _ _ x F2 tr xx xx x xx A1 tr xxx xx x xx Fruits B2 x xxx xx xx x B3 tr xxx xx x xx D1 x xxx xx xx x Key: _ = not detectable tr- trace x-detectable in small amounts xx-present in average yields xxx- present in high yields (major compound)
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Bark 2 Fruits Solvent peaks 3
4 1 5
2 Leaves
3
1 4 5
Figure 4.2: Representative GC-MS chromatograms of the bark, fruits and leaves.
Non-polar plant extracts are usually composed of essential oils which are complex mixtures of up to sixty volatile, low molecular weight, aromatic components.22 On that regard, R. melanophloeos contains relatively few essential oil components which could be justified by the fact that essential oils are known to be selective in their distribution occurring mainly in members of the Labitae, Verbanaceae and Lamiaceae families.7 Previous research has shown that the nature and composition of essential oils vary with the extraction method used and that they are best extracted by hydrodistillation.23 Thus, the use of solvent extraction in this study may also account for the observation of few components with relatively large molecular weights. A comparison of the fruits, leaves and bark, shows variations in the accumulation of the five compounds (Table 4.3). The bark has an obviously different chemical profile while the fruits and leaves have a similar profile with caryophyllene oxide (compound 2) as the major compound. Bark samples either lack most of the non-polar compounds that are found in the leaves and fruits or have these compounds in very low concentrations. This observation concurs with the extraction yield results where the bark recorded the lowest percentage yields
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of non-polar compounds. Results from the antibacterial activity tests (Section 4.2) will determine if this variation has an effect on the bioactivity of extracts.
Generally essential oils are characterised by their major compounds (components that occur in high concentrations), and they are known to show a very wide variation that is both qualitative and quantitative between plants.24 A closer look at the leaf samples shows random plant to plant variation and a possible geographical trend. The chemical profile of plants from localities A and B are comparable and differ from those of plants from localities C, D, E, and F. Plants from localities C, D, E and F accumulate compound 2 (caryophyllene oxide) as the major compound and the other four compounds in minor to average yields. Whereas plants from localities A and B either lack some of the compounds or they occur in trace or minor concentrations.
4.1.4 Screening of H2O extracts for free amino acids content
In addition to having a structural and nutritive role, plant amino acids have important biological activities primarily associated with the nervous system. 25 Some amino acids (especially non-protein) are associated with neuroactive and anticancer properties, as well as the ability to enhance insulin production. For example γ-amino butyric acid (GABA) has neuroactive properties and canavanine has anticancer properties. Reports on the toxicity of some amino acids with a potential of causing clinical disorders or poisoning necessitate their screening in medicinal plants.26 As part of the chemical variation study on R. melanophloeos, the leaves, bark and fruits were screened for the presence of free protein (standard) and non-protein (non-standard) amino acids using GC-MS as discussed in Section 3.1.5.2. Derivatised amino acids were identified using an EZ- Varian amino acid library by comparison of their mass fragments and retention times with those of standards. Quantification was achieved by plotting calibration curves for the amino acids using standard concentrations (a plot of total ion counts (TIC) against standard concentrations).
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Both protein and non-protein amino acids were detected from the leaves, fruits and bark in quantities ranging between 0.05 and 296 nmol/ cm3. The concentrations are relatively low compared to those previously reported from other plants.26 Mostly standard amino acids were detected and none of those amino acids associated with toxicity were detected, having a good bearing on the uses of the plant. A comparison of the three plant parts shows slight qualitative variation with most of the amino acids occurring in all plant parts and others being selective. For example cystathionine was detected only in the leaves and the two amino acids α-amino butyric acid and methionine were detected only in the fruits and not in the leaves and bark. Remarkable quantitative variation between the three plant parts is noted as well. For most amino acids, the fruits recorded the highest concentrations and the bark recorded the lowest. For example for alanine the concentrations are 79, 9.0 and 5.4 nmol/ cm3 for the fruits, leaves and bark respectively. Table 4.4 shows concentrations of the identified amino acids in the leaves, fruits and the bark.
Available ethno-botanical information on the medicinal uses of R. melanophloeos suggests that it used for neuroactive disorders. Traditionally, bark infusions are often administered to persons who are depressed and feel like crying and for treating palpitations possibly caused by stress and anxiety.10 Since some amino acids are associated with neuroactive properties, the occurrence of amino acids in this plant could provide a tentative justification for its traditional medicinal uses for neuroactive conditions.26 Though partly contradictory to the medicinal uses of the plant where the bark is the most commonly used, the higher concentrations in the fruits are particularly significant for its nutritional value as a chewable fruit.
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Table 4.4: Free amino acids detected in H2O extracts of the leaves, fruits and bark from R. melanophloeos.
Amino Acid Abbreviation Retention Unique Concentration (nmol/ cm3)
time (s) mass Leaves Fruits Bark (m/z) Alanine ALA 55.1 130 9.0 79.6 5.4 Sarcosine SAR 58.3 73 nd 0.2 nd Glycine GLY 60.7 116 tr 27.7 0.2 α-amino butyric acid ABA 66.6 144 nd 1.9 nd Valine Val 72.0 116 4.7 14.9 3.2 Β-amino butyric acid Β-AiB 75.9 116 8.8 8.9 9.3 Norvaline (IS) Nor 79.4 158 100 100 100 allo-Isoleucine aILE 84.3 172 10.2 30.7 10.0 Leucine LEU 87.5 130 5.3 14.3 3.5 Threonine THR 100.2 101 1.1 60.3 tr Γ-amino butyric acid GABA 102.2 130 tr tr tr Serine SER 102.5 60 7.0 100.5 6.8 Proline PRO 105.9 156 7.4 296.7 3.7 Asparagine ASN 112.2 69 tr 37.4 tr Aspartic acid ASP 145.8 216 tr 107.3 tr Methionine MET 147.0 101 nd 5.4 nd Glutamic acid GLU 167.8 84 2.6 210.2 8.9 Phenylalanine PHE 168.2 148 13.6 27.1` 15.1 Α-amino adipic acid AAA 186.6 98 6.2 4.6 0.9 Α-aminopimellic acid APA 202.6 112 6.8 nd 2.6 Glutamine GLN 205.5 84 6.7 30.5 3.2 Glycine-proline* GPR 232.5 114 15.2 13.6 13.2 Lysine LYS 246.6 170 21.3 39.4 21.4 Histamine HIS 257.6 81 19.9 19.8 21.2 Hydroxylysine HLY 267.5 129 17.3 nd 2.5 Tyrosine TYR 274.4 107 10.7 51.8 7.5 Proline- PHP 286.7 156 4.1 0.05 5.4 hydroxyproline* Tryptophan TRP 291.0 130 19.3 50.7 18.9 Cystathionine CTH 335.5 56 20.2 nd nd Cystine C-C 322.2 74 7.1 12.5 3.6 n=11 for leaves, 6 for bark and 6 for fruits, nd= non detectable, tr- concentrations below detection limit i.e. cannot be quantified. IS = internal standard * Dipeptide
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4.1.4.1 Geographical variation on amino acid content
To investigate geographical variations, the mean concentrations of 14 amino acids were calculated for five of the localities (A, B, C, D and F). Figure 4.3 shows a comparison of the mean amino acid concentrations. Some variation in the mean concentration of some amino acids is observed. For example the mean concentrations for alanine which is a standard amino acid are 5.0, 4, 10, 15 and 17 nmoles/ cm3 for localities A, B, C, D and F respectively. Somehow for some amino acids the mean concentrations for Sicunusa (A) and Nhlangano (B) plants are lower than those from the other three locations. However, a comparison of the means through ANOVA showed no significant geographical variation (p ˃ 0.05). This means that any observed variations could be random plant to plant variations that cannot be accounted for by the difference in collection sites.
Mean amino acid concentrations of leaf samples from different locations
3 30 25 A 20 B 15 10 C 5 D 0 MeanConcentration nmol/cm
Val F ALA AiB - aILE LEU Β PRO GLU PHE GLN LYS GPR HIS TRY TRP
Figure 4.3: A comparison of the leaf mean concentrations of 14 amino acids of plants collected from four localities.
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4.1.5 RP-HPLC analysis of H2O and MeOH extracts
As part of the chemical variation, bark, fruit and leaf samples from R. melanophloeos were analysed in HPLC to screen for the occurrence of polar phytochemicals as discussed in Section 3.1.6. Both the methanol and water extracts of plants from the six localities were analysed to investigate similarities and differences between the three plant parts and plants from different localities. HPLC is a method of choice for the separation and authentication of crude plant extracts as an HPLC chromatogram gives the fingerprint and substantial information on the chemical composition of an extract.
4.1.5.1 RP-HPLC screening of H2O extracts
The aqueous extracts of the leaves, fruits and bark show an almost comparable chemical profile. Based on the retention time and UV spectra, three compounds (P 01, P 02, and P 03) are observed, accumulating in all three plant parts at varying levels. Fig 4.4 shows representative chromatograms of the aqueous extracts and Fig 4.5 shows the corresponding UV spectra of the three major compounds. More chromatograms are shown in Appendix 3. Compound P 01, has a retention time of 2 minutes and its UV spectra shows maximum absorption at 225 nm. Based on its absorption, this compound is likely to be a saponin since saponins typically absorb at short wavelengths. 27 The bark accumulates higher levels of this compound as depicted by the peak intensities and the fruits accumulate the lowest levels. Compound P 02 elutes at around 3 minutes and shows two absorption bands at 225 and 277 nm. This accumulates in all three plant parts as well with high levels in the bark and the lowest in the fruits. Compound P 03 elutes at around 24 minutes and has three absorption bands at 228, 254 and 349 nm. This accumulates in the leaves and fruits in relatively higher intensities compared to P 01 and P 02. The accumulation of P 03 in the bark is ambiguous as a small stump appears at around 24 minutes which could be assumed to be a P 03 peak at very low levels not detectable under the analysis conditions. Notably the leaves and fruits differ also from the bark in that their chromatograms show the occurrence of smaller peaks around P 03. These compounds have the same UV spectra as P03
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and could be assumed to be decomposition products of P 03 or compounds of the same class.
0.80 Fruits
P03 0.60
AU 0.40 P 01 P 02 0.20
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
0.80 Leaves
0.60 24.416 P03
AU P 01 0.40 P 02
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
0.80 P 01 Bark
0.60 2.149 3.170
AU 0.40 P 02 P03 0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
Figure 4.4: Representative chromatograms of H2O extracts of the leaves, fruits and bark R. melanophloeos.
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P01
P02
P03
Figure 4.5: UV spectra for the three major peaks from H2O extracts of the leaves, fruit and bark samples from R. melanophloeos.
4.1.5.2 RP-HPLC screening of methanol extracts
Representative chromatograms for the methanol extracts of the leaves, fruits and bark are shown in Fig 4.6 and corresponding UV spectra of the two main compounds are shown in Fig 4.7. It should be noted that chromatograms of the leaves and bark are of the same scale, and the fruit chromatogram has been expanded. The chemical profiles of the three plant parts differ remarkably both qualitatively and quantitatively. Similarities are only in the occurrence of two compounds eluting at 2 minutes (P 04) and 24 minutes (P 05). P 04 appears in the bark and leaves and is not detectable in the. Its UV spectrum shows maximum absorptions at 239 and 273 nm and is similar to that of P 02 from aqueous extracts. Its retention time, suggests that it is a very polar compound likely to be slightly extractable in methanol and more extractable in water hence it is observed in both extracts. Compound P 05 elutes at around 24 minutes and appears clearly in the fruits and leaves. In the bark it appears as a broad peak that is not well resolved, which could mean that it is a mixture of several related compounds not separable under the analysis conditions or it is a sugar. Judging from its UV absorption bands (255 and 348 nm), this compound is likely to be a flavonoid as flavonoids are characterised by two absorption bands from 240 to 285 nm and from 300 to 550 nm wavelength.28 Again the UV spectrum of P 05 is comparable to that of P 03 from the water extracts. In the leaves another compound elutes at
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15 minutes with a UV spectrum similar to that of P 05. These two are most probably compounds with the same structural backbone bone with different substituents. For example flavonoid compounds with OH substituents in different carbons.
0.10 Fruits 0.08 P05 0.06 AU 0.04
0.02
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
1.50 Leaves
1.00 P05 AU
0.50 P04
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
Bark 1.50
1.00 P05 AU P04
0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
Figure 4.6: Representative HPLC chromatograms of MeOH extracts of the leaves, fruits and bark.
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2.083 Peak 1 3.00 239.2 273.4
2.00 P04 AU
1.00
0.00 21.850 Peak 3 255.7
348.4 1.00 P05 AU
0.50
220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 nm
Figure 4.7: UV spectra of the two major compounds observed in the MeOH extracts of the leaves, fruits and the bark.
HPLC analysis managed to reveal differences that were otherwise not observed from TLC analysis. Generally the leaves, fruits and bark show slight qualitative and quantitative variations in their chemical profile of water and methanol extracts accounted for by the storage of secondary metabolites in different plant parts and in varying concentrations. Based on the retention time and UV spectra, three major compounds were observed to accumulate in the three plant parts in varying concentrations. Two of these accumulate in both the MeOH and H2O extracts and one is restricted to the water extracts. These compounds are associated with saponin and flavonoid structures and they lack any benzoquinone skeleton which typically shows absorptions at 290 and 430 nm.29 Clearly plants from different localities show the same chemical compositions as observed from their identical chromatograms in Appendix 3
4.1.6 Summary on the chemical variation study
The chemical variation study (extraction yields, TLC, GC-MS and HPLC) has confirmed the accumulation of several groups of phytochemicals in the leaves, fruits and bark. Some similarities and differences in their chemical profiles were established. Since the bioactivity of plant extracts is often correlated to their
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chemical composition, the next section reports on the antibacterial activity of the different solvent extracts of the leaves, fruits and the bark.
4.2 Antibacterial activity screening of crude extracts
MIC values of the PE, EtOAc and MeOH extracts of the leaves, fruits and bark were determined as an evaluation of their antimicrobial activity against six bacterial strains as discussed in Section 3.2. Extracts were further subjected to a bio-autography assay so as to localize individual bioactive compounds. A comparison of the activity of plants from different localities was done to investigate possible geographical variations in antibacterial activity.
4.2.1 Determination of MIC values
The MIC value is a quantitative measure of antimicrobial activity defined as the lowest concentration at which bacterial growth is inhibited. This value is not only influenced by the method used but also by the microorganisms tested, the extraction method and the degree of solubility of each test-compound.30 For crude extracts antimicrobial activity ranges between 8 and 1 mg/ cm3 and MIC values below 1 mg/ cm3 are considered exceptionally good.31
R. melanophloeos extracts generally showed good antibacterial activity against the six bacterial strains tested (B. cereus, E. coli, E. faecalis, K. pneumoniae, P. aeruginosa and S. epidermidis). However, the response for each bacterial strain tested was different and from the results extracts exhibited stronger activity against gram-positive strains compared to the gram-negative ones. Typically gram-negative bacteria are less susceptible because their cell walls have an outer lipo-polysaccharide membrane which prevents easy diffusion of compounds.32 The MIC values of all extracts (raw data) are shown in Appendix 4. These are summarized in Tables 4.5a to 4.5c which show the mean MIC values and standard deviation for the different extracts of the leaves, fruits and bark extracts against each of the six pathogens. The mean MIC values were calculated for each extract (PE, EtOAc, MeOH) of each plant part (leaf, bark, fruit) against each of the six pathogens.
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4.2.1.1 Antibacterial activity of the leaves
The mean MIC values of the PE, EtOAc and MeOH extracts of the leaves are shown in Table 4.5a. These showed good antibacterial activity against all the six pathogens. The strongest activity was exhibited by ethyl acetate extracts against S. epidermidis (mean MIC = 0.20 mg/ cm3) and lowest activity was exhibited by petroleum ether extracts against K. pneumoniae (mean MIC = 5.5 mg/ cm3). Generally ethyl acetate extracts exhibited the strongest activity (mean MIC ranging between 0.20 and 3.00 mg/ cm3) and petroleum ether extracts showed the lowest activity (mean MIC ranging between 0.61 and 5.5 mg/ cm3). From the sensitivity of pathogens towards the different extracts, it appears the non-polar and polar extracts have different modes of action. For the PE and EtOAC extracts the sensitivity decreases from S. epidermidis, B. cereus, E. faecalis, P. aeruginosa, E. coli and K. pneumoniae. Yet for the MeOH extracts sensitivity decreases from E. faecalis, B. cereus, S. epidermidis, E. coli, K. pneumoniae and P. aeruginosa.
Table 4.5a: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of leaf samples.
Pathogen Mean MIC mg/ cm3 PE EtOAc MeOH Control S. epidermidis 0.61 0.20 0.88 0.16x10-3 B. cereus 0.79 0.28 0.83 0.16x10-3 E. faecalis 0.88 0.29 0.80 0.63 x10-3 P. aeruginosa 1.15 0.55 1.88 0.31 x10-3 E. coli 1.25 0.67 1.00 0.63 x10-3 K. pneumoniae 5.50 3.00 1.05 0.63 x10-3 Average 1.56 0.83 1.06 0.5 x 10-3 Each value = mean activity of 10 samples against the specified pathogen Average = mean activity of 10 samples against all six pathogens calculated from absolute values Control = ciprofloxacin 0.01 mg/cm3
4.2.1.2 Antibacterial activity of the bark
The mean MIC values of the PE, EtOAc and MeOH extracts of the bark are shown in Table 4.5b. These showed good antibacterial activity with ethyl acetate extracts exhibiting the highest activity and the methanol extracts exhibiting the least
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activity. The strongest activity was exhibited by ethyl acetate extracts against B. cereus (mean MIC = 0.09 mg/ cm3) and the lowest activity shown by methanol extracts against P. aeruginosa (mean MIC = 3.40 mg/ cm3). Pathogen susceptibility was different for the non-polar and polar extracts. For ethyl acetate and petroleum ether extracts, pathogen susceptibility decreases from B. cereus, S. epidermidis, E. faecalis, E. coli, K. pneumoniae and P. aeruginosa. While for methanol extracts pathogen susceptibility decreases from S. epidermidis, E. faecalis, B. cereus and E. coli, K. pneumoniae and P. aeruginosa.
Table 4.5b: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of bark samples.
Pathogen Mean MIC value (mg/ cm3) PE EtOAc MeOH B. cereus 0.17 0.09 1.00 S. epidermidis 0.18 0.13 0.50 E. faecalis 0.67 0.19 0.67 E. coli 1.45 0.93 1.00 K. pneumoniae 1.43 0.95 1.10 P. aeruginosa 0.80 1.55 3.40 Average 0.76 0.64 1.28 Each value = mean activity of 10 samples against the specified pathogen Average = mean activity of 10 samples against all six pathogens calculated from absolute values
4.2.1.3 Antibacterial activity the fruits
The mean MIC values of the different extracts of the fruits are shown in Table 4.5c. Generally the fruits showed exceptionally good antibacterial activity with the highest activity exhibited by ethyl acetate extracts against B. cereus (mean MIC = 0.05 mg/ cm3) and the lowest activity exhibited by methanol extracts against P. aeruginosa (mean MIC = 2.67 mg/ cm3). As observed with the leaves and bark, the ethyl acetate extracts were the most active and methanol extracts were the least active. For the ethyl acetate and petroleum ether extracts pathogen susceptibility decreased from B. cereus, S. epidermidis, E. faecalis, K. pneumoniae, E. coli and P. aeruginosa. While for the methanol extracts the
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susceptibility decreased from S. epidermidis, K. pneumoniae, B. cereus, E. faecalis, E. coli and P. aeruginosa.
Table 4.5c: Mean MIC values (mg/ cm3) of PE, EtOAc and MeOH extracts of fruit samples.
Pathogen Mean MIC (mg/ cm3) PE EtOAc MeOH B. cereus 0.14 0.05 2.00 S. epidermidis 0.15 0.12 0.96 E. faecalis 0.30 0.11 2.33 K. pneumoniae 0.90 0.50 1.45 E. coli 1.17 0.67 2.33 P. aeruginosa 0.92 2.00 2.67 Average 0.58 0.57 2.00 Each value = mean activity of 6 samples against specific pathogen. Each sample was tested in duplicate Average = mean activity of 6 samples against all six pathogens calculated from absolute values
4.2.1.4 Total biological activity
To give a clear comparison of the efficacy of the three plant parts, the total activity was calculated (Table 4.6) as the total mass (mg) of plant extract prepared from 1◦g powdered plant material divided by the MIC value (mg/ cm3). A high total activity virtually means that a small mass of plant material can be diluted to a low concentration but still elicit the anticipated antibacterial activity.9 For the fruits the highest total activity was calculated for the ethyl acetate and petroleum ether extracts (189 and 129 cm3/ g respectively). In both cases the values recorded were almost 13 times higher than that of the bark and leaves. On the other hand for the bark and leaves, the highest total activity was calculated for the methanol extracts (160 and 156 cm3/ g respectively). However a high total activity for methanol extracts is less significant as methanol extracts primary metabolites, (which are less active) alongside the bioactive compounds. On average, the fruit samples clearly recorded the highest total activity. This implies that in addition to being more potent, the fruits are more efficacious rendering them the most viable plant part to use for medicinal purposes.
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Table 4.6: Total activity of R. melanophloeos bark, fruit and leaf extracts.
Extractant Plant Yield (Total activity cm3/ g) part (mg/g) Bc Ef Se Ec Pa Kp Mean PE B 7.3 43 11 41 5 9 5 19 F 35.7 255 119 238 31 39 40 120 L 17.7 18 20 29 14 16 3 17 EtOAc B 7.6 84 40 58 8 5 8 34 F 27.4 548 249 228 41 14 55 189 L 12.0 43 42 60 18 22 4 31 MeOH B 155.1 310 231 78 155 46 141 160 F 124.0 62 53 129 53 46 85 72 L 155.7 181 195 177 156 83 148 156 Key: Bc = B. cereus, Ef = E. faecalis, Ec = E. coli, Pa = P. aeruginosa, Kp = K. pneumoniae, B = bark, F = fruits, L = leaves
4.2.1.5 Geographical variation on antibacterial activity
The mean MIC data for ethyl acetate extracts of the leaves of plants collected from localities B, C, D and F are compared to assess if there is any variation between plants from different localities. The mean MIC values for each location were calculated and the data is presented in Table 4.7. There is some consistency in the activity of plants across the different locations. For instance the mean MIC value for P. aeruginosa is 0.50 mg/ cm3 in all five localities. Analysis of variance (ANOVA) in Microsoft Excel 2007 showed that there is no significant variation in the activity of plants from different localities (p ˃ 0.05). This implies that even though there may be random variation in the activity of individual plants it cannot be accounted for by differences in the collection site. This has a good bearing on the medicinal use of R. melanophloeos plants from a wide geographical distribution.
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Table 4.7: Mean MIC values (mg/ cm3) of leaf EtOAc extracts from 4 different localities.
Locality *Mean MIC (mg/ cm3)
E. S. B. P. E. K. faecalis epidermidis cereus aeruginosa coli pneumoniae Nhlangano (B) 0.38 0.25 0.19 0.50 0.50 5.00
Bhunya (C) 0.13 0.19 0.11 0.50 1.11 2.00
Fonteyn (D) 0.19 0.19 0.50 0.50 0.38 3.00
KZN (F) 0.38 0.19 0.25 0.50 0.50 2.00
Overall mean 0.29 0.20 0.28 0.50 0.67 3.00
Std deviation 0.17 0.06 0.18 0 0.57 1.51
*P-value 0.54 0.80 0.15 0 0.38 0.11
*Mean MIC, each value is the mean activity of plants collected from the same specified locality against a specific pathogen. *P-value was calculated from ANOVA, (p ≤ 0.05) implies statistically different means
4.2.2 Bio-autography assay An important characteristic of plant extracts is their complex chemical composition, containing different groups of bioactive compounds which either work individually or in unison to effect bioactivity. Often bio-autography is effectively used in identifying single bioactive compounds from complex crude plant extracts.8 R. melanophloeos extracts (PE, EtOAc and MeOH) of the bark, fruits and leaves were subjected to bio-autography. These were tested against B. cereus to localize individual bioactive compounds as discussed in Section 3.2.2. From all TLC plates no zones of inhibition were observed implying that no individual compound is responsible for the observed bacterial growth inhibition. This therefore implies that different components could be working in synergy to account for the good antibacterial activity observed. Synergy is defined as the interaction of different components of an extract to effect bioactivity. It is an important characteristic of phytomedicines and explains the difficulty often encountered in isolating single active components as well as the efficacy of apparently low doses of active ingredients in herbal mixtures.33 However, from experience it is not uncommon for bio-autography assays to give false negatives especially with non-polar
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phytochemicals.30 So, the possibility of individual bioactive compounds cannot be overruled especially when considering the small amounts of crude extracts spotted. Hence increase in the concentrations of extracts spotted could be useful in confirming these results.
4.2.3 Summary on the antibacterial activity of crude extracts
This study demonstrates that R. melanophloeos extracts from different localities exhibit good antibacterial activity against pathogens associated with ailments of the respiratory tract, stomach and skin. This provides an important basis for justifying the traditional medicinal uses of the plant against infectious diseases. The highest activity was observed with the EtOAC extracts of the fruits against S. epidermidis (MIC = 0.003 mg/ cm3) and lowest activity was observed with PE extracts of the leaves against K. pneumoniae (MIC ˂ 8 mg/ cm3). Of particular interest is the high activity exhibited against S. epidermidis and B. cereus. S. epidermidis is one of the nasty opportunistic pathogens responsible for most blood stream nosocomial infections especially in patients admitted in Intensive Care Units (ICU).29 Based on the plant‟s activity, infections from this pathogen could possibly be avoided with the traditional use of R. melanophloeos extracts. B. cereus on the other hand is notorious for developing resistance towards most available commercial antibiotics, and from the observed activity this plant could be a good candidate in the search for natural antimicrobial agents against drug resistant bacterial strains.34
However, the observations in this study contradict previous reports on the poor activity exhibited by water extracts of the bark against E. coli, P. aeruginosa and S. epidermidis from the plate hole diffusion assay.35 The discrepancy could arise from the use of different bioassay methods. Available evidence suggests that diffusion methods are sometimes not reliable especially with non-polar compounds whose hydrophobic nature prevents uniform diffusion.30 Whereas the microdilution assay used in this study, is considered as one of the more sensitive assays.36 The different extraction techniques and extraction solvents used could also account for the discrepancy. In this study sequential extraction with organic solvents was
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employed compared to a single step water extraction used previously. Some experiments comparing the two methods revealed that sequential extractions provide extracts with stronger antibacterial and antioxidant activity.37 Even more, water extracts tend to exhibit poor antimicrobial activity as compared to organic solvent extracts.36
The effectiveness of medicinal plants is often affected by several variables including biochemical factors within individual species, plant part extracted and external factors such as climate, geographical location, season and growth conditions.5 A comparison of the antibacterial activity of the leaves, fruits and bark reveal minor random variations in their activity. For instance, PE and EtOAc extracts of the fruits exhibit the highest activity compared to the corresponding extracts of the bark and leaves. For these extracts high extraction yields were recorded by the fruit samples and from TLC analysis more components were extracted from the fruit samples. In essence this could explain the high activity observed with the PE and EtOAc extracts of the fruits. The accumulation of chlorophylls which reportedly affect bioactivity in the leaves could account for their low activity as well. However, MeOH extracts of the leaves exhibit the highest activity towards all pathogens as compared to the corresponding extracts of the bark and fruits. From TLC analysis the MeOH extracts of the leaves, fruits and bark had shown a similar chemical profile though HPLC revealed some minor quantitative and qualitative differences. Since synergistic interactions were noted for the extracts, the difference in antibacterial activity could be attributed to differences in quantities of various components. Despite the minor differences, the activity of the different plant parts is comparable and overall the fruits exhibited the highest or strongest activity followed by the bark and the leaves exhibited the lowest. This observation is in accordance with the traditional medicinal uses of the plant where the bark and fruits are commonly used against infectious ailments.10
The comparable MIC values of extracts of the bark, fruits and leaves suggest that they can be used interchangeably for the same ailments. Due to overexploitation an unsustainable harvesting, R. melanophloeos is listed as one of 34 plants reportedly becoming scarce and as such prioritized for conservation in the Eastern
110 Chapter4: Results and Discussions
Cape Province. 38 In view of the antibacterial activity of the three plant parts, harvesting of the leaves and fruits in substitution of the bark may be encouraged to promote conservation of the plant. Compared to debarking, harvesting of the leaves and fruits evidently inflict less damage to plants. Consequently substitution of the bark and bulbs with leaves, fruits or twigs is considered as an effective conservation strategy for medicinal plants. Accompanied with strict phytochemical and pharmacological evaluation of different plant parts, it could promote sustainable harvesting and simultaneously provide similar medicinal benefits.21
The antibacterial activity of plant extracts is also affected by the extraction solvent used as different solvents extract different types of compounds depending on their polarity and solubility.36 A comparison of the PE, EtOAc and MeOH extracts shows that EtOAc extracts exhibit the strongest activity in all three plant parts. While for the fruits and bark MeOH extracts exhibit the least activity and for the leaves PE extracts are the least active. This implies that in R. melanophloeos the non-polar and medium polar compounds are the most active. This could justify the poor activity exhibited by aqueous extracts of the bark reported by Steenkamp as water extracts only the polar phytochemicals.35 The high activity of EtOAC extracts could be attributed to the substantial amounts of benzoquinones and the activity of PE extracts could be attributed to the components of essential oils as observed in TLC and GC-MS analysis.
Even though no single bioactive compound was identified or localized from the bio-autography, an attempt was made to isolate major compounds from the fruits. This is presented and discussed in the next section.
4.3 Structure elucidation of isolated compounds
Isolations were done from the fruiting bodies since these showed the best activity in the biological tests, and these are a major focus of many of the ethnobotanical uses. To the best of our knowledge only embelin (a benzoquinone) and related benzoquinones have previously been isolated from the fruits. In this study a total of eight compounds were isolated as discussed in Section 3.3. These were
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subjected to different spectroscopic techniques (FT-IR, 1H-NMR, 13C-NMR and 2D-NMR experiments (COSY, HMBC, HSQC) for structure elucidation. This section reports on the structural elucidation of the isolated compounds. It should be noted that structure elucidation for compounds TL 03 and TL 07 is not presented in this section because TL 03 could not be effectively purified and very low yields were obtained for TL 07.
4.3.1 Structure elucidation of compound TL 01
This was isolated as a purple solid (4 mg) from the polar fraction of the fruits (methanol soluble fraction). It has very poor solubility and is only partly soluble in
DMSO. TLC analysis in TLC 3 showed a single spot (Rf = 0.7) which stains purple- blue on visualization with vanillin-sulfuric acid. The bright purple colour of this compound is typical of benzoquinones which are often characterised by very bright colours. 39 The spectral data (Appendix 5) as well is characteristic of a 1,4- benzoquinoid skeleton with an alkyl side chain. FT-IR (neat) spectrum showed major absorption bands at 3344 cm-1 (O-H), 2920 and 2851 cm-1 (C-H), 1600 cm-1 as well as 1529 cm-1and 1375 cm-1 (aromatic C-H). The proton NMR (1H-NMR 400
MHz (CD3)2SO) demonstrates chemical shifts at δ ppm, 7.49 (d, J = 8.1 Hz, 1H), 7.10 (dd, J = 15.8, 8.0 Hz, 1H), 5.45 (s, 1H), 4.23 (bs, 2H), 1.18 (m, 14H), 0.79 (m,
3H). Carbon NMR (101 MHz, (CD3)2SO) shows chemical shifts at δppm 184.2, 182.8, 135.1, 132.2, 127.2, 119.9, 49.1, 29.6 – 29.1 and 14.4. The chemical shifts observed around 180 ppm correspond to resonances of carbonyl carbons and those around 130 ppm correspond to resonances within an aromatic ring. Because there was not sufficient sample for 2D NMR experiments, the connectivities of fragments could not be determined. However, the available spectral data is consistent with those of known 1,4-benzoquinones with long alkyl side chains.29 Thus the compound can tentatively be identified as an alkyl benzoquinone and a possible skeleton is presented in Fig 4.8.
112 Chapter4: Results and Discussions
1 Table 4.8: H NMR (400 MHz, (CD3)2CO) data for TL 01
*Assignment Chemical shift Multiplicity, J (Hz) No. of H Possible fragments a 7.49 d,8.1 1 -CH (aromatic) b 7.10 dd, 15.8, 8.0 1 -CH (aromatic) c 5.45 s 1 =CH or OH d 4.23 s 2 -CH2 or 2x OH e 1.18 unresolved multiplet 14 (-CH2)14 f 0.79 distorted triplet 3 -CH3 *Assignments based on 13C NMR spectral data.
O
b c a R= alkyl chain R
O
Figure 4.8: A 1,4-benzoquinone skeleton consistent with the spectral data for compound TL 01.
4.3.2 Structure elucidation of compound TL 02
This compound was isolated from the methanol soluble fraction as a white amorphous solid soluble in methanol and water. TLC analysis (in TLC 3) showed a single spot with Rf = 0.7 staining yellow on visualization with vanillin-sulfuric acid. The FT-IR (in MeOH) spectrum showed major absorption bands at 3260 cm-1 (OH), 2953 and 2844 cm-1 (C-H), and 1647 cm-1 (C=O). The 1HNMR spectrum was dominated by solvent residual peaks which could be accounted for by rather low sample quantities. For that reason the compound could not be characterised any further.
4.3.3 Structure elucidation of compound TL 04
This compound (2.6 mg) was isolated from the non-polar (hexane soluble) fraction of the fruits as a white sticky solid. TLC analysis in Hex-CHCl3 (100:1) showed a
113 Chapter4: Results and Discussions
single spot (Rf = 0.4) appearing as white fluorescence under UV366 nm and not visible on visualization with vanillin-sulfuric acid. The FT-IR spectrum (in CHCl3) showed major absorption bands at 3456 cm-1(C=C), 2917 cm-1and 2849 cm-1 (C- H), 1709 cm-1 (C=O), 1365 cm-1 as well 1215 cm-1 (C-H). Proton NMR (1H NMR
400 MHz, CDCl3) showed chemical shifts at δppm 5.32 (m, 1H), 3.67 (s, 2H), 1.99 (s, 2H), 1.54 (s, 12H), 1.23 (m, 32 H), 0.85 (t, J = 6.8 Hz, 6H). Carbon NMR (13C
NMR 101 MHz, CDCl3) spectrum demonstrated resonances at δppm 172.4, 134.9, 31.6, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 24.3, 14.1). The carbon NMR spectrum confirms the carbonyl group indicated by the FT-IR spectrum as well as the unsaturation observed from the HNMR spectrum. Even though the structure could not be fully resolved, the preliminary spectral data indicates that the compound is typically a low molecular weight, non-polar terpenoid with no aromatic rings and hydroxyl functional groups. It should be noted that the spectral data for compound TL 05 is identical to that of TL 04 thus it is not presented.
4.3.4 Structure elucidation of compound TL 06
This compound (7 mg) was isolated as a white amorphous solid from the non- polar fraction (hexane soluble). TLC analysis in CHCl3-MeOH (100:1), showed a single spot (Rf = 0.2) not visible under UV and staining blue on visualization with vanillin-sulfuric acid. The FT-IR (CHCl3 ) spectrum showed major absorption bands at 2927 and 2840 cm-1 (C-H), 2253 cm-1 (C≡C), 1708 cm-1 (C=O). Clearly this indicates the lack of aromaticity and hydroxyl functional groups which is further confirmed by both the 1H NMR and 13C NMR spectra. The 1H NMR spectrum indicates the presence of a long alkyl chain, two terminal methyl group as well as a double bond which is confirmed by 2 carbon resonances at δc 129.0 and 127 ppm in the 13C NMR spectrum. The 13C NMR also indicates the presence of two carbonyl carbons at δc 173 and 179 ppm. Even though the FT-IR spectrum indicates the presence of an alkyne functional group, it could not be confirmed from the H-NMR and C-NMR spectra hence it was ruled out as an impurity. The compound was subjected to COSY, HSQC and HMBC experiments to determine the connectivity of proton and carbon resonances. On basis of the available NMR
114 Chapter4: Results and Discussions
data (Table 4.9), TL 06 can tentatively be identified as pentacos-4-ene-6,7-dione
(C25H46O2) shown in Fig 4.9.
Table 4.9: 1H NMR (400 MHz), 13C NMR (100 MHz) and 2D NMR {1H-NMR (300 MHz) and 13C-NMR (75 MHz)} data for compound TL 06
*C assignment δc (ppm) δH (ppm), J (MHz) COSY HMBC 1 173.2 - - - 2 178.5 - - 3, 4 3 33.7 2.30 t (7.5) 4 5, 2 4 24.41 1.61 dd (14.0, 6.8) 3, 5 3, 5, 2 5-18 29.0 - 31.9 1.23 m 4 3 19 22.7 1.23 m 20 - 20 14.1 0.86 t (7.5) 19 - 21 129.7 *5.34 m - 23 22 127.9 *5.32 m 23 - 23 27.2 1.99 dd (15.2, 6.6) 22, 24 21, 1, 25 24 22.6 1.23 m 23 - 25 19.2 0.86 t (7.5) 24 23 *Signals unclear due to overlapping *Assignments determined from HSQC, HMBC and COSY experiments.
O 21 1 2 23 3 22 5 O CH 4 24 3 7 6 9 8 11 10 13 12 15 14 17 16 19 18
3CH 20
Figure 4.9: Compound TL 06, pentacos-4-ene-6,7-dione.
115 Chapter4: Results and Discussions
4.3.5 Structure elucidation of compound TL 08
Compound TL 08 (2 mg) was isolated through PTLC from the non-polar fraction of the fruits as an amorphous yellow solid. TLC analysis in Hex-EtOAc (2:3) showed a single spot with Rf = 0.8 staining red in vanillin-sulfuric acid. The FT-IR (in -1 CHCl3) spectrum showed absorption bands at 3384 cm (C-OH), 2925 and 2854 cm-1 (C-H), 1599 cm-1 (C=O) as well as 1375 and 1230 cm-1 (C=C). 1H NMR (400
MHz, CDCl3) δppm 0.86 (t, 39H), 1.26 (m, 168H),1.56 (m, 73H), 2.00 (d, 22H), 2.15 (s, 4H), 2.45 (t, 8H), 2.60 (s, 3H), 2.76 (s, 4H), 3.47 (s, 1H), 5.33 (d, 10H). The HNMR spectrum is almost similar to that of compound TL 06 which means that these are closely related compounds. This compound is likely to be one of the non-polar compounds observed from GC-MS analysis. The spectral data suggest that it could be a low molecular weight terpenoid. Due to extensive overlapping in the NMR spectra, the structure could not be fully resolved. The FT-IR and proton NMR spectra are shown in Appendix 5.
116 Chapter4: Results and Discussions
References
1 Nyika A. (2009). The Ethics of Improving African Traditional Medical Practice: Scientific or African Traditional Research Methods? Acta Tropica 112: 32-36.
2 Gurib-Fakim A. (2006). Medicinal Plants: Traditions of Yesterday and Drugs of Tommorrow. Molecular aspects of Medicine 27: 1-93.
3 Van Wyk, B-E., Van Oudtshoorn, B., Gericke, N. (1997). Medicinal plants of South Africa. Briza Publications, Pretoria. Pg 208.
4 Szabolcs N. (2004). Seperation Strategies of Nyika A. (2009). The Ethics of Improving African Traditional Medical Practice: Scientific or African Traditional Research Methods? Acta Tropica 112: 32-36.
4 Gurib-Fakim A. (2006). Medicinal Plants: Traditions of Yesterday and Drugs of Tommorrow. Molecular aspects Plant Constituents – Current Status. Journal of Chromatography B 812: 35-51.
5 Burwa L.V., Van Staden J. (2007). Effects of Collection Time on the Antimicrobial Activity of Harpephyllum caffrum Bark. South African Journal of Botany 73: 242-247.
6 Walton N.J., Brown D.E. (1999). Chemicals from Plants: Perspectives on Plant Secondary Products. Imperial College Press, London. pg 91-186.
7 Mann J., Davidson B.S., Hobbs J.B., Banthorpe D.V., Harbone J.B. (1994). Natural products, their chemistry and biological significance. Longman group UK. Pg 1-40.
8 Mendonça-Filho R.R. (2002). Bioactive Phytocompounds: New Approaches in the Phytosciences. In Ahmad I, Aqil F., Owais M., Modern Phytomedicine: Turning Medicinal Plants into Drugs. Wiley- VCH Verlag. Pp 1-24.
9 Eloff J.N. (2004). Quantification of the Bioactivity of Plant Extracts during Screening and Bioassay guided Fractionation. Phytomedicine 11: 370-371.
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10 Hutchings A., Scott A.H., Lewis G., Cunningham A. (1996). Zulu Medicinal Plants: An Inventory. University of Natal Press, Scottsville. pp 227-228.
11 Turner C.E., Williamson D.A., Straud P.A., Talley D.J. (2004). Evaluation and Comparison of Commercially Available Aloe vera L. Products Using Size Exclusion Chromatography with Refractive Index and Multi-angle Laser Light Scattering Detection. International Immunopharmacology 4: 1727-1737.
12 Hamman J.H. (2008). Composition and Application of Aloe vera Gel. Molecules 13: 1599-1616.
13 Schmourlo G., Mendonça-Filho R.R., Alviano C.S., Costa S.S. (2005). Screening of Antifungal Agents Using Ethanol Precipitation and Bioautography of Medicinal and Food Plants. Journal of Ethnopharmacology 96: 563-568.
14 Neuwinger H.D. (2000). African Traditional Medicine: A Dictionary of Plant Use and Applications with Supplement Search System for Diseases. Medpharm Scientific Publications, Germany. Pg 434.
15 Heinrich M., Barnes J., Gibbons S., Williamson E.M. (2004). Fundamentals of Pharmacognosy and Phytotherapy. Churchill Livingstone, Spain. Pp 3- 24.
16 Wagner H., Bladt S. (1996). Plant Drug Analysis. A Thin Layer Chromatography Atlas. 2nd edition. Springer-Verlag, New York. Pg 166.
17 Joshi R., Kamat J.P., Mukherjee T. (2007). Free Radical Scavenging Reactions and Antioxidant Activity of Embelin: Biochemical and Radiolytic Studies. Chemico-Biological Interactions 167: 125-134.
18 Vincken J-P., Heng L., De Groot A., Gruppen H., (2007). Saponins: Classification and Occurrence in the Plant Kingdom. Phytochemistry 68: 275- 297.
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19 Hostettmann K., Marston A., Ndjoko K., Wolfender J–L., (2000). The Potential of African Plants as Sources of Drugs. Current Organic Chemistry 4, 973-1010.
20 Van Wyk B-E, Van Oudtshoorn B., Gericke N., (1997). Medicinal Plants of South Africa. Briza Publications Pretoria. pg 208.
21 Zschocke S., Rabe T., Taylor J.L.S., Jager A.K., Van Staden J. (2000) Plant Part Substitution – A Way to Conserve Endangered Medicinal Plants? Journal of Ethnopharmacology 71: 281-292.
22 Burt S. (2004). Essential Oils: Their Antibacterial Properties and Potential Application in Foods - A Review. International Journal of Food and Microbiology 94: 223-253.
23 Okoh O.O., Sadimenko A.P, Afolayan A.J. (2010). Comparative Evaluation of the Antibacterial Activities of the Essential Oils of Rosmarinus officinalis L. Obtained by Hydrodistillation and Solvent Free Microwave Extraction. Food Chemistry 120: 308-312.
24 Bakkali F., Averbek S., Averbek D., Idaomar M. (2008). Biological Effects of Essential Oils – A Review. Food and Chemical Toxicology 46: 446-475.
25 Van Wyk B-E., Albrecht C. (2008). A Review of the Taxonomy, Ethnobotany, Chemistry and Pharmacology of Sutherlandia frutescens (Fabaceae). Journal of Ethnopharmacology 119: 620-629.
26 Yu-Haey K, Fumio I., Fernand L. (2003). Neuroactive and Other Free Amino Acids in the Seed and Young Plants of Panax ginseng. Phytochemistry 62: 1087-1091.
27 Oleszek W.A. (2002). Chromatographic Determination of Plant Saponins. Journal of Chromatography A 967: 147-162.
28 De Rijke E., Out P., Niessen W.M.A., Ariese F., Gooijer C., Brinkman U.A.Th. (2006). Analytical Seperation and Detection Methods for Flavonoids. Journal of Chromatography A 1112: 31-63.
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29 Drewes S.E., Khan F., Van Vuuren S.F., Viljoen A.M. (2005). Simple 1,4- Benzoquinones with Antibacterial Activity from the Stems and Leaves of Gunnera perpensa. Phytochemistry 66: 1812-1816.
30 Klančnik A., Piskernik S., Jeršek B., Možina S.S. (2010). Evaluation of Diffusion and Dilution Methods to Determine the Antibacterial Activity of Plant Extracts. Journal of Microbiological Methods 81: 121-126.
31 Van Vuuren S.F. (2008). Antimicrobial Activity of South African Plants. Journal of Ethnopharmacology 119: 603-613.
32 Fennell C.W., Lindsey K.L., McGaw L.J., Sparg S.G., Stafford G.I., Elgorashi E.E., Grace O.M., Van Staden J. (2004). Assessing African Medicinal Plants for Efficacy and Safety: Pharmacological Screening and Toxicology. Journal of Ethnopharmacology 94: 205-217.
33 Williamson E.M. (2001). Synergy and Other Interactions in Phytomedicines. Phytomedicine 8 (5): 401-409.
34 Hajji M., Jarraya R., Lassoued I., Masmoudi O., Damak M., Nasri M. (2010). GC-MS and LC-MS Analysis, Antioxidant and Antimicrobial Activity of Various Solvent Extracts from Jalapa mirabilis Tubers. Process Biochemistry 45: 1486-1493.
35 Steenkamp V., Fernandes A.C., Van Rensburg C.E.J. (2007). Antibacterial Activity of Venda Medicinal Plants. Fitoterapia 78: 561-564.
36 Eloff J.N. (1998). Which Extractant Should be Used for the Screening and Isolation of Antimicrobial Components from Plants? Journal of Ethnopharmacology 60: 1-8.
37 Hayouni E.A., Adedrabba M., Bouix M., Hamdi M. (2007). The Effects of Solvents and Extraction Method on the Phenolic Contents and Biological Activity in vitro of Tunisia Quercus coccifera L. and Juniperus phoenicea L. Fruit Extracts. Food Chemistry 105: 1126-1134.
38 Dold A.P., Cocks M.L. (2002). The Trade in Medicinal Plants in the Eastern Cape Province, South Africa. South African Journal of Science 98: 589-597.
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39 Abraham I., Joshi R., Paedasani P., Pardasani R.T. (2011).Recent advances in 1,4 Benzoquinone Chemistry. Journal of Brazil Chemical Society 22,3 : 385-421.
121
CHAPTER 5…
CONCLUSIONS AND RECOMMENDATIONS
Introduction
This chapter gives a concise summary of the results and main outcomes that have risen from this study. Recommendations for future work are presented as well.
5.1 Conclusions
The set objectives of the study were explored and from the obtained results the following conclusions can be drawn:
Substantial amounts of non-polar, medium polar, and polar phytochemicals are extractable from the leaves, fruits and bark of R. melanophloeos. The highest yields are extractable with methanol and compared to the bark and fruits the percentage yields of leaf samples are the highest. The high percentage yields of methanol extracts could suggest high levels of polar phytochemicals or it could be attributed to methanol being a broad spectrum solvent capable of extracting high molecular weight compounds like sugars and tannins. Significant for the medicinal uses of the plant is the accumulation of alcohol precipitable solids (APS) associated with wound healing and anti-diabetic properties. Substantial amounts (higher than those normally reported in the aloe industry) are extractable from the leaves, fruits and the bark. Compared to the leaves and the bark, higher yields were recorded from the fruits. Accumulation of non-polar to polar phytochemicals as well as the APS‟s shows random plant to plant variation within and between plant populations. This could be accounted for by differences in plant age and developmental stage.
122 Chapter 5: Conclusions and Recommendations
TLC analysis of the three plant parts revealed the accumulation of non- polar phytochemicals which are components of essential oils and confirmed previous literature reports on the accumulation of benzoquinones, tannins, flavonoids and saponins. The accumulation of different groups of phytochemicals show both qualitative and quantitative variations between different plant parts. The chemical profiles of polar extracts (methanol and water) of all plant parts accumulate the same groups of compounds (tannins, saponins and flavonoids) with variations in concentrations. However, with the non-polar and medium-polar extracts the chemical profile of the leaves and bark are more comparable while the fruits accumulate extra compounds which are missing in the leaves and bark. Random plant to plant variation within populations and between populations occurs especially in the concentrations of compounds. However, no distinct geographical trend in the accumulation of compounds was noted, which has a good bearing on the medicinal use of the plant in different communities for the same ailments.
GC-MS analysis of non-polar (petroleum ether) extracts confirms the accumulation of non-polar, volatile low molecular weight compounds making up essential oils observed from TLC analysis. Based on the retention time and mass spectra three (out of five) compounds were tentatively identified as (-)-spathulenol, caryophyllene oxide, and α-cardinol. The chemical composition of the leaves and fruits showed similarities with caryophyllene oxide accumulating as the major compound. As expected, with all five compounds high levels were noted in the fruit and leaves with trace amounts in the bark. Typical of essential oil components, the five compounds showed remarkable plant to plant qualitative and quantitative variation between and within populations.
The accumulation of protein and non-protein amino acids in all three plant parts was confirmed from GC-MS analysis of aqueous extracts. A total of 29 free amino acids were identified and quantified from the leaves, fruits and bark. Although the concentrations are typically low, the fruits
123 Chapter 5: Conclusions and Recommendations
accumulate higher amino acid concentrations compared to the bark and leaves. As amino acids are associated with immunomodulatory properties, their accumulation is important in justifying the traditional medicinal uses of R. melanophloeos for ailments of the neuroactive system. The non- detection of amino acids associated with toxicity particularly has a good bearing on the plant‟s medicinal value. Amino acid accumulation in all plant parts shows no significant geographical variation.
RP-HPLC analysis of methanol and water extracts further revealed minor differences in the chemical profile of the leaves, fruits and bark. Three possible major compounds with high response factors were observed. Two of these occur in both the water and methanol extracts and the third compound occurs only in the water extracts. The UV spectra of the three compounds suggest that they are different from the benzoquinones reported as major compounds in Myrsinaceae plants. The accumulation of these compounds shows quantitative variations between the different plant parts, and no geographical variation is evident as plants from different localities show the same chemical profile with slight variations in concentrations.
Contrary to previous antimicrobial tests on R. melanophloeos extracts, this study demonstrates that crude extracts of the bark, fruit and leaf extracts exhibit good antibacterial activity against B. cereus, E. faecalis, S. epidermidis, E. coli, K. pneumoniae and P. aeruginosa. No geographical variation is evident in the antibacterial activity of extracts. The observed activity provides an important basis for justifying the traditional medicinal uses of the plant against skin, stomach and respiratory infections. The highest activity is exhibited against gram positive strains and the fruits show superior activity as compared to the bark and leaves. Compared to the non- polar and polar phytochemicals, the medium polar phytochemicals (extractable with ethyl acetate) exhibit the highest activity in all plant parts. From the bio-autography assay, no individual compound was identified as being active implying synergistic interaction of compounds. The crude
124 Chapter 5: Conclusions and Recommendations
extracts, mostly fruit ethyl acetate hypothetically possess a potential for clinical applications and further pharmacological and toxicity evaluation could be necessary to confirm this hypothesis.
From the fruit methanol extract, about six compounds were isolated and purified. Two of these were isolated from the polar fraction and four from the non-polar fraction. From spectral analysis, one compound (non-polar) was successfully characterised as pentacos-4-ene-6, 7-Dione. Although the other 3 non polar compounds could not be fully characterised their preliminary spectroscopic data suggest the presence of a terpenoid skeleton and are highly likely to be the components of essential oils detected from GC-MS analysis. Interestingly, preliminary spectroscopic analysis of the polar compounds suggests that only one them possesses a 1,4-benzoquinone skeleton which is characteristic of the major compounds in the family.
5.2 Recommendations
From the outcomes of the study the following recommendations for future work can be made:
The crude extracts especially the fruits have shown good antibacterial activity and hypothetically possess a potential in the development of standardised phytomedicines. Further studies on toxicity, mode of action and possibly time-kill assays (death kinetics assays) are necessary to further confirm the efficacy and safety of the plant.
Since the plant is often used in mixtures with other plants, studies on their synergy would also add value to the established medicinal properties of the plant.
125
APPENDIX 1
Thin layer chromatography visualisation reagents Visualisation reagents for chemical detection of TLC plates, their preparation procedure and colour changes with different groups of phytochemicals. 1
Visualizing reagent Preparation Groups of Observed colours procedure phytochemicals detected
Vanillin in Sulphuric To 1g vanillin add 5 Essential oils red, blue, purple, acid (VS) cm3 sulfuric acid and brown, make up to 200 cm3 with ethanol. Spray Flavonoids yellow plate and heat at 110 °C for about 5 minutes Saponins blue, blue-violet, or until colours just appear. Tannins strong red
3 Chromic acid To 50 cm H2O add Carbohydrates blue – black (appear 41.2 cm3 concentrated as white spots on a
H2SO4 in ice, cool and yellow background for
add 5 g Na2Cr2O7 overbaked plates) make up to 100 cm3. Spray plate in fumehood and bake in oven until colours become visible
1. Wagner H., Bladt S. (1996). Plant Drug Analysis. A Thin Layer Chromatography Atlas. 2nd edition. Springer-Verlag Heidelberg, New York. pg 166.
126
APPENDIX 2...
______
GC-MS chromatograms of the leaves, fruits and bark samples collected from 6 different localities.
GC-MS chromatograms of the leaves
BL2 CL3
BL3 CL4
DL1 CL1
127
DL3 FL1
DL5 FL2
FL3 EL1
128 Appendix 3
GC-MS chromatograms of the bark
AB1 DB3
BB2 FB1
FB2 CB1
129 Appendix 3
GC-MS chromatograms of the fruits
AF1
BF2
130
APPENDIX 3...
HPLC Chromatograms of the methanol and water extracts of the leaves, bark and fruits of plants collected from six different localities.
HPLC chromatogram of acetone
2.00 AU 1.00
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
HPLC chromatograms of water extracts of the leaves
BL2
0.60 4.686 26.351 0.40 AU
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
CL4
0.60 22.085 0.40 AU
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes DL2
0.40 24.718 AU 0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
131 Appendix 4
EL1
1.00 21.751 2.625 AU 1.920 0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
FL1
0.20 21.008 2.934 AU 0.10
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
HPLC chromatograms of water extracts of the bark
BB2 0.60
0.40 2.018 AU
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes CB4
0.60 2.033
0.40 3.027 AU
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes DB2 0.80
0.60 2.149 3.170
AU 0.40
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
132 Appendix 4
EB1 0.60
0.40 2.018 AU
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes FB1 0.06
0.04 AU
0.02
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
HPLC chromatograms of water extracts of the fruits
AF1
0.30
0.20 AU
0.10
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes BF2
0.80
0.60
AU 0.40
0.20
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
133 Appendix 4
HPLC chromatogram of methanol extracts of the leaves, fruits and bark
HPLC chromatograms of methanol extracts of the leaves
AL1 3.00
2.00 1 AU
1.00
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes
BL1
1.00 22.320 AU 1.994 0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes CL1
1.00 2.053 22.951 AU 0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
134 Appendix 4
DL5
0.80 2.276
0.60 22.758
AU 0.40
0.20
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes FL1
1.00 1.855 21.486 AU 0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes HPLC chromatograms of methanol extracts of the bark
AB1
3.00
2.00 AU
1.00
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
BB3
1.50
1.00 AU
0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
135 Appendix 4
CB2 1.50
1.00 2.138 AU
0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
DB3
1.00 2.258 AU
0.50
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
EB1
2.00 AU
1.00
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes FB1
3.00
2.00 AU
1.00
0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
136 Appendix 4
HPLC chromatograms of methanol extracts of the fruits
AF1 0.10
0.08
0.06 AU 0.04
0.02
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
BF2
0.10
0.08
0.06 AU 0.04
0.02
0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes
137
APPENDIX 4...
Minimum inhibitory concentration (MIC) values (mg/ cm3) of petroleum ether, ethyl acetate, and methanol extracts of the leaves, bark and fruits from Rapanea melanophloeos. MIC values (mg/ cm3) of petroleum ether, ethyl acetate, and methanol extracts of fruit samples Extractant Sample *MIC (mg/ cm3) name E. S. B. P. E. K. faecalis epidermidis cereus aeruginosa coli pneumoniae Petroleum A1 0.13 0.13 0.25 0.50 2.00 >8.00 ether B2 0.25 0.13 0.13 1.00 1.00 2.00 B3 1.00 0.19 0.38 1.00 1.00 0.50 B5 0.13 0.13 0.02 2.00 1.00 0.50 B6 0.13 0.13 0.02 0.50 1.50 0.50 D1 0.13 0.13 0.01 0.50 0.50 1.00 Average 0.30 0.15 0.14 0.92 1.17 0.90 Std deviation 0.35 0.24 0.15 0.58 0.52 0.65
Ethyl A1 0.01 0.02 0.03 2.00 0.50 0.50 acetate B2 0.04 0.12 0.10 2.00 1.00 0.50 B3 0.50 0.38 0.13 2.00 0.50 0.50 B5 0.06 0.02 0.02 2.00 0.50 0.50 B6 0.04 0.19 0.03 2.00 1.00 0.50 D1 0.03 0.0001 0.003 2.00 0.50 0.50 Average 0.11 0.12 0.05 2.00 0.67 0.50 Std deviation 0.19 0.15 0.05 0.00 0.26 0.00
Methanol A1 4.00 1.00 2.00 2.00 4.00 1.50 B2 2.00 0.50 2.00 2.00 2.00 >8.00 B3 2.00 0.75 2.00 4.00 2.00 2.00 B5 2.00 2.00 2.00 4.00 2.00 2.00 B6 2.00 1.00 2.00 2.00 2.00 2.00 D1 2.00 0.50 2.00 2.00 2.00 2.00 *Average 2.33 0.96 2.00 2.67 2.33 1.45 Std deviation 0.81 0.56 0.00 1.03 0.82 0.76 *Each value is the mean activity of at least 2 independent tests. *Average calculated from absolute values.
138 Appendix 5
MIC values (mg/ cm3) of petroleum ether, ethyl acetate, and methanol extracts of the leaves. Extractant Sample MIC (mg/ cm3) name E. S. B. P. E. K. faecalis epidermidis cereus aeruginosa coli pneumoniae A1 2.00 0.75 0.50 1.00 1.00 8.00 Petroleum B3 1.00 0.50 3.00 1.00 2.00 4.00 ether B4 2.00 0.75 0.50 2.00 2.00 >8.00 C2 0.25 1.00 0.50 1.00 2.00 8.00 C3 0.25 0.50 0.19 1.00 1.00 8.00 D1 0.50 0.75 0.50 2.00 1.00 4.00 D5 0.38 0.50 0.25 1.00 1.00 4.00 E1 1.00 0.38 1.00 0.50 0.50 >8.00 F1 1.00 0.50 0.50 1.00 1.00 4.00 F2 0.50 0.50 1.00 1.00 1.00 4.00 Average 0.89 0.61 0.79 1.15 1.25 5.50 Std deviation 0.66 0.38 1.64 0.47 0.54 2.07 A1 0.50 0.25 0.25 0.50 1.00 4.00 Ethyl B3 0.25 0.25 0.13 0.50 0.50 6.00 acetate B4 0.50 0.25 0.50 0.50 0.50 4.00 C2 0.13 0.13 0.13 0.50 0.22 2.00 C3 0.13 0.25 0.08 0.50 2.00 2.00 D1 0.13 0.13 0.50 0.50 0.75 4.00 D5 0.50 0.25 0.50 0.50 0.25 2.00 E1 0.50 0.13 0.25 1.00 0.50 2.00 F1 0.13 0.25 0.25 0.50 0.50 2.00 F2 0.13 0.13 0.25 0.50 0.50 2.00 Average 0.29 0.20 0.28 0.55 0.67 3.00 Std deviation 0.18 0.06 0.16 0.16 0.52 1.41 A1 0.50 1.00 0.31 2.00 1.00 1.50 Methanol B3 1.00 1.00 1.00 1.00 1.00 1.00 B4 1.00 1.00 1.00 1.00 1.00 1.00 C2 0.50 0.75 1.00 1.00 1.00 1.00 C3 1.00 1.00 0.50 2.00 1.00 1.00 D1 1.00 1.00 1.00 4.00 1.00 1.00 D5 0.75 1.00 0.50 2.00 1.00 1.00 E1 0.75 0.13 1.00 2.00 1.00 1.00 F1 1.00 1.00 1.00 2.00 1.00 1.00 F2 0.50 1.00 1.00 1.00 1.00 1.00 Average 0.8 0.89 0.83 1.8 1.00 1.05 Std deviation 0.3 0.28 0.28 0.92 0.00 0.16
139 Appendix 5
MIC values (mg/ cm3) of petroleum ether, ethyl acetate, and methanol extracts of the bark.
Extractant Sample MIC (mg/ cm3) name E. S. B. P. E. K. faecalis epidermidis cereus aeruginosa coli pneumoniae A1 0.04 0.19 0.13 0.50 1.50 >8.00 Petroleum B3 0.13 0.13 0.13 2.00 1.00 0.50 ether B4 0.11 0.13 0.13 0.50 2.00 >8.00 C2 2.00 0.19 0.19 1.00 1.00 2.00 C3 4.00 0.50 0.50 1.50 1.00 2.00 D1 0.10 0.25 0.13 0.50 2.00 2.00 D5 0.13 0.13 0.13 0.50 2.00 2.00 E1 0.04 0.03 0.06 0.50 1.00 1.00 F1 0.04 0.13 0.13 0.50 2.00 1.00 F2 0.13 0.13 0.13 0.50 1.00 1.00 Average 0.67 0.18 0.17 0.80 1.45 1.44 Std deviation 1.31 0.12 0.12 0.54 0.50 0.62 A1 0.01 0.13 0.02 2.00 1.00 1.00 Ethyl B3 0.04 0.13 0.38 2.00 1.00 0.50 acetate B4 0.04 0.13 0.16 1.00 1.00 3.00 C2 0.75 0.50 0.06 1.00 1.00 1.00 C3 1.00 0.38 0.13 1.00 1.00 1.00 D1 0.09 0.01 0.04 1.50 0.75 0.50 D5 0.03 0.01 0.04 1.00 1.00 1.00 E1 0.01 0.01 0.03 2.00 0.50 0.50 F1 0.01 0.01 0.02 2.00 1.00 0.50 F2 0.01 0.01 0.03 2.00 1.00 0.50 Average 0.20 0.13 0.09 1.55 0.92 0.95 Std deviation 0.36 0.17 0.11 0.50 0.17 0.76 A1 1.00 0.50 1.00 2.00 1.00 1.00 B3 1.00 0.50 1.00 2.00 1.00 1.00 Methanol B4 0.75 0.50 1.00 4.00 1.00 1.00 C2 0.50 0.50 1.00 4.00 1.00 2.00 C3 0.50 0.50 1.00 4.00 1.00 1.00 D1 0.50 0.50 1.00 4.00 1.00 1.00 D5 0.50 0.50 1.00 4.00 1.00 1.00 E1 0.50 0.50 1.00 4.00 1.00 1.00 F1 1.00 0.50 1.00 2.00 1.00 1.00 F2 0.50 0.50 1.00 4.00 1.00 1.00 Average 0.68 0.50 1.00 3.40 1.00 1.10 Std deviation 0.23 0.00 0.00 0.97 0.00 0.32
140
APPENDIX 5...
IR and NMR Spectra of some isolated Compounds
TL 01 IR spectrum
TLTL 01 06
80
O-H
% Transmittance% C-H
40
C-H C=O
1000 1500 2000 2500 3000 3500 4000
-1 Wavenumber cm
141 Appendix 6
TL 01 1HNMR spectrum
TL 01 13CNMR spectrum
142 Appendix 6
TL 02 IR spectrum
TL 02
80 C-H
C=O %Transmittance
OH
40 1000 1500 2000 2500 3000 3500 4000 4500
-1 Wavenumber cm
143 Appendix 6
TL 04 IR Spectrum
TL 0409 105
100
95
C=O C=C %Transmittance C-H 90 C-H
85 1000 1500 2000 2500 3000 3500 4000 4500
-1 Wavenumber cm
TL 04 1HNMR
144 Appendix 6
TL 04 13CNMR
TL 06 IR spectrum
110
108
106
104
102
100
98 % Transmittance % 96 C≡C C-H 94 C=O
92
90 1000 1500 2000 2500 3000 -1 Wavenumber cm
145
723 Appendix 6
TL 06 HNMR
TL 06 13CNMR spectrum
146 Appendix 6
TL 06 COSY
H5-18, 24
H-20, 25 H21-22 H3 H23 H4
TL 06 HSQC
147 Appendix 6
TL 06 HMBC
TL 08 IR spectrum
TL 0806
100
%Transmittance C-H
C=O
80 C-H
1000 1500 2000 2500 3000
-1 Wavenumber cm
148 Appendix 6
TL 08 1HNMR spectrum
TL 08 1H-NMR
149