PHYTOCHEMICAL COMPOSITION and ANTIOXIDANT and ANTIMICROBIAL ACTIVITIES of Solanum Retroflexum LEAF EXTRACTS By

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PHYTOCHEMICAL COMPOSITION AND ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF Solanum retroflexum LEAF EXTRACTS By

DAJI GRACE ABOSEDE STUDENT NUMBER: 201505331

A Dissertation submitted to the Faculty of Science, University of Johannesburg, South Africa In partial fulfilment of the requirements for the award of a Master’s Degree in Technology: Food Technology

Supervisor: Dr. B. C. Dlamini Co-Supervisor: Dr. N. E. Madala September, 2017.

EXECUTIVE SUMMARY

Solanum retroflexum is a herbal plant that is consumed in some parts of the Venda region in Limpopo Province, South Africa. In this study, the phytochemical composition, antioxidant and antimicrobial activity of leaf extracts from the plant were investigated. Extraction of the metabolites was carried out with pressurized hot water extraction (PHWE) and different concentrations of aqueous methanol (40 %, 60 %, 80 %, methanol/water, v/v). Ultra-performance liquid chromatography coupled to quadruple- time-of-flight mass spectrometer (UPLC-qTOF-MS) technique was used to achieve an untargeted metabolite fingerprinting of the S. retroflexum leaf extracts.

In total, 30 phytochemicals including alkaloids, flavonoids and cinnamic acids derivatives were identified and characterised from the aqueous methanolic leaf extracts. The identified compounds were from different classes namely, phenolics (22), flavonoids (6) and alkaloids (2). The different concentrations of aqueous methanol resulted in very similar phytochemical composition. As expected, chlorogenic acids were identified in S. retroflexum leaf extracts. Interestingly, both trans- and cis- isomers of chlorogenic acid were identified in this study. Among the flavonoids, quercetin and kaemferol were present, while two alkaloids, Solasonine and Solamargine were identified in the aqueous methanol leaf extracts. With PHWE, optimum extraction occurred at the highest temperature (250 °C). A total of 6 health-promoting metabolites were extracted and these included Quercetin-3-rutinoside, Kaempferol-3-0-rutinoside, Kaempferol-3-0-glucoside, 3- Caffeoylquinic acid, 5-Caffeoylquinic acid and 3, 4-di-Caffeoylquinic acid. Quercetin rutinoside was extracted at all extraction temperatures, while alkaloids were absent in the PHWE extracts.

Solanum retroflexum aqueous methanol leaf extracts showed strong scavenging activities against DPPH and ABTS radicals. Similarly, the FRAP assay showed some antioxidant activity of the leaf extracts. The concentration of methanol did not substantially affect the antioxidant activity with all the assays, while the highest concentration of total phenol content did not exhibit high antioxidant activity in this study.

The antimicrobial activity of S. retroflexum aqueous methanol leaf extracts was investigated with the disc diffusion and minimal inhibitory concentration assays. About 14 microorganisms constituting of 6 Gram-negative and 8 Gram-positive bacteria were used

in the study. In general, the S. retroflexum leaf extracts showed moderate inhibition against 6 (B. cereus, M. smegmatis, S. aureus, S. epidermis, K. pneumonia and P. vulgaris) of the investigated microorganisms. As expected, Gram-positive bacteria were more susceptible to the extracts than Gram-negative bacteria. Among the Gram-positive microorganisms, B. cereus was the most susceptible to the extracts followed by S. aureus, K. pneumonia and M. smegmatis. Better inhibition occurred with samples extracted with the highest concentration (80 %) of methanol. The minimum inhibitory concentration (MIC) as determined by micro dilution method was found to be 4-16 mg/mL.

This study shows that S. retroflexum leaves contain phytochemical compounds that significantly contribute to the antioxidant and antimicrobial activity that are similar to those found in other Solanum plants. The extraction solvent affects the extract yield with aqueous methanol yielding more compounds than PHWE. The concentration of methanol does not affect the type of metabolites extracted. Understanding the type of metabolites in S. retroflexum will be useful in encouraging the use of the plant as food and its application in other foods to control foodborne bacteria.

DECLARATION

I, Daji Grace Abosede hereby declare the composition of this dissertation and the work herein described was carried out entirely by me unless otherwise cited or acknowledged. It has not been submitted for degree purpose at any other University or institution. I further declare that all cited sources are acknowledged by list of referencing.

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DEDICATION

I dedicate this dissertation to God Almighty for his grace and mercy that He has bestowed upon me, my husband, our daughter and my parents who together have helped me to sustain my sense of wonder through a rather long scientific career.

ACKNOWLEDGEMENTS

First, my utmost gratitude and honour goes to God Almighty for His grace and mercy upon my life during the period of my study at the esteemed University of Johannesburg.

I would like to express my deepest gratitude to my supervisors Dr B.C. Dlamini and Dr N.E. Madala for their inspiring guidance, support, expertise and valuable advice. Words are not enough to express how grateful I am and I could not wish for nothing better. Thanks for being part of my success story. God bless all. Besides my supervisors, I would like to sincerely appreciate Dr V. Mavumengwana, Prof P. A. Steenkamp, Dr E. Kayitesi, Dr P.B. Njobeh, and Associate Prof O.C. Nwinyi for their encouragement, mentorship and insightful comments.

Importantly, I wish to render my profound appreciation to my lovely husband Mr. A.O. Idris and our daughter Miss Esther Ebunoluwa Lerato Idris for emotional understanding and always cheering me up during the quest of this journey. I am indeed a million times better than I was before I started this journey. May God bless you abundantly.

Special thanks to my parents MWO. (Rtd) and Deac. Mrs. Folorunsho and Glory Daji for their love, prayers and support and for bringing me to this earth of great discovery. I also wish to thank my in-laws Mr. and Mrs. Adisa and Abimbola Idris for your words of encouragement and best wishes. My Siblings Mrs. M. Idris, Mr. V.K. Daji, Mr. A. Idris, Mr. O.N. Daji, Mr. M.H. Idris, Mr. E.O. Daji and Mr. J.O. Daji for always supporting me in love and prayers.

Furthermore, I appreciate my friends and colleagues for your companionship, assistance and Guidance: Miss. S. Moyo, Mrs. N.C. Uche-Okereafor, Miss. T.E. Sebola, Mr. O.A. Adebo, Miss. J.A. Adebiyi, Mrs. I. Olotu, Mr P. Akanji and Mr. S. Gbashi. And my colleagues at Biochemistry Department: Mrs. R.M. Ramabulana, Miss. K. Masike, Miss. P.S. Mudau, Miss. M. Makola and Mr. E. Ncube, thank you for making my stay worthwhile. God bless you beyond measures.

Last but not the least, I wish to thank the University of Johannesburg and the National Research Foundation (NRF) for providing financial support for this study.

PRESENTATIONS AND PUBLICATIONS

Part of the work in this dissertation has been presented in International conference.

CONFERENCE ORAL PRESENTATION:

Daji, G.A., Steenkamp, P.A., Dlamini, B.C. and Madala, N.E. (2016). Phytochemical composition of Solanum retroflexum with the aid of Ultra-performance liquid chromatography hyphenated to quadruple time-of-flight mass spectrometry (UPLC-qTOF- MS). Oral presentation, 25th South African Society of Biochemistry and Molecular Biology. University of Fort Hare, 10th-14th July 2016.

ARTICLES PREPARED FOR PUBLICATION

Daji, G.A., Steenkamp, P.A., Dlamini, B.C. and Madala, N.E. (2017). Phytochemical composition of Solanum retroflexum with the aid of Ultra-performance liquid chromatography hyphenated to quadruple time-of-flight mass spectrometry (UPLC-qTOF- MS). Manuscript to be submitted for publication in Journal of Food Quality Hindawi.

Daji, G.A., Madala, N.E., and Dlamini, B.C. (2017). Total phenolic, flavonoid, tannin content, antioxidant and antimicrobial activity of Solanum retroflexum leaves extracts.

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TABLE OF CONTENTS

EXECUTIVE SUMMARY ...... i DECLARATION ...... iii AFFIDAVIT ...... iv DEDICATION ...... v ACKNOWLEDGEMENT ...... vi PRESENTATIONS AND PUBLICATIONS ...... vii TABLE OF CONTENTS ...... viii LIST OF FIGURES ...... xii LIST OF TABLES ...... xiii LIST OF ABBREVIATIONS ...... xiv LIST OF SYMBOLS AND UNITS…...... xvi DISSERTATION OUTLINE………………………………………………………...... xvii

CHAPTER ONE ...... 1

1.0 GENERAL INTRODUCTION ...... 1 1.1 PROBLEM STATEMENT ...... 2 1.2 HYPOTHESES ...... 3 1.3 OBJECTIVES ...... 4 1.4 REFERENCES ...... 4

CHAPTER TWO ...... 9

2.0 LITERATURE REVIEW ...... 9 2.1 SOLANUM PLANTS ...... 9 2.1.1 Solanum retroflexum ...... 11 2.2 PHYTOCHEMICALS ...... 11 2.2.1 Alkaloids ...... 12 2.2.2 Phenolics ...... 13 2.2.2.1 Flavonoids ...... 14 2.2.2.2 Tannins ...... 16

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2.2.3 Saponins ...... 17 2.2.4 Terpenes ...... 17 2.2.5 Steroids ...... 18 2.3 PLANTS AS ANTIOXIDANT AGENTS ...... 19 2.4 ADVANCES IN THE EXTRACTION OF PHYTOCHEMICALS ...... 20 2.5 ANTIMICROBIAL ACTIVITY OF PHYTOCHEMICALS ...... 21 2.6 FOOD APPLICATION OF NATURAL ANTIMICROBIAL COMPOUNDS ...... 23 2.7 FUTURE PROSPECTS OF PHYTOCHEMICALS AS SOURCES OF ANTIMICROBIAL COMPOUNDS ...... 24 2.8 REFERENCES ...... 24

CHAPTER THREE ...... 38

PHYTOCHEMICAL COMPOSITION OF Solanum retroflexum ANALYSED WITH THE AID OF ULTRA-PERFORMANCE LIQID CHROMATOGRAPHY HYPHENATED TO QUADRUPOLE-TIME-OF-FLIGHT MASS SPECTROMETRY (UPLC-qTOF-MS) ... 38 Abstract ...... 38

3.1 Introduction ...... 38

3.2 Materials and Methods ...... 40 3.2.1 Plant Collection ...... 40 3.2.2.1 Metabolite Extraction with Methanol ...... 41 3.2.2.2 Metabolite extraction with Pressurised hot water ...... 41 3.2.3 Ultra-High Performance Liquid Chromatography ...... 42 3.2.4 Mass Spectrometry Acquisition Parameters ...... 42 3.3 Results and Discussion ...... 43 3.3.1 Methanol extraction of phytochemicals ...... 43 3.3.2 Chlorogenic acids ...... 44 3.3.3 Characterisation of Caffeoylquinic acids ...... 46 3.3.4 Characterisation of Feruloylglycoside ...... 47 3.3.6 Characterisation of p-coumaroylquinic acid and Coumaroyl-hexose ...... 48 3.3.7 Characterisation of Feruloylquinic Acids ...... 48

3.3.8 Characterisation of Flavonoid Derivatives ...... 51 3.3.9 Characterisation of Alkaloid Derivatives ...... 52 3.4 Pressurised hot water extraction ...... 54 3.5 Conclusion ...... 55 REFERENCES ...... 56

CHAPTER FOUR ...... 64

TOTAL PHENOLIC, FLAVONOID AND TANNIN CONTENTS AND ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF Solanum retroflexum LEAF EXTRACTS. . 64 4.1 Introduction ...... 65 4.2 Materials and Methods ...... 66 4.2.1 Collection of plant materials ...... 66 4.2.2 Chemicals and Solvents ...... 67 4.2.3 Bacteria strains and Growth condition ...... 67 4.2.4 Preparation of Extracts ...... 67 4.3 Determination of Total Phenolic Content (TPC) ...... 68 4.4 Determination of Total Flavonoid Content (TFC)...... 68 4.5 Determination of Tannin Content (TC) ...... 69 4.6 Antioxidant Activities ...... 69 4.6.1 DPPH Assay: ...... 69 4.6.2 FRAP Assay: ...... 69 4.6.3 ABTS Assay: ...... 70 4.7 Antimicrobial Assays ...... 70 4.7.1 Disc Diffusion Assay ...... 70 4.7.2 Minimum inhibitory concentration (MIC)...... 71 4.7.3 Statistical Analysis: ...... 71 4.8 Results and Discussion ...... 72 4.8.1 Total phenolic, flavonoid and tannin content ...... 72 4.8.3 Correlation between Total phenolic, Flavonoid Tannin Content and Radical Scavenging Activity ...... 78 4.9 Antimicrobial activity of Solanum retroflexum leaf extracts ...... 78 4.10 Conclusion ...... 83 REFERENCES ...... 84

5.0 GENERAL DISCUSSION AND CONCLUSIONS ...... 92 5.1.1 Phytochemical composition of S. retroflexum leaf extracts ...... 95 5.1.2 Antioxidant and antimicrobial activity of S. retroflexum leaf extracts ...... 98 5.2 Conclusions and Recommendation ...... 100 REFERENCES ...... 101

LIST OF FIGURES

Figure 2.1: Solanum retroflexum plant…………………………………………………...11

Figure 2.2: Chemical structures of selected plant derived Alkaloids ...... 13

Figure 2.3: Chemical structures of selected plant derived Phenolics ...... 14

Figure 2.4: Chemical structures of selected plant derived Flavonoids ...... 15

Figure 2.5: Chemical structures of Gallic acid and Theaflavin ...... 16

Figure 2.6: Chemical structures of selected plant derived Saponin ...... 17

Figure 2.7: Chemical structures of selected plant derived Terpenes ...... 18

Figure 2.8: Chemical structures of selected plant derived Steriods ...... 19

Figure 3.1: UPLC-qTOF-MS Chromatogram of 40 % (A), 60 % (B) and 80 % (C) leaf extracts showing peaks detected in the negative and positive ionization mode ...... 43

Figure 3.2: UPLC-qTOF-MS Chromatogram shows single ion chromatogram (A) and the base peak in chromatogram (B) of chlorogenic acid detected in the negative ionization mode ...... 44

Figure 3.3: Mass spectra showing fragmentation pattern of different positional isomers of 3CQA (A), 4CQA (B) and 5CQA (C) ...... 45

Figure 3.4: Chemical strutures of identified metabolites from Solanum retroflexum…….50

Figure 3.5: Mass spectra showing fragmentation pattern of Solasonine (A) and Solamargine (B)…………………………………………………………………………...53

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LIST OF TABLES

Table 2.1: Biological activities of compounds extracted from Solanum plants ...... 10

Table 3.1: UHPLC data of the compounds identified in Solanum retroflexum leaf extracted with methanol: Water ...... 49

Table 4.1: Observation of total phenolic, flavonoid and tannin content of Solanum retroflexum leaf extract at different percentages of methanol ...... 73

Table 4.2: Antioxidants activity of Solanum retroflexum leaf extracts ...... 76

Table 4.3: Pearson`s Correlation coefficient (r) between Radical Scavenging assay with total phenolic, flavonoid and tannin content ...... 78

Table 4.4: Assessment of Disc Diffusion assay of extracts isolated from Solanum retroflexum ...... 80

Table 4.5: Assessment of Minimum inhibitory concentrations of extracts isolated from Solanum retroflexum ...... 81

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LIST OF ABBREVIATIONS

ANOVA Analysis of variance

ABTS 2, 2- Azinobis (3-Ethyl-Benzothiazoline-6-Sulfonic Acid)

AlCl3 Aluminium Chloride

CO2 Carbon dioxide diCQA Dicaffeoylquinic acid

DNP Dictionary of natural products

DPPH 2, 2-Diphenly-1- Picrylhydrazyl dw Dry weight

EI Electron Ionisation

ESI Electrospray Ionisation

FAO Food and Agriculture Organisation

FRAP Ferric Reducing Antioxidant Assay

GC Gas Chromatography

HCl Hydrochloric Acid

HPLC High performance liquid chromatography

KMI KNApSAcK metabolite information

UHPLC Ultra High Performance Liquid Chromatography

MeOH Methanol

MS Mass spectrometry

MIC Minimum Inhibitory Concentration Assay n number

Na2CO3 Sodium Carbonate

NaNO2 Sodium Nitrite

NH4Cl Ammonium Chloride

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NI- No Inhibition

PDA Photodiode array

PHWE Pressurised hot water extraction qTOF Quadrupole time-of-flight

R2 Coefficient of determination rpm Revolution per minute

ROS Reactive Oxygen Species

Rt Retention time sd standard deviation

TEAC Trolox Equivalent Antioxidant Capacity

TFC Total Flavonoid Content

TPC Total Phenolic Content

TPTZ 2, 4, 6-Tripyridyl-S-Triazine

Trolox 6-Hydroxy-2, 5, 7, 8-Tetramethylchroman-2-Carboxylic Acid

TTC Total Tannin Content

UPLC-qTOF-MS Ultra-performance Liquid Chromatography/ Quadrupole Time-of-

Flight Mass Spectrometry

UV Ultraviolet

WHO World Health Organisation

LIST OF SYMBOLS AND UNITS

% Percentage

± Plus or minus

Da Daltons oC Degree Celsius cm Centimetre g Gram

> Greater than hr Hour

< Less than

μl Microlitre

μm Micromolar

L/H Liter per Hour

M Molar mg Milligrams mL Milliliter mg/ mL Milligram/millilitres mM Millimolar m/z Mass to charge ratio nm Nanometer min Minutes pH Power of hydrogen concentration

Psi Pounds per square inch

S Second

V Volts

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DISSERTATION OUTLINE

This dissertation is on phytochemical composition, antioxidant and antimicrobial activities of Solanum retroflexum. A brief outline of the chapters presented in this dissertation is provided below. Chapter One: General introduction This chapter provides relevant background information as well as the problem statement. This chapter also states the hypothesis, aim and objectives of the study.

Chapter Two: Literature review This chapter presents an overview of Solanum plants with emphasis on S. retroflexum. The phytochemical compounds identified in Solanum plants are reviewed followed by their antimicrobial and antioxidant properties. Later the extraction of phytochemicals from plants and the safety concerns of phytochemicals are briefly reviewed.

Chapter Three: phytochemical composition of solanum retroflexum with the aid of ultra-performance liquid chromatography hyphenated to quadruple time-of-flight mass spectrometry (UPLC-qTOF-MS). This chapter identifies the phytochemical compounds in S. retroflexum leaves. Two extraction solvents, aqueous methanol at different percentages and water at different temperatures was used to extract metabolites that were later identified using UPLC-qTOF- MS. The work presented in this chapter will be submitted to the Journal of Food Quality Hindawi for publication.

Chapter Four: Total phenolic, flavonoid, tannin contents and antioxidant and antimicrobial activity of Solanum retroflexum leaf extract. This chapter reports on the investigation of the total phenolic, flavonoid, tannin content, antioxidant activity Solanum retroflexum leaf extracts. Later, the investigation of the antimicrobial activity of the extracts against selected bacteria responsible for foodborne illness is reported.

Chapter Five: General Discussion and Conclusion This chapter evaluates the methodology used in the research. Later, it contains the general discussion of findings, conclusions and recommendations of the study.

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CHAPTER ONE

1.0 GENERAL INTRODUCTION

The genus Solanum belongs to the family Solanaceae which is otherwise known as wonder-berry or sun-berry. It consists of many essential fruits and vegetables which include potatoes, tomatoes, aubergines, paprika, chillies, cape gooseberries, green and red pepper (Knapp et al., 2004). Other important food plants such as potato (Solanum tuberosum), the aubergine or eggplant (Solanum melongena), tomato (Solanum lycopersicum) and the lulo or naranjilla (Solanum quitoense Lam.) also fall under this family (Frusciante et al., 2000; Samuels et al., 2015; Herraiz et al., 2016). Solanum plants are largely known for their protective health benefit (Pardhi et al., 2010; Yang and Ojiewo et al., 2013). For example, Solanum nigrum possesses medicinal properties such as anti-microbial, anti-oxidant, cytotoxic properties, anti-ulcerogenic and hepatoprotective activity (Jainu and Devi et al., 2006; Arulmozhi et al., 2012). The health benefits of Solanum plants are largely attributed to the presence of phytochemical compounds.

A phytochemical can be defined as bioactive secondary plant metabolite is found in vegetables, fruits, grains and other plant foods (Motilva et al., 2013). They are produced by plants as a natural defence mechanism against pathogens, predators and herbivores (Puupponen-Pimiä et al., 2008). Phytochemicals from plants have been used as raw material for traditional medicine since ancient times (Oikeh et al., 2013). The concentration of phytochemicals in plants is affected by a number of factors such as plant species, the age of the plant and duration of exposure to ultraviolet (UV) light (Szakiel, Paczkowski and Henry, 2010). Phytochemicals that have been reported in Solanum plants include alkaloids, flavonoids, tannins, saponins and glycosides (Kusirinisin et al., 2009). These compounds have been isolated in various parts (leaves, fruit, stem and roots) of Solanum plants (Yousaf et al., 2013).

According to World Health Organisation (WHO), antimicrobial resistance to synthetic drugs will cause a post antibiotic era in which common infections and minor injuries, which have been treatable for decades are once again almost incurable with antibiotics (WHO, 2014). This highlights the need for alternative and safer interventions for the treatment of foodborne illness. Several studies have reported the presence of antimicrobial and antioxidant properties of

Solanum plants. Solanum nigrum for example, has been shown not only to exhibit antimicrobial activity but also anti-cancer, anti-tumor, antioxidant and hepaprotective properties (Harikrishnan et al., 2011; Jainu and Devi, 2006; Arulmozhi et al., 2012). In addition, anthocyanins in S. tuberosum extracts have been reported to exhibit antimicrobial, antioxidant and anticancer properties (Bontempo et al., 2013). The presence of antioxidants in plants is associated with reduced adverse effects caused by free radicals in living systems (Rahal et al., 2014). In addition, dietary antioxidants can reduce the risk of cardiovascular diseases and systematic inflammation (Liu et al., 2003 ; Devaraj et al., 2009; Colarusso et al., 2016).

Recent advances in instrumentation and extraction techniques of plant chemical substances have elicited interests of scientist in screening phytochemicals, particularly in the development of new drugs (Mungole et al., 2010). Similarly, screening of plants that can potentially be used as natural antimicrobials in foods have increased (Lucera et al., 2012). The latter has partly imposed the need for “greener” or environmentally friendly methods for the extraction of phytochemicals from plants. As a result, techniques such as pressurised hot water extraction (PHWE) and the use of ultra-high-performance liquid chromatography coupled to mass spectrometry have been investigated for analysis of food polyphenols (Motilva et al., 2013; Liu et al., 2013).

The phytochemical profile of S. retroflexum has not been documented in literature despite the fact that S. retroflexum leaves are consumed in some parts of South Africa and likely in other region of the world. Therefore, there is a need to investigate the bioactive compounds, associated antimicrobial and antioxidant properties of S. retroflexum. Understanding the phytochemical composition and the antimicrobial effects of S. retroflexum leaves may be used as a basis for encouraging commercial propagation of the plant and ultimately its application in food as antimicrobial compound.

1.1 PROBLEM STATEMENT

Foodborne disease outbreaks continue to have devastating health and economic consequences in both developed and developing countries. According to the World Health Organisation, foodborne diseases kill approximately 2 million people annually (WHO Report, 2014). Infections caused by human pathogenic microorganisms are generally treated with commercial

antimicrobial drugs (Bax et al., 2007). However, apart from having side effects, there has been increase in the resistance of pathogenic microorganisms to commercial drugs. These has forced scientists to investigate alternative sources of antimicrobial substances such as medicinal plants (Parekh and Chanda, 2007). Furthermore, consumers’ awareness has recently increased, leading to an increased demand of foods preserved with natural antimicrobials instead of synthetic chemical additives. Natural antioxidants are substances that are capable of neutralizing free radicals or their actions. They act at different stages which include prevention, interception and repair (Devasagayam et al., 2004). Polyphenols are secondary metabolites that occur naturally in fruits, vegetables, cereals and beverages. Epidemiological studies suggest that moderate consumption of diets rich in plant polyphenols offers some protection against development of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases (Pandey and Rizv, 2009). Polyphenols and other food phenolics is the subject of increasing scientific interest because of their possible beneficial effects on human health.

Solanum plants have a long history of medicinal usage, probably due to the fact that they are rich in phytochemicals (Latha and Kannabiran, 2006). There is however, paucity of knowledge on the phytochemical composition and antimicrobial and antioxidant properties of Solanum retroflexum. Since this plant is consumed in South Africa, a better understanding of its health promoting benefits can potentially be used to improve human health.

1.2 HYPOTHESES

Aqueous methanol and PHWE of S. retroflexum leaves will contain substantial amounts of phytochemicals comparable to other Solanum plants. This is because other Solanum plants such as Solanum nigrum have been reported to contain saponins, tannins, alkaloids, flavonoids and steroids in their leaf extracts (Kumar et al., 2016).

Solanum retroflexum aqueous methanol leaf extracts will exhibit antioxidant activity using the DPPH, ABTS and FRAP assays. The presence of phytochemicals such as phenolic acids and flavonoids in most Solanum plants leaf extracts has been found responsible for their antioxidant activities (Abdennacer et al., 2015; Jiang et al., 2016).

Solanum retroflexum aqueous methanol leaves extracts will exhibit antimicrobial activity against selected foodborne pathogens. This is because other Solanum plants contain phytochemical compounds that inhibit the growth of bacteria. For example, the presence of flavonoids is thought to inactivate microbial adhesion and cell envelope in some bacteria (Plaper et al., 2003). In addition, fat-soluble flavonoids are thought to disrupt microbial membranes, alter membrane fluidity and negatively affect the respiratory chain (Haraguchi et al., 1998; Mishra and Jha, 2009).

1.3 OBJECTIVES

 To determine the phytochemical composition of S. retroflexum leaf extracted with pressurised hot water and aqueous methanol (40 %, 60 % and 80 %, methanol/water, v/v).  To determine the antioxidant activity of S. retroflexum aqueous methanol leaf extracts (DPPH, ABTS, FRAP assays).  To determine the antimicrobial activities of S. retroflexum aqueous methanol leaf extracts against selected foodborne pathogens.

1.4 REFERENCES

Abdennacer, B. (2015). Determination of phytochemicals and antioxidant activity of methanol extracts obtained from the fruit and leaves of Tunisian Lycium intricatum Boiss. Food Chemistry, 174(1), 577–584.

Arulmozhi, V. (2012). Protective effect of Solanum nigrum fruit extract on the functional status of liver and kidney against ethanol induced toxicity. Journal of Biochemical Technology, 3, 339-343.

Bax, R. (2007). Development of a twice daily dosing regimen of amoxicillin/ clavulanate. International Journal of Antimicrobial Agents, 30( 2), 118–121.

Bontempo, P. (2013). Antioxidant, antimicrobial and anti-proliferative activities of Solanum tuberosum L. var. Vitelotte. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association, 55, 304–12.

Colarusso, L. (2016). Dietary antioxidant capacity and risk for stroke in a prospective cohort study of Swedish men and women. Nutrition, 33, 234-239.

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Frusciante, L. (2000). Evaluation and use of plant biodiversity for food and pharmaceuticals. Fitoterapia, 71(202), 66–72.

Haraguchi, H., Tanimoto, K., Tamura, Y., Mizutani, K. and Kinoshita, T. (1998). Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata. Phytochemistry, 125–129.

Harikrishnan, R. (2011). Solanum nigrum enhancement of the immune response and disease resistance of tiger shrimp, Penaeus monodon against Vibrio harveyi. Aquaculture, 318(1-2), 67–73.

Herraiz, F. J. (2016). The first de novo transcriptome of pepino (Solanum muricatum): assembly, comprehensive analysis and comparison with the closely related species S. caripense, potato and tomato. BMC Genomics, 17(321), 1–17.

Jainu, M. and Devi, C. S. S. (2006). Antiulcerogenic and ulcer healing effects of Solanum nigrum (L.) on experimental ulcer models: Possible mechanism for the inhibition of acid formation. Journal of Ethnopharmacology, 104(1-2), 156–163.

Jiang, Q. Y. (2016). Effect of Funneliformis mosseae on the growth, cadmium accumulation and antioxidant activities of Solanum nigrum. Applied Soil Ecology, 98, 112–120.

Kumar, P. (2016). Studies on phytochemical constituents and antimicrobial activities of leaves, fruits and stems of Solanum nigrum L . Asian Journal of Plant Science and Research, 6(4), 57–68.

Kusirisin, (2009). Antioxidative activity, polyphenolic content and anti-glycation effect of some Thai medicinal plants traditionally used in diabetic. Medicinal chemistry, 5(2), 2009.

Knapp, S. Bohs, L., Nee, M. and Spooner, D.M. (2004). Solanaceae – a model for linking genomics with biodiversity. Comparative and Functional Genomics 5, 285-291.

Latha, P. S. and Kannabiran, K. (2006). Antimicrobial activity and phytochemicals of Solanum trilobatum Linn. African Journal of Biotechnology, 5(23), 2402–2404.

Liu, J. (2013). Simultaneous Determination of Isoflavones, Saponins and Flavones in Flos Puerariae by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time- of-Flight Mass Spectrometry. Chemical and Pharmaceutical Bulletin, 61(9), 941–951.

Liu, R. H. (2003). Health benefits of fruit and vegetables are from additive and synergistic combinaions of phytochemicals. The American Journal of Clinical Nutrition, 78, 3–6.

Lucera, A. (2012). Food applications of natural antimicrobial compounds. Frontiers in Microbiology, 3(8), 1–13.

Mishra, A. and Jha, B. (2009). Isolation and characterization of extracellular polymeric substances from micro-algae dunaliella salina under stress. Bioresource Technology 100, 3382-3386.

Motilva, M., Serra, A. and Macià, A. (2013). Analysis of food polyphenols by ultra high- performance liquid chromatography coupled to mass spectrometry: An overview. Journal of Chromatography A, 1292, 66–82.

Mungole, A. J. (2010). Preliminary phytochemical screening of Ipomoea obscura (L) -a hepatoprotective medicinal plant. International Journal of PharmTech Research, 2(4), 2307–2312.

Oikeh, E. I. K., Oriakhi. and Omoregie, E. S. (2013). Proximate analysis and phytochemical screening of Citrus sinensis fruit wastes. Bio-scientist, (1), 164–170.

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Yousaf, Z., Wang, Y. and Baydoun, E. (2013). Phytochemistry and Pharmacological Studies on Solanum torvum. Journal of Applied Polymer Science, 3(4), 152–160.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 SOLANUM PLANTS

Solanum is a large genus of flowering plants that include two food crops of high economic importance, tomato and potato as well as numerous plants cultivated for their ornamental flowers and fruits (Chiarini and Barboza, 2009; Herraiz et al., 2016). Solanum species also show a wide range of growth characteristics such as annual and perennial, vines, sub shrubs, shrubs and small trees. The family is distributed throughout tropical and temperate regions of the world, with centre of diversity occurring in central of South America and Australia (Edmonds and Chweya et al., 1997). In South Africa, the fruits (very ripe) of other Solanum species, such as Solanum nigrum, are used to make jam (Yang and Ojiewo et al., 2013). Green (unripe) berries contain high concentration of toxins and they can be extremely poisonous (Teklehaimanot et al., 2015). The toxicity of Solanum plants, however, varies widely depending on the variety (Kaur et al., 2010). In China, where Solanum plants are fairly common, they have been used as food since early time and are termed “an ancient famine food” (Sarma and Sarma et al., 2011).

Plants from the genus Solanum are economically important agricultural plants that are widely known for their medicinal uses. This is because they contain naturally occurring metabolites known as phytochemicals (Moehninsi et al., 2015). Table 2.1 shows biological activities of compounds isolated from various Solanum plants. Apart from medicinal properties, Solanum plants have a healthy nutritional profile in form of minerals including calcium, iron and phosphorus, an appreciable amount of proteins, vitamin A and C, fat, fibre as well as sufficient amounts of methionine (Lu et al., 2011). Moreover, the seeds can provide appreciable amounts of vitamin C and carotene (Fernandez-Orozco et al., 2013). The berries contain sufficient amounts of iron, calcium and vitamin B (Moehninsi et al., 2015). The high nutritional value of Solanum plants play a significant role in preventing malnutrition in rural areas (Akubugwo et al., 2007).

Table 2.1: Biological activities of compounds extracted from Solanum plants.

Solanum specie Plant part Biological activity Compound References

Solanum torvum Whole plant Anti-microbial, Flavonoid, (Yousaf et al., 2013) Antioxidant, Alkaloid, Saponin Antiviral and Anti- Tannin and inflammatory. Glycoside.

Solanum Leaves, Anti-oxidants, Flavonoids, (Liu et al., 2012; lycopersicum stems and Ascorbic acids, Phenolic, Vitamin Nzanza et al., 2012) root Lycopene, Lutein and A, B, C and D. Beta-Carotene. Solanum Leaves, Anti-microbial, Tannin, (Shilpha et al., 2015; trilobatum Flowers, Antioxidant, Anti- Flavanoids, Premalatha et al., Stem and inflammatory, Phenolic, 2013) Fruit. Hepatoprotective Alkaloids and activity, Anti-tumour Glycoside. and Anti-ulcerogenic.

Solanum nigrum Leaves, Antioxidant, Anti- Phenolics, (Ramesh et al., 2015; Fruits and viral, Anti-tumour, Flavonoids, Muthuvel et al., Seed Anti-cancer, Anti- Alkaloids, 2014) inflammatory, Anti- Glycoalkaloids, bacteria, Diuretic, Glycoproteins, Anti-pyretic, Anti- Polysaccharides tumorigenic, Anti- and Polyphenolic. Bacteria and Hepatoprotective effect.

2.1.1 Solanum retroflexum

Solanum retroflexum (Solanaceae), also known as “sun-berry”, in some places, particularly where the latter species has been introduced. The common name sun-berry is used sometimes for the poisonous European Black Nightshade. S. retroflexum is a herbacious plant that was promoted by the plant breeder Luther Burbank at the beginning of the 21st century, as “a new food plant from a poisonous family” (Edmonds and Chweya et al., 1997). According to the breeder, Solanum retroflexum is a hybrid of S. guineense and S. villosum. The plant is erect, bushy and can grow up to 60 cm tall. Its stem is pale green and it produces globular berries of the same colour (Figure 2.1). Solanum retroflexum is consumed in some parts of Limpopo province, South Africa. The plant can also be found in some regions in Europe and sporadically in North and South America (Edmonds and Chweya et al., 1997).

Figure 2.1: Solanum retroflexum plant (Adapted from Google, 2015).

2.2 PHYTOCHEMICALS

Phytochemicals are defined as bioactive secondary plant metabolites that are found in vegetables, fruits, grains and other plant foods (Abdennecer, 2015). They are associated with reducing the risk of major chronic diseases possibly through their anti-cancer, anti-tumour, antimicrobial, anti-oxidant and hepaprotective properties they possess (Harikrishnan et al., 2011;

Jainu and Devi, 2006; Arulmozhi et al., 2012). Phytochemicals are also used as flavouring and colouring agents in foods for example phenolics and flavonoids (Ameh and Eze, 2010; Adeyemi et al., 2014). A large number of phytochemicals has been identified in plants, however, this review will focus on a few.

2.2.1 Alkaloids

Alkaloids are the largest group of secondary metabolites made largely of ammonium compounds (Zia et al., 2011). They comprise mainly of a nitrogen base synthesised from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring with most containing oxygen atom. The compounds have basic properties such as antibacterial and are alkaline in nature, turning red litmus paper blue (Khamtache-abderrahim et al., 2006). The majority of alkaloids exist in solid form such as atropine, while some exist as liquids. Alkaloids react with acid to form crystalline salts without the production of water (Zia et al., 2011). The solutions of alkaloids are intensely bitter and perhaps this is why they function in the defence of plants against invading insects, bacteria and herbivores (Mao et al., 2017). They are also used as stimulants (Ng et al., 2015), narcotics (Pothier and Galand, 2005) and poisons (Niitsu et al., 2013) due to their potent antibacterial and antioxidant activities. In nature, alkaloids are found in large proportions in the seeds and roots of plants and often in combination with plant organic acids (Ukachukwu et al., 2015). Alkaloids have been identified in Solanum nigrum (Ramesh et al., 2015), Solanum tuberosum (Al-hay-Ashaal et al., 2010), Solanum esculentum (Cirlini et al., 2017) and Solanum trilobatum plants (Shilpha et al., 2015). Some most important alkaloids in plants include the addictive stimulants such as morphine, codeine, caffeine and berberine (Figure 2.2).

Figure 2.2: Chemical structures of selected plant derived alkaloids (Adapted from Doughari, 2009).

2.2.2 Phenolics

Phenolics, phenols or polyphenolics (or polyphenol extracts) are chemical components that occur ubiquitously as natural colour pigments responsible for the colour of fruits and plants (Adeyemi et al., 2014). Plant phenolics are mostly synthesised from phenylalanine via the action of phenylalanine ammonia lyase (PAL) (Ververidis et al., 2007). They play a significant role in plant defence against pathogens, herbivores and predators (Puupponen-Pimiä et al., 2008). In humans, phenolics such as gallic, vanillic and cinnamic acids are used in the control of pathogenic infections (Alkan and Yemenicioglu, 2016). Phenolics essentially represent a host of natural antioxidants used as nutraceuticals. They are found in apples, green-tea and red-wine and

have the ability to combat cancer, anti-inflammatory agents and are also thought to prevent heart ailments to an appreciable degree (Raiola et al., 2016). Solanum plants such as tomatoes (Solanum lycopersium) are rich in phenolic compounds (flavonoids and phenolic acids) (Raiola et al., 2016). Examples of phenolics include caffeic acid, chlorogenic acid, rutin and flavon (Figure 2.3).

Figure 2.3: Chemical structures of selected plant derived phenolic (Adapted from Doughari, 2009).

2.2.2.1 Flavonoids

Flavonoids are an important group of polyphenols widely distributed among the plant flora (Kumar et al., 2015). Structurally, the basic flavonoid ring contains 15 carbon atoms 15C. Numerous reports support their use as antioxidants or free radical scavengers (Godstime et al., 2014). The compounds are derived from parent compounds known as flavans (Faggio et al.,

2017). Over 4000 flavonoid compounds are known to exist and some of them are pigments in higher plants. Quercetin and kaempferol are the common flavonoids present in nearly 70% of plants (Ukachukwu et al., 2015). Pharmacologically important plant derived flavonoids are flavones, anthocyanidin, dihydroflavone and flavan (flavanol) and these are presented in Figure 2.4 below.

Figure 2.4: Chemical structures of selected plant derived flavonoids (Adapted from Doughari, 2009).

2.2.2.2 Tannins

Tannins are phenolic compounds of high molecular weight and they are widely distributed in plants (Zia et al., 2011). Tannins are soluble in water and alcohol and they are mainly found in the root, bark, stem and outer layers of plant tissues (Doughari et al., 2009). They are acidic in solution probably due to the presence of phenolic or carboxylic group (Doughari et al., 2009). Tannins form complexes with proteins, carbohydrates, gelatin and alkaloids thus affecting the functional properties of these compounds (Assefa et al., 2008). They are divided into hydrolysable tannins and condensed tannins. Hydrolysable tannins, upon hydrolysis, produce gallic acid (Figure 2.5) and ellagic acid (Huang et al., 2005). Depending on the type of acid produced, hydrolysable tannins are called gallotannins or egallitannins (Lee et al., 2010). On heating, tannins form pyrogallic acid. Tannins are used as antiseptic and this activity is due to presence of the phenolic group (Yalavarthi and Thiruvengadarajan et al., 2013). Tannins are present in Solanum xanthocarpum (Patel et al., 2010) Solanum nigrum (Harikrishnan et al., 2010), Solanum tuberosum (Harikrishnan et al., 2010), Solanum esculentum (Friedman et al., 2004) and Solanum trilobatum (Shilpha et al., 2015).

Figure 2.5: Chemical structures of Gallic acid and Theaflavin (Adapted from Doughari, 2009).

2.2.3 Saponins

The term saponin is derived from Saponaria vaccaria (Quillaja saponaria), a plant, with abundant saponins and was once used as soap (Aghel et al., 2010). Saponins, therefore possess ‘soaplike’ behaviour in water, that is, they produce foam. On hydrolysis, an aglycone (sugar removed) is produced, which is called sapogenin (Aldo et al., 2017). The two major groups of saponins are steroid saponins and triterpene saponins (Vincken et al., 2007). Saponins are extremely poisonous, as they cause heamolysis of blood and are known to cause cattle poisoning (Godstime et al., 2014). They possess a bitter and acrid taste, besides causing an irritation to the mucous membrane (Jiang and Xiong 2016). Saponins have been identified in Solanum xanthocarpum (Patel et al., 2010), Solanum nigrum (Seikou et al., 2008), Solanum tuberosum (Burmeister et al., 2011), Solanum esculentum (Torres and Andrews, 2006) and Solanum trilobatum plants (Latha and Kannabiran, 2006).

Saponin Figure 2.6: Chemical structure of selected plant derived saponin (Adapted from Doughari, 2009).

2.2.4 Terpenes

Terpenes are amongst the most widespread and chemically diverse groups of natural products. They are flammable unsaturated hydrocarbons that exist in liquid form and they are commonly found in essential oils, resins or oleoresins (Kraujaliene et al., 2016). The triterpenes include steroids, sterols, and cardiac glycosides with anti-inflammatory, sedative, insecticidal or cytotoxic activity. Common triterpenes: amyrins, ursolic acid and oleanic acid sesquiterpene like monoterpenes, are major components of many essential oils (De Sousa et al., 2011). A number

of sesquiterpene lactones have been isolated and broadly they have antimicrobial, particularly antiprotozoal and neurotoxic activities (De Sousa et al., 2011). The sesquiterpene lactone, palasonin, isolated from Butea monosperma has anthelmintic activity, inhibits glucose uptake and depletes the glycogen content in Ascaridia galli (Doughari et al., 2012). Terpenes are also found in Solanum incanum (Wambugu et al., 2011).

Figure 2.7: Chemical structures of selected plant derived terpenes (Adapted from Doughari, 2009).

2.2.5 Steroids

Plant steroids (or steroid glycosides) also referred to as ‘cardiac glycosides’ are one of the most naturally occurring plant phytoconstituents that have found therapeutic applications as arrow poisons or cardiac drugs (Chan et al., 2003). The cardiac glycosides are basically steroids with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection into man or animal. Steroids (anabolic steroids) have been observed to promote nitrogen retention in osteoporosis and in animals with wasting illness

(Madziga et al., 2010). Diosgenin and cevadine (from Veratrum veride) are examples of plant steroids (Madziga et al., 2010). Steroids are present in Solanum esculentum (Weissenberg et al., 2001) and Solanum nigrum (Akilan et al., 2014).

Figure 2.8: Chemical structures of selected plant derived steriods (Adapted from Doughari, 2009).

2.3 PLANTS AS ANTIOXIDANT AGENTS

An antioxidant is a substance that inhibits the process of oxidising chain reactions either by delaying or by inhibiting the oxidation of molecules (Alenisan et al., 2017). Oxidation is one of the key processes which produce free radicals in food, chemical and living systems (Wojtunik- kulesza et al., 2016). Free radicals in human contribute to more than one hundred disorders including AIDS, central nervous system injury, arthritis, gastritis, cancer and atherosclerosis (Wojtunik-kulesza et al., 2016). In humans, catalase and hydroperoxidase enzymes transform hydrogen peroxide and hydroperoxide to non-radical forms and operate as natural antioxidants (Lin et al., 2015). However, the body generates insufficient natural antioxidant defence to help in the removal of free radicals (Rahman et al., 2007). The disparity between intracellular reactive oxygen species (ROS) and intracellular antioxidants is a notable factor that contributes to over a hundred diseases (Liou and Storz, 2010). This is because antioxidants play an important role in health maintenance and prevention of chronic diseases such as cardiac and cerebral ischema

(Aslani and Ghobadi, 2016). There is, therefore a growing interest in the study of the antioxidant activity of secondary metabolites from plants, particularly compounds with higher potency and lower toxicities than synthetic sources (Oloyede et al., 2016). Available synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), tertiary butylated hydroquinone and gallic esters have been associated with negative health effects (Antonopoulou et al., 2016).

Antioxidant properties have been associated with phytochemicals that are necessary for the prevention of many diseases (Rahman et al., 2007). They may be proteins, enzymes, while others are low molecular weight compounds such as flavonoids, vitamins, carotenoids and other phenolic compounds (Gahruie and Niakousari, 2017; Alenisan et al., 2017). Another major class of antioxidants is the amino acids and amidothiols (Richard and Everette, 2009). These include the amino acid cysteine, antiurolithic drug (N- (2- mercaptopropionyl) glycine), radio protective drug (amifostine), antiarthritic drug (penicillamine), antihypertensive drug (captopril) and endogenous tripeptide glutathione (Lushchak et al., 2011). The mode of action of antioxidants, such as phenolic compounds is due to their propensity to chelate metals (Komolafe et al., 2014). Phenolics possess hydroxyl groups that bind iron and copper. The ability of phenolics to inhibit lipid peroxidation by trapping the lipid alkoxyl radical is dependent on the structure of the molecule and the number and position of the hydroxyl group in the molecule (Anyachukwu et al., 2016).

Recently, the food industry has focused on finding natural antioxidants that can be used as substitutes to replace synthetic compounds (Taghvaei and Jafari, 2015). Antioxidants are often added to foods to prevent radical chain reactions of oxidation. They act by inhibiting the initiation and propagation step leading to the termination of the reaction and a delay in oxidation process (Gülçin et al., 2010). Therefore, there is growing trend in consumer preferences for natural antioxidants, all of which has given more impetus to explore natural source of antioxidants.

2.4 ADVANCES IN THE EXTRACTION OF PHYTOCHEMICALS

Recent advances in the development of new drugs particularly in extraction techniques and instrumentation have elicited interests of scientist in screening phytochemicals (Mungole et al.,

2010). Similarly, screening of the plants that can potentially be used as natural antimicrobials in foods has increased (Lucera et al., 2012). Metabolite fingerprinting is a reliable technological approach in the generation of information from chromatographic spectra of bioactive compounds that has been utilised in biochemical studies of plants (Dias et al., 2012). Extraction plays a pivotal role in metabolite fingerprinting of plants as the quality of analyte has often been linked to the extraction technique utilised (Kopka et al., 2004). The extraction method must produce reliable, reproducible and consistent results on both the quantitative and qualitative aspects of data generated (Premalatha et al., 2013). Conventional extraction methods such as the use of organic solvents have been used in the extraction of phytochemicals (Dhanani et al., 2017; Wang et al., 2017). However, there are concerns on the potential health threatening effects associated with them. In addition, these methods are time consuming, laborious and they require large amount of hazardous and environmentally unfriendly organic solvents (Dhanani et al., 2017). These challenges have motivated the adoption of “greener” and more effective techniques such as pressurised hot water extraction (PHWE) (Chye et al., 2010). PHWE is the most utilised extraction technique with potential to overcome this setback. It is affordable and not time consuming (Liau et al., 2017). PHWE promises better selectivity as its solvation power can be manipulated over a wide spectrum of polarities (Mokgadi et al., 2013). Importantly, it can give results that are better than those of conventional extraction methods in terms of cost and environmental friendliness (Liau et al., 2017). Further, PHWE utilises water as extraction solvent, which is readily available and compatible with most chromatographic instruments (Plaza and Turner, 2015; Shang et al., 2017). The technique gives a true reflection of what the public consumes since water is used as extraction medium for daily consumption and preparation of medicinal plants.

2.5 ANTIMICROBIAL ACTIVITY OF PHYTOCHEMICALS

Antimicrobial resistance is a growing problem in the world and causes millions of deaths every year (WHO, 2014). The World Health Organisation (WHO) further states that antimicrobial resistance is threatening the effective prevention and treatment of a range of infections caused by bacteria, parasite, virus and fungi. The common types of drug-resistant bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vacomycin-resistant Staphylococcus aureus (VRSA), extended spectrum beta-lactamase bacteria (ESBL), vacomycin resistant

Enterococcus (VRE) and multidrug resistant Acinetobacter baumannii (MRAB) (Chen et al., 2017; Ahmed et al., 2018).

The problems of antibiotics resistance are due to the overuse and misuse, as well as lack of development of new antibiotics drugs by the pharmaceutical industry because of economic incentives and strict regulatory requirement (Qiao et al. 2018). The overuse and misuse of antibiotics foster the rapid emergence of antibiotic-resistance bacteria (ARB) and antibiotics resistant genes (ARGS), this reduces their medicinal potential against animal pathogens and humans. Unfortunately, WHO classified antimicrobial resistance as a worldwide public health crisis that must be managed with high sense of urgency (WHO, 2015) (Durão et al. 2018).

The natural protection of plants against bacteria, fungi, pathogenic insects or protozoa has found applications in food and human medicine and these health benefits are attributed to the presence of phytochemicals (Guo et al., 2017). The indiscriminate use of antibiotics that has led to drug resistance in many bacteria has necessitated the search for alternative to antibiotics (Low et al., 2016; Guo et al., 2017). According to WHO, about 80 % of people living in developing countries have use traditional medicine (Galen et al., 2014; Gene et al., 2016). Different parts of medicinal plants are utilised for the treatment of various human ailments such as cholera, wounds, ulcers and bowels (Dzoyem et al., 2016). The leaves of Solanum trilobatum have been reported to alleviate phlegmatic rheumatism, asthma, arrest blood vomiting, high blood glucose level and several kinds of leprosy (Premalatha et al., 2013). Solanum nigrum leaves are also reported to possess antiviral, antibacterial, anti-inflammatory, anti-tumour, anti-cancer and hepatoprotective effect (Premalatha et al., 2013). Solanum tuberosum plant contains sequestered glycosides which are released upon injury or infection (Yvonne et al., 2017). These glycosides may be hydrolysed by glycosidase to yield more active aglycones that exhibit antimicrobial properties against invading pathogens (Milner et al., 2011). Phenolic compounds may be oxidised to highly reactive antimicrobial quinines and free radicals (Pinacho et al., 2015). With respect to antimicrobial properties, the most important bioactive constituents of plants are phenolics, flavonoids, tannins and alkaloids (Mohamed et al., 2013).

Different mechanisms of microbial inhibition by phytochemicals have been suggested. These include interfering with microbial metabolic activities, inducing cellular membrane perturbation,

inhibition of microbial growth and modulation of signal transduction or gene expression pathways (Godstime et al., 2014). Plant extracts such as phenolic acids may deactivate the activities of some organisms adhered to the cell lining of the bladder which could invariably result in lowering the incidence of urinary tract infection caused by E. coli (Godstime et al., 2014). Essential oils may exhibit inhibition or inactivation of the enzymes involved in the production of cellular energy and synthesis of structural components (Zsuzsanna Schelz1 et al., 2010). They may also cause destruction or inactivation of genetic material (Gupta et al. 2017).Tannins have been shown to suppress bacterial cell proliferation and thus causing a reduction in the occurrence of many diseases (Lucera et al., 2012). Saponins are known to alter the permeability of cell walls and may exert toxicity on organised tissue (Lu et al., 2013).

2.6 FOOD APPLICATION OF NATURAL ANTIMICROBIAL COMPOUNDS

The use of natural antimicrobial compounds in biomedical and food applications is in line with the current trend of giving value to natural and renewable resources (Lucera et al., 2012). Antimicrobials can be directly added into product formulation, incorporated into the packaging material or coated on its surface so as to inhibit the growth of microorganisms in food (Lucera et al., 2012). Spice extracts (clove and rosemary) have recently been shown to be highly effective against microbial growth on raw chicken meat stored for 15 days (Zhang et al., 2015). Coating skinless frankfurters with phytochemicals reportedly reduced Listeria monocytogenes counts by up to 5 log CFU/frankfurter after 42 days storage at 4 °C (Upadhyay et al., 2015).

In general, the efficacy of some plant antimicrobials added to foods may be reduced by certain food components (Glass and Johnson et al., 2004). For example, essential oils have been reported to be effective against foodborne pathogens and spoilage microorganisms associated with meat (Mytle et al., 2006; Ahn et al., 2007). However, other studies have reported very low or no antimicrobial activity against L. monocytogenes or Salmonella in beef or chicken that has been treated with essential oils (Uhart et al., 2006; Firouzi et al., 2007). This highlights the need to test the efficacy of plant antimicrobials in food or food model media.

2.7 FUTURE PROSPECTS OF PHYTOCHEMICALS AS SOURCES OF ANTIMICROBIAL COMPOUNDS

Most of the natural products that are currently in development have come from either plant or microbial sources. Scientific reports have pointed out that relatively little of the world’s plant biodiversity has been extensively screened for bioactivity and that very little of the estimated microbial biodiversity has been available for screening (Doughari et al., 2009). Hence, more extensive collections of plants could provide many novel compounds for use in drug discovery assays. With advances in fractionation techniques to isolate and purify natural products (e.g. counter-current chromatography (Doughari et al., 2009)) and in analytical techniques to determine structures (Singh and Barrett et al., 2006), screening of natural product mixtures is now more compatible with the expected time-scale of high-throughput screening campaigns. It is estimated that more than 5000 individual phytochemicals have been identified in fruits, vegetables and grains, but a large percentage still remains unknown and needs to be identified before we can fully understand the health benefits of phytochemicals (Liu et al., 2003).

In conclusion, Solanum plants possess a wide variety of phytochemicals with potential antimicrobial activity. These phytochemicals use different mechanisms to inhibit the growth of foodborne pathogens. Phytochemicals have been applied to food to control foodborne pathogens. However, their use in food may be hampered by the presence of food components. Therefore, further studies are needed to investigate the effect of adding phytochemicals in the production of safe and wholesome foods.

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CHAPTER THREE

PHYTOCHEMICAL COMPOSITION OF Solanum retroflexum ANALYSED WITH THE AID OF ULTRA-PERFORMANCE LIQID CHROMATOGRAPHY HYPHENATED TO QUADRUPOLE-TIME-OF-FLIGHT MASS SPECTROMETRY (UPLC-qTOF-MS)

Abstract

Solanum retroflexum (Nightshade) is an edible plant that is consumed in some regions of South Africa. Its leaves are a good source of vitamins, proteins and minerals. It appears that, there is no scientific report about the phytochemical composition of S. retroflexum. Here, ultra-performance liquid chromatography coupled to quadruple-time-of-flight mass spectrometer (UPLC-qTOF- MS) technique was use to achieve an untargeted metabolite fingerprinting of this plant. A total of 30 phytochemicals, including alkaloids, flavonoids and cinnamic acids derivatives were identified from the methanolic leaf extracts. With Pressurised Hot Water Extraction (PHWE), only six metabolites were identified and the extraction yield increased proportionately with increasing temperature. The highest extraction temperature (250 °C) resulted in qualitative yields of molecules such as Quercetin-3-rutinoside, Kaempferol-3-0-rutinoside, Kaempferol-3-0-glucoside, 3-Caffeoylquinic acid, 5-Caffeoylquinic acid and 3, 4-di-Caffeoylquinic acid. Interestingly, none of the health-threatening phytochemicals was found with PHWE. This difference in extractability of metabolites suggests that the metabolites have different physicochemical properties such as polarity. The present study confirms the presence of phytochemical compounds in S. retroflexum similar to other Solanum plants. Key words: Phytochemicals, Solanum retroflexum, pressurised hot water and UPLC-qTOF-MS.

3.1 Introduction

Naturally, plants contain secondary metabolites that play a vital role in protection against pathogens, herbivores and predators (Puupponen-Pimiä et al., 2008). A number of phytochemicals have been identified in plants and these include phenolic acids, flavonoids, tannins, saponins, alkaloids, steroids and many others (Patel, 2017; Lavola et al., 2017). The phytochemicals found in plants are believed to inhibit the growth of pathogens that infect

humans and they confer health benefits largely due to their antioxidant properties (Ikram et al., 2015). The health benefits associated with phytochemicals include anticancer, antitumor and hepaprotective properties (Harikrishnan et al., 2011; Jainu and Devi, 2006; Arulmozhi et al., 2012).

Solanum plants have been widely explored for their phytochemical constituents. These bioactive compounds have been isolated from various parts such as the leaves, fruit and roots (Yousaf et al., 2013; Piana et al., 2016; Kumar et al., 2016). The presence of various phytochemicals in Solanum plants has allowed for their use in medicine and food as functional foods or dietary supplements (Rahman and Kang, 2009; Neacsu et al., 2015). The type of phytochemicals found in Solanum plants vary with species, plant part and the extraction method (Paulsen et al., 2010). The presence of tannins, flavonoids, alkaloids, glycosides, steroids and terpenoids has been reported in S. nigrum L. leaves, fruit and stem (Kumar et al., 2016). Phenolic compounds such as chlorogenic acid, caffeic acid, rosmarinic acid, gallic acid and flavonoids have been identified together with alkaloids in S. corymbiflorum leaves (Piana et al., 2016). The presence of some compounds such as alkaloids in food can cause toxicity to humans depending on dosage. Low doses of glycoalkaloids intake reportedly cause gastrointestinal disturbance such as vomiting, diarrhoea and abdominal pain, while high doses lead to acute intoxication and more severe symptoms such as rapid pulse, neurological disorder and in severe cases coma and death (Yvonne et al., 2017; Ruprich et al., 2009).

Extraction of phytochemicals from plants is generally done with conventional extraction methods such as the use of organic solvents (Nguyen et al., 2015; Chigayo et al., 2016). However, there are concerns about the potential health threatening effects of organic solvents (Felhi et al., 2017). In addition, these methods are time consuming, laborious and require large amount of hazardous and environmentally unfriendly organic solvents (Felhi et al., 2017). Since extraction plays a pivotal role in metabolite fingerprinting of plant extracts, improved extraction methods that produce reliable, reproducible and consistent results have been proposed (Premalatha et al., 2013). These include the use of pressurised hot water extraction (PHWE) technique and application of ultra-high-performance chromatography coupled to mass spectrometry (Alarcón-Flores et al., 2013; Matshediso et al., 2015). PHWE uses water as extraction solvent and it is considered a “green” or environmentally friendly method. It can also

produce a good extract yield probably because its solvation power can be manipulated over a wide spectrum of polarities (Sun et al., 2012). Coupling the HPLC with MS does not only offer excellent sensitivity and selectivity but also allows for determination of unknown and known phytochemical compounds (Zhao et al., 2016; Wu et al., 2016). Quadruple-Time-of-Flight Mass Spectrometry (QTOF-MS) fingerprinting method is one of the improved methods that has been proved to generate stable and reproducible results, particularly in the identification of chlorogenic acid (Ncube et al., 2014).

It appears that there is no scientific report on the phytochemistry of Solanum retroflexum despite the fact that the people in Venda region, Limpopo province, South Africa consume the leaves as food. Therefore, the aim of this research was to investigate the phytochemical composition of S. retroflexum leaf extracts with the aid of ultra-performance liquid chromatography hyphenated to quadruple-time-of-flight mass spectrometry (UPLC-qTOF-MS).

3.2 Materials and Methods 3.2.1 Plant Collection

Fresh Solanum retroflexum leaves were bought from small scale farmers in the Venda region of Limpopo province, South Africa, from September – October, 2015. The leaves were air dried under shade and stored at ambient temperature in airtight containers for further use.

3.2.2 Sample preparation Seven different types of sample extracts were used. Three samples were obtained by using 40 %, 60 % and 80 % methanol aqueous solution as solvent while four others were obtained by using PHWE at 50, 100, 150 and 250 °C.

3.2.2.1 Metabolite Extraction with Methanol

Extraction of metabolites from S. retroflexum leaves was performed using a procedure documented by Abu-Reidah et al. (2015) with slight modifications. Briefly, the leaves were ground using a pestle and mortar. The powdered leaf material (2 g) was mixed with 40 %, 60 % and 80 % aqueous methanol and shaken for 15 minutes using an ultrasonic cleaning bath. The homogenates were then centrifuge at 5000 rpm for 10 minutes using a bench top swinging bucket centrifuged at 4 °C. Thereafter, the supernatant was reduced to approximately 1 mL using a Buchi rotary evaporator at 55 °C under reduced pressure. The extracts were further dried to completeness using a heating block at 55 °C overnight. The dried extracts were reconstituted in 50 % methanol, filtered through 0.22 μm syringe filters and stored at -20 °C prior to an untargeted UHPLC-qTOF-MS analysis.

3.2.2.2 Metabolite extraction with Pressurised hot water

Pressurised hot water extraction of metabolites from S. retroflexum was done by a makeshift laboratory scale PHWE unit according to the method reported by Khoza et al. (2015). The extraction was carried out at different temperatures (50, 100, 150 and 250 °C) with a pressure of 1000 ± 200 psi maintained using the back-pressure valve. The extraction solvent (pure water) was pumped at a flow rate of 5.0 mL/min for approximately 10 minutes. For the extraction, 3 g of homogenised ground plant materials was mixed with 2 g of diatomaceous earth (Sigma Aldrich, Germany), a dispersing agent and placed in an extraction cell located inside an oven with automatically regulated temperature (± 1 °C). The extracts were collected into sealed Falcon tubes up to the 50 mL mark through the outlet coil immersed in a cooling water bath. They were filtered using a 0.22 µm nylon syringe filter into a 2 mL HPLC vial and preserved at -20 °C prior to analysis.

3.2.3 Ultra-High Performance Liquid Chromatography

For the chromatographic separation of the analyte, 2 μL of the extracts was injected and separated using an Acquity UHPLC equipped with an Acquity BEH C18 reverse phase column (150 mm × 2.1 mm, 1.7 μm) (Waters Corporation, MA, USA). The following chromatographic conditions were employed: mobile phase A consisted of 0.1 % formic acid in deionised water and mobile phase B consisted of 0.1 % formic acid in acetonitrile (Romil Pure Chemistry, Cambridge, UK). The initial conditions were 98 % A at a flow rate of 0.4 mL/min and kept constant for 1 min. A gradient elution was introduced to change the chromatographic condition to 97 % at 3 min for 1 min, 92 % at 4 min and held for 21 min, then changed to 50 % A at 25 min for 1 min, 5 % at 26 min for 2 min and finally returned to initial conditions of 98 % A at 28 min for 2 min. The total run time was 30 min. Chromatographic separation was monitored using a photodiode-array detector (PDA) collecting 20 spectra per second between the 200 and 500 nm range. Furthermore, chromatographic elution was also monitored using an electrospray ionisation quadruple time of flight mass spectrometer (ES1-qTOF –MS) detector.

3.2.4 Mass Spectrometry Acquisition Parameters

For MS, an ESI-qTOF –MS detector was used to monitor the separation of the analyte post- PDA detection. The mass spectrometer was operated in both positive and negative ionisation modes to scan the range of 100-1000 Da with a scan time of 0.2 s. The capillary voltage of 3.5 kV, sample cone potential of 30 V, multichannel plate detector potential of 1600 V, source temperature of 120 °C, desolvation temperature of 450 °C, cone gas flow of 550 L/h and desolvation gas flow of 550 L/h were the optimal experimental conditions. The MS data was acquired using series of collision energies in order to generate fragmentation data (MSE).

3.3 Results and Discussion

3.3.1 Methanol extraction of phytochemicals

Different concentrations of methanol (40 %, 60 % and 80 %, methanol/ water, v/ v) were used in this study to understand different degrees of the extractive potency of different concentrations of the solvent. Screening of metabolites was achieved by an untargeted UPLC-qTOF-MS at positive and negative ionisation modes. For efficient metabolite identification, MS fragmentation patterns were used in conjugation with standards. The methanol extracts of Solanum retroflexum generated using aqueous methanol resulted in very similar phytochemical composition (Figure 3.1). A total of 30 metabolites were identified and these include chlorogenic acids (CGAs), flavonoids and alkaloids as shown in (Table 3.1).

Relative Peak Intensity

3.3.2 Chlorogenic acids

Chlorogenic acids are phenolic acids that result from the esterification of quinic acid (QA) and cinnamic acid (CA) derivatives including caffeic, sinapic and ferulic acid (Khoza et al., 2016). In this study, chlorogenic acids were identified chromatographically with the aid of our recently developed in-source collision induced dissociation method. Generally, CGAs result in very similar fragments characterised by the following fragment ions: Q1 [quinic acid-H], C1 [caffeic acid-H], Q2 [quinic acid-H2O] and C2 [caffeic acid-CO2] (Che et al., 2016). Four peaks at m/z 353, with region-isomers and geometric isomers of Caffeoylquinic acid (CQAs) were detected in the extracts of S. retroflexum leaves (Figure 3.2). The CQAs derivatives, the most common form of CGA, are formed from the esterification of quinic acid to one or more caffeic acid unit. As shown in Figure 3.3, the metabolites from CGAs and their isomers (positional) can be differentiated mainly by fragmentation patterns into 3 CQA, 4 CQA and 5 CQA (Clifford et al., 2008).

Relative Peak Intensity

Chromatographically, it is easy to identify CGA and also differentiate between the trans and cis isomers because of the order in which they elute (Ncube et al., 2014). Interestingly, another important factor to note is that both trans- and cis- isomers have very similar fragmentation patterns (Feng et al., 2016). In this study, both the trans and cis isomers were identified (Table 3.1). This can be attributed to too much exposure of the leaves to UV rays since the S. retroflexum came from the northern part of South Africa that is known to be very hot. Ultraviolet rays are known to convent trans to cis isomer (Romero-pe and Lamuela-ravento, 1996). The presence of these cis isomers is important since they are known to possess biological activity such as antioxidant activity, antimicrobial activity, anti-viral and anti-diabetic activity (Willems et al., 2016; Plazas et al., 2014).

The presence of CGAs in Solanum leaves has previously been reported in the Solanaceae family but not in S. retroflexum. For instance, CGAs were identified in Lycium intricatum methanolic leaves extract (Abdennacer et al., 2015). Solanum corymbiflorum leaves extracted with ethanol were also found to contain high amounts of CGAs (Piana et al., 2016). Previous research has shown that CGAs exhibit many biological activities such as anti-bacterial, anti-viral and antioxidant activities (Upadhyay and Rao, 2013). In addition, anti-inflammatory activities of CGAs possibly by modulating a number of important metabolic pathways have been reported elsewhere (Liang and Kitts, 2015). Chlorogenic acids are also found in beverages such as tea, coffee and wine. This could be responsible in part for the health promoting benefits of such food products (Liang and Kitts, 2015).

Apart from the CQAs, other mono-acyle CGA that are not ferulic but coumaric and ferulic were identified (Figure 3.4). Also, di-acyl CGAs which are very important molecules with good biological activity such as being natural antioxidants in food or non-food products (Suárez- Quiroz et al., 2014), were observed.

3.3.3 Characterisation of Caffeoylquinic acids

Molecules 1, 2 and 3 were identified as 3CQA, 4CQA and 5CQA, respectively (Table 1). Molecule 1 at retention time 9.8 min produced a precursor ion at m/z 353.081 was identified as cis-3CQA. This molecule produced fragments ions at m/z 179, 191, 135 characteristic of quinic acylated at 3 positions with Caffeoylquinic acids, thus 3CQA (Carlotto et al., 2015). Molecule 2

and 3 was identified as trans-4CQA and 5CQA with a precursor ion at 191, 179, 173, 135. Molecule 4 and 5 were identified as cis-4CQA and 5CQA. Amongst the CGAs, the CQAs have the widest occurrence and are well researched (Ncube et al., 2014; Mondolot et al., 2006). Another interesting observation was the fact that cis-5-CQA (Figure 3.2) appeared in a relatively high intensity compared to the cis-3-CQA. It has also been reported that the same molecule was shown to be induced by activators of plant defence and primary responses (Farah and Donangelo, 2008).

3.3.4 Characterisation of Feruloylglycoside

Feruloylglycosides have a molecular weight (Mr) of 356 and they produce a precursor ion at m/z 355. Here, only one molecule was identified and it produced a base peak at m/z 175 and 160

([ferulic acid-H-H2O]) (Table3.1). The fact that feruloylglycoside molecule is biosynthesised in Solanum retroflexum leaves suggests a very interesting biochemical characteristic of this plant (Mhlongo et al., 2015).

3.3.5 Characterisation of di-caffeoylquinic acid and caffeoylquinic acid- glucoside

Both di-caffeoylquinic acid and caffeoylquinic acid glucoside were identified with a precursor ion at m/z 515 (Table 3.1). However, based on their accurate masses and fragmentation patterns these molecules can be distinguished (Carlotto et al., 2015). Here, molecules 7-13 were annotated as either di-CQA or CQA-glycoside. Based on the accurate mass, the di-CQA were detected with an average m/z of 515.1463 (C25H23O12) and the CQA glycosides were found to have an average m/z of 515.1292 (C22H27O14) based on previous reports (Clifford et al., 2005; Iwashina and Matsumoto, 2013). Molecule 7 and 8 were identified as trans- and cis-CQA- glycoside. The CQA glycoside produced distinctive ions at m/z 341 ([caffeoyl glucoside- H]-) or/ and 323 (caffeoyl glucoside – H – H2O] -) which were not present in the diCQA MS spectra. Recently, the hierarchical fragmentation schemes of similar molecules have been reported (Clifford et al., 2003). In their work, it was noted that CQA forms a glycoside through an ether bond at either C-3 or C-4 on the aromatic caffeoyl ring. Molecules 9-13 were identified as di-

CQAs (3, 4 di-CQA (9), trans- and cis-3, 5 di-CQA (10, 11) and trans- and cis-4, 5 di-CQA (12, 13).

3.3.6 Characterisation of p-coumaroylquinic acid and Coumaroyl-hexose p-coumaroylquinic acid has a Mr of 338 and precursor ions at m/z 337 were detected in the leaf extracts of S. retroflexum (Table 3.1). These ion peaks were identified as 3-pCoQA (14), trans 5- pCOQA (15), cis-5-pCOQA (16) and 4-p-COQA (18). The presence of p-CoQA has been found to accumulate in other plants such as legume forages and birch trees (Jaiswal et al., 2014; Nurmi et al., 1996). Molecule 17 was identified as Coumaroyl-hexose with a precursor ion at m/z 339 and product ions at 223, 164 and 149.

3.3.7 Characterisation of Feruloylquinic Acids

Feruloylquinic acids have a Mr of 368. Similar to pCoQA and CQA, molecules harbouring ferulic acid moieties were identified. Four peaks were successfully identified as trans-5-FQA (19), cis-5-FQA (20), 4-FQA (21) and 3-FQA (22) respectively (Table 3.1).

Table 3.1: UHPLC data of the compounds identified in Solanum retroflexum leaf extracted with aqueous methanol and PHWE

Mol. Retention time Mass Compound name Diagnostic m/z ions no. (min)

1. 9.8 353 3-CQA 191, 179, 135 2. 5.6 353 trans -4-CQA 191, 135, 173, 179 3. 5.1 353 trans -5-CQA 191, 135 4. 6.0 353 cis-4-CQA 191, 135, 173, 179 5. 5.9 353 cis-5-CQA 191, 135 6. 6.5 355 Feruroylglycoside 160,175 7. 5.3 515 trans-5-CQA glycoside 191, 135 8. 6.4 515 cis-5-CQA glycoside 191, 135 9. 8.9 515 3,4-diCQA 191, 179, 173, 135 10. 9.1 515 cis-3,5-diCQA 191, 179, 135 11. 9.3 515 trans-3,5-diCQA 191, 179, 135 12. 9.5 515 trans-4,5-diCQA 191, 179, 135 13. 10.5 515 cis - 4,5 -diCQA 191, 179, 135 14. 5.3 337 3-p-CoQA 177 15. 6.8 337 trans-5-p-CoQA 191 16. 7.5 337 cis-5-p-CoQA 191 17. 8.7 339 coumaroyl-hexose 223, 164, 149 18. 7.4 337 4-p-CoQA 173 19. 7.4 367 trans-5-FQA 191 20. 8.0 367 cis - 5 - FQA 191 21. 10.7 367 4-FQA 173 22. 10.3 367 3- FQA 193, 134 23. 8.1 609 Quercetin-3-rutinoside 300 24. 9.1 447 Kaempferol-3-glucoside 284 25. 8.4 463 Quercetin-7-glucoside 300 26. 8.8 593 Kaempferol-3-0-rutinoside 285 27. 9.0 623 Isorhamnetin-3-0-rutinoside 315 28. 8.7 447 Quercetin-3-rhamnoside 300 29. 20.3 884 Solasonine 722, 576, 414 30. 20.1 868 Solamargine 722, 576, 414 *Note: CQA– Caffeoylquinic acid; diCQA– di- Caffeoylquinic acid; p-CoQA– p-Coumaroylquinic acid; FQA– Feruloyquinic acids; Mol no– Molecule number; Min- Minutes; m/z– Mass-to-charge-ratio; KMI– KNApSAcK Metabolites Information; DNP– Dictionary of natural products.

3.3.8 Characterisation of Flavonoid Derivatives

A total of six flavonoids 23, 24, 25, 26, 27 and 28 were detected and characterised in S. retroflexum by QTOF-MS method presented herein (Table 3.1). Molecule 23 at retention time 8.12 min produced a precursor ion at m/z 609 and its fragmentation pattern was found consistent to that of quercetin-rutinoside in the literature (Makita et al., 2016). Briefly, the product ions of molecule 23 were primarily due to the elimination of a rutinoside sugar moiety, which resulted in an intense peak at m/z 300 (Regos et al., 2009). Importantly, quercetin is reported to be the main dietary flavonoid that is connected to various properties that suppress certain chronic diseases (Makita et al., 2016). This has made it useful for the treatment of certain chronic diseases (Suganthy et al., 2016; Mascaraque et al., 2014). Furthermore, quercetin has other biological potential such as protecting liver cells and suppressing haemoglobin oxidation because of its antioxidant activity (Sánchez-gallego et al., 2010; Yousef et al., 2010; Arya et al., 2010). Molecule 24 was identified as kaemferol-3-glucoside with a precursor ion at m/z 447 [M-H] ˉ (Table 3.1). It produced fragment ions at m/z 284 obtained due to the loss of a hexose moiety as reported in literature (Nishimura et al., 2016). Molecule 25 was identified as quercetin-7- glucoside with a precursor ion at m/z 463 [M-H] ˉ and it produced a fragment ion at m/z 300 as documented in the literature (Ramabulana et al., 2016). Molecule 26 was identified as kaempferol-3-0 rutinoside with a precursor ion at m/z 593 [M-H] ˉ and a fragment ion at m/z 285, indicating the elimination of a rutinoside moiety. Such a fragment pattern has been reported elsewhere (Vagiri et al., 2010). Molecule 27 was identified as Isorhamnetin-3-0-rutinoside with a precursor ion at m/z 623.1607 [M-H] ˉ and it produced a fragment ion at m/z 315, representing an isorhamnetin aglycone due to the elimination of a rutinoside moiety (Makita et al., 2016). Molecule 28 was identified as quercetin-3-rhamnoside with a precursor ion at m/z 447 [M-H] ˉ and it produced a fragment ion at m/z 300.

Flavonoids occur in foods as 0-glycosides with sugar bound at C3 position. The structures of Quercetin-3-rutinoside, Kaempferol-3-glucoside, Quercetin-7-glucoside, Kaempferol-3-0- rutinoside, Isorhamnetin-3-0-rutinoside and Quercetin-3-rhamnoside identified flavonoid glycosides show that almost all of them contain either kaemferol, quercetin or isorhamnetin core moieties (Figure 3.4). These indicate the presence of a wide spectrum of sugar moieties attached to these flavonoid core structures. The sugar moiety is important in the bioavailability of

flavonoid glycosides in humans. The total number of sugar moieties, their position and structure influences the antioxidant activity of such flavonoids (Litvinov et al., 2007).

Polyphenol metabolites such as flavonoids have been reported to possess various medicinal and pharmacological activities (Sharma et al., 2006). These properties have been suggested to be dependent on the structural configuration of the flavonoid molecule (Ramabulana et al., 2016). Interestingly, some of these flavonoids (such as quercetin) have been utilised in clinical trials (Tai et al., 2006). High levels of quercetin and kamferol in Solanum lycopersicum have been reported to exhibit antioxidant and pharmacological properties (Toku et al., 2003). Flavonoids isolated from Solanum torvum have also been shown to have important antimicrobial activity. The antibacterial activities might be due to their ability to complex with bacterial cell wall, thereby inhibiting microbial growth (Govindaraju et al., 2010; Sivapriya et al., 2011). Kaemferol, quercetin and isorhamnetin aglycone are the most common flavonoids that are abundant in plant tissues (Makita et al., 2016).

3.3.9 Characterisation of Alkaloid Derivatives

The S. retroflexum methanol leaf extracts were found to contain two alkaloids. Molecule 29 and 30 with precursor ions at m/z 884 and 868 were identified as Solasonine and Solamargine, respectively (Table 3.1). The mass spectra of these molecules are shown in (Figure 3.5). The difference in the detected m/z ratios in Alkaloid mixtures are a result of variation in their aglycones. However, their corresponding glycosides contain either solatriose or chacotriose as a sugar moiety. Thus, solasonine has a molecular ion at m/z 884 and solamargine has one at m/z 868. In addition, the fragment ions at m/z 722, 576 and 141 were identical and they elute very close to each other.

Glycoalkaloids (GAs) function chemically as defence against herbivores, microorganisms and competing plants. As a survival strategy, plants need to store sufficient amount of these defence compounds at a strategic site such as the bark, leaves, flower, seeds or fruits (Iijima et al., 2010). Therefore, the presence of steroidal glycoalkaloid in S. retroflexum leaves should not be neglected because of its potential toxicity to humans. Solasonine and Solamargine have also been identified in the leaves of Solanum incanum (Al Sinani S et al., 2016). In their study, it was reported that the concentration of these alkaloids declined during the plants developmental stages with the least levels recorded at maturity. Other Solanum plants such as Solanum nigrum have been reported to produce Solasonine and Solamargine (Chen et al., 2010).

It has been documented that low doses of GAs intake can cause gastrointestinal disturbance such as vomiting, diarrhoea and abdominal pain, while at high doses the toxicity of GAs leads to acute intoxication and more severe symptoms including rapid pulse, low blood pressure, neurological disorder and in severe cases coma and death (Ruprich et al., 2009).

3.4 Pressurised hot water extraction

With pressurised hot water extraction, an eco-friendly extraction technique, optimum extraction occurred at the highest temperature (250 °C). Unlike what occurred with methanol, only six health promoting metabolites were identified with PHWE (Table 3.1). These include 3CQA, trans-5-CQA, 3, 4-diCQA, Quercetin-3-rutinoside, Kaempferol-3-glucoside and Kaempferol-3- 0-rutinoside. One notable compound, which was seen at all temperatures (50, 100, 150 and 250 °C) is quercetin rutinoside. It is worth noting that quercetin was the most abundant flavonoid aglycone identified in methanol extracts too. The importance of rutin in the diet of humans has been previously discussed. Interestingly, Alkaloids were absent at all extraction temperatures with PHWE. This was thought to be due to the fact that alkaloids dissolve better in alcohol. With regard to human, this might justify why the S. retroflexum leaves are not toxic when prepared as food. It also highlights the importance of the PHWE technique for extracting metabolites in food plants and its better selectivity, which is due the fact that its solvation power can be manipulated over a wide spectrum of polarities (Sun et al., 2012).

3.5 Conclusion

The present study represents the first comprehensive phytochemical composition of Solanum retroflexum using the described UHPLC-qTOF-MS method. A total of 30 different metabolites were identified with methanol extraction and these include alkaloids, flavonoids and cinnamic acid derivatives. With PHWE, only six metabolites, which were also extracted with methanol, were identified. However, glycoalkaloids, which were identified in the methanol extracts, were not found in the PHWE. The concentration of solvent and extraction temperature did not affect the type of compounds extracted. The present study confirms the presence of phytochemical compounds in S. retroflexum similar to other Solanum plants. The results also show that although PHWE is an attainable green technique for the extraction of different metabolites, it does not always yield high amounts of polyphenols compared to methanol extraction. Further studies must be done to optimise extraction of phytochemicals from S. retroflexum leaves using the PHWE technique. In food and medicinal applications, PHWE would be the choice extraction solvent rather than alcoholic solvent such as methanol because of its safety and environmental friendliness as the extraction solvent is water (which is non-toxic, non-flammable and renewable). In addition, water is cheap, readily available and does not generate harmful byproducts.

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CHAPTER FOUR

TOTAL PHENOLIC, FLAVONOID AND TANNIN CONTENTS AND ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF Solanum retroflexum LEAF EXTRACTS.

Abstract

The purpose of this work was to determine the total phenolic, flavonoid and tannin content of Solanum retroflexum leaves extracted with different concentrations of aqueous methanol (40 %, 60 % and 80 %, methanol/water, v/v). Further, the antioxidant and antimicrobial activities of the extracts were determined. The results show that the total phenolic content of S. retroflexum leaves is substantially higher (approx. 4.57 mg GAE/g) than in other Solanum plants such as S. nigrum and S. tuberosum. However, the total flavonoid (approx. 3.03 mg QE/g) and tannin content (approx. 2.90 mg CE/mg) of S. retroflexum seem to be substantially lower than that of other Solanum plants. Solvent concentration affected the extract yield with more compounds extracted at the highest concentration of methanol. With regard to antioxidant activity, S. retroflexum leaf extracts exhibited some antioxidant activity in vitro when investigated with the DPPH, ABTS and FRAP assays. In general, solvent concentration did not affect the antioxidant activity. The aqueous methanol extracts of S. retroflexum showed moderate antimicrobial activity against 6 out of the 14 microorganisms investigated in this study. The susceptible microorganisms included B. cereus, M. smegmatis, S. aureus, S. epidermis, K. pneumonia and P. vulgaris. The minimum inhibitory concentration (MIC) as determined by the Micro Dilution method was 4-16 mg/mL. This study provides the baseline information for the possible utilisation of S. retroflexum aqueous methanolic leaf extracts in food applications and in the control of infectious diseases.

Keywords: Solanum retroflexum, Antioxidant activity, Antimicrobial activity, Total phenolic, Total flavonoid and Tannin content.

4.1 Introduction

An interest has developed in the last decade to search for phytochemicals of native and naturalised plants for pharmaceutical and nutritional purposes (Zarabska-Bozejewicz et al., 2015). Plant derived products have great potential as a source of phytochemicals (Tuttolomondo et al., 2014; Ouelbani et al., 2016) and their application in food products is gaining momentum. Different plant parts are rich in various types of bioactive compounds, many of which are discarded as by-product by the food industry. Recently, mainly due to undesirable side effects (such as toxicity and carcinogenicity) of synthetic additives, interest has considerably increased for the search for naturally occurring antioxidants and antimicrobial compounds suitable for use in food and/or medicine (Aguiar et al., 2016; Jiang and Xiong, 2016). As such, current research has focused on many plant species in order to find new natural bioactive compounds.

The genus Solanum has an important economic value with many cultivated species being utilized as a source of edible vegetables and fruits (Muthoni et al., 2012). In addition, the genus is well known for its medicinal properties (Anandan et al., 2015; Pereira et al., 2014). Due to Solanum complexity, the genus has attracted research interest of many taxonomists. Pharmacological studies of Solanum lycopersicum (tomato) show the beneficial effects of tomato consumption in the prevention of chronic diseases, such as cardiovascular disease and cancer (Erba et al., 2013). Furthermore, Solanum plants are known for their nutritional and commercial properties (Gómez- Romero et al., 2010). Although several other Solanum plants have been investigated for their phytochemical properties in many research projects, no reports have been documented on the phytochemical properties of Solanum retroflexum leaves. This is despite the fact that S. retroflexum leaves are consumed as food in some parts of South Africa and that several Solanum plants have been found possessing a large number of compounds with health benefits. For example, Solanum nigrum leaves are a good source of antioxidants (De Sousa et al., 2013; Jiang et al., 2016). In humans, research has shown that endogenous antioxidants or exogenous antioxidants supplied by the diet can function as free radical scavengers and improve human health (Peñalvo et al., 2016). Antioxidants achieve this through retarding the oxidation process and thus preventing oxidative damage of DNA and other essential cell components thereby mitigating against degerative conditions such as ageing, cardiac and cerebral ischema (Amir Aslani and Ghobadi, 2016).

Plants are also sought for their antimicrobial properties, because antibiotic resistance is becoming a worldwide public health concern, especially in terms of foodborne illness (Al-zoreky and Al-Taher, 2015). Synthetic preservatives such as parabens (propyl parabens, methyl, butyl and ethyl), butylated hydroxanisole (BHA) and butylated hydroxytoluene (BHT) are suspected to cause negative health effects, such as cancer in humans. As alternatives, plant derived compounds seem to have less such effects. According to literature, the antimicrobial and antioxidant activities of plant by-product has considerably increased (Silva-Beltrán et al., 2015). The current trend in agribusiness sector is to evaluate, recover and find better uses for plant by- product such as leaves, stem peels and seeds. Solanum plants contain bioactive substances that could be potential sources of antimicrobial, antiviral and antioxidant compounds, giving them economic value in nutraceuticals and food industry. In this context, the aim of this study was to investigate the total phenolic, flavonoid and tannin contents and antioxidant and antimicrobial activities of Solanum retroflexum leaf extracts.

4.2 Materials and Methods

4.2.1 Collection of plant materials

Solanum retroflexum leaves were bought from small scale farmers in the Venda region of Limpopo province, South Africa, between September and October 2015. The leaves were air dried under the shade and stored at ambient temperature in airtight containers for further use.

4.2.2 Chemicals and Solvents

Solvents used in this study include UPLC/MS grade methanol, purchased from Romil, MicroSep, South Africa. Ultrapure water was obtained from a Milli-Q Gradient A10 system (Millipore, Billerica, MA, USA), 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tris (2-pyridyl)-1,3,5- triazine (TPTZ), Folin-Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchroma-2-carboxylic acid (Trolox), Sodium carbonate, Sodium nitrate, Sodium hydroxide, Vanillin, HCl, Catechin, Iron chloride, Acetate buffer, Potassium sulphate, Resazurin sodium salt, Gallic acid, Aluminium chloride, Quercetin, Streptomycin, Mueller Hinton agar, Nutrient broth and DMSO (dimethyl sulfoxide) were purchased from Sigma Aldrich.

4.2.3 Bacteria strains and Growth condition

Bacillus cereus (ATCC 10876), Bacillus subtilis (Biotechnolgy and Food Technology, UJ) Enterococcus faecalis (ATCC 7080), Mycobacterium smegmatis (Biotechnolgy and Food Technology, UJ), Staphylococcus aureus (ATCC 6571), Staphylococcus epidermidis (ATCC 12228), Enterobacter aerogenes, Enterobacter cloacae (ATCC 13047), Escherichia coli (Biotechnolgy and Food Technology, UJ), Klebsiella oxytoca (ATCC 13182), Klebsiella pneumoniae (ATCC 13883), Proteus mirabilis (ATCC 12453), Proteus vulgaris (ATCC 33420) and Pseudomonas aeruginosa (ATCC 15442) were employed in the experiments. The strains were sub-cultured in nutrient agar and allowed to grow overnight in an incubator at 37 °C. Biochemical tests and morphological characterisation was used to confirm the bacteria.

4.2.4 Preparation of Extracts

Extraction of metabolites from S. retroflexum leaves samples was performed using a procedure reported elsewhere (e.g Abu-Reidah et al., 2015) with slight modifications. Dried leaves of S. retroflexum were grounded using a pestle and mortar to a fine powder. The powdered leaf material (2 g) was mixed with 20 mL of 40 %, 60 % and 80 % aqueous methanol and sonicated for 15 minutes using an ultrasonic cleaning bath (SB- 120DT, Loyal Key Group, Hong Kong). The homogenates were then centrifuged at 5000 rpm for 10 minutes using a bench top swinging bucket centrifuge at 4 °C. Thereafter, the supernatant was dried using a rotary evaporator at 55

°C under reduced pressure. The extracts were further dried to completeness using a heating block at 55 °C overnight under continuous airflow. The dried extracts were reconstituted in 1 mL methanol at different percentages. The resulting extracts were then maintained at -20 °C to avoid degradation before subsequent analysis. Extractions were done in triplicate.

4.3 Determination of Total Phenolic Content (TPC)

The total phenolic content was determined with the Folin-Ciocalteu method as reported by Simirgiotis and Schmeda-Hirschmann (2010) with some modifications. Extracts (10 mg) were dissolved in 1 mL of various concentrations of methanol/water (40:60; 60:40; 80:20 v/v). Thereafter, 10 μL of each sample was mixed with 50 μL Folin-Ciocalteu reagent and 500 μL of distilled water. The mixture was incubated at room temperature (25 °C) for 3 minutes. After that,

200 μL of 100 g/L solution of sodium carbonate (Na2CO3) was added. The mixture was shaken and incubated for 30 mins in the dark at room temperature followed by measuring the absorbance at 750 nm using a Milton Roy 601 UV-Vis spectrophotometer. A calibration curve was constructed with the standard gallic acid (R² = 0.9913) and the results were expressed as milligrams of gallic acid equivalent (GEA) per gram dry weight.

4.4 Determination of Total Flavonoid Content (TFC)

Determination of total flavonoid content in extracts was performed using a method based on the formation of complex flavonoid-aluminium (Fujieda et al., 2012) with slight modifications. Extracts (10 mg) were dissolved in 1 mL of various concentrations of methanol/water (40:60; 60:40; 80:20 v/v). Sample solutions (30 μL) were mixed with 20 μL of 1 M sodium nitrite

(NaNO2). Five minutes later, 20 μL aluminium trichloride (AlCl3) (10 %, w/v) was added, followed by the addition of 100 μL sodium hydroxide (NaOH) to give a total volume of 170 μL. The absorbance was measured at 450 nm using a Milton Roy 601 UV-Vis spectrophotometer. Quercetin was used as a standard (R² = 0.9910). Results were expressed as mg quercetin equivalents ̸ g dry weight of plant.

4.5 Determination of Tannin Content (TC)

Tannin content was determined following the procedure outlined elsewhere (e.g. Furstenburg et al., 1994). Samples (10 mg) of the leaf extracts were dissolved in 1 mL of 40 %, 60 % and 80 % aqueous methanol (MeOH). Exactly 1 mL of each extracts were transferred to test tubes containing 5 mL of vanillin-HCl reagent, vortexed and incubated at room temperature (25 °C) for exactly 20 minutes. For colour blank determination, 1 mL of each extracts were transferred to test tube containing 5 mL of 4 % hydrochloric acid (HCl) in methanol, vortexed and incubated at room temperature. The vanillin-HCl, reagent was used as reagent blank. The absorbance was measured at 500 nm with a spectrophotometer. The results were expressed as mg catechin equivalents (CE) per 100 mg.

4.6 Antioxidant Activities

4.6.1 DPPH Assay: The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed according to the procedure reported in the literature with some modification (Wollinger et al., 2016). A DPPH solution (0.4 mM) in methanol was stirred for 20 minutes until all particles were dissolved and the absorbance of the solution was adjusted to 1.1 at 490 nm. Thereafter, 40 µL of the plant extracts (40 %, 60 % and 80 %) were mixed with 160 µL of DPPH solution and incubated for 10 minutes in the dark at room temperature (25 °C). The reduction of the DPPH radical was measured by monitoring the decrease of absorption at 490 nm and the radical scavenging was calculated and expressed as µM Trolox equivalent/ g sample in dry mass using a standard curve (R² = 0.9986).

4.6.2 FRAP Assay: The ferric reducing ability of the leaf extracts was assessed following the method described elsewhere (e.g. Wootton-Beard et al., 2011). Freshly prepared FRAP reagent: 2.5 mL of 10 mM 2, 4, 6-tris (2-pyridyl)-1, 3, 5-triazine (TPTZ) solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3 and 25 mL of 0.3 M acetate buffer, pH 3.6. Aliquots (40 µL) of studied extracts (40 %, 60 % and 80 %) were mixed with 0.2 mL distilled water and 1.8 mL FRAP reagent was added. In parallel, a solution containing 0.24 µL ultrapure distilled water and 1.8 mL FRAP reagent was prepared as negative control. The absorbance was measured at 595 nm and the

results were expressed as µM Fe (II) equivalent/g sample in dry weight using a standard curve (R² = 0.9995).

4.6.3 ABTS Assay: 2,2- Azinobis (3- Ethyl-Benzothiazone-6-Sulfonic Acid) test was performed using the method describe elsewhere (e.g. Evans and Britton, 1987) with slight modification. Briefly, the ABTS+ solution was obtained by mixing ABTS+ (7 mM) at pH 7.4 with potassium sulphate (2.5 mM). The solution was stored in the dark at room temperature (25 °C) for 12 hours and then used within 16 hours. Samples (20 µL) of each diluted aqueous methanol extracts (40:60; 60:40; 80:20 v/v) were mixed with 180 µL of the ABTS+ solution. The samples were left for 5 min at room temperature followed by measuring the absorbance at 750 nm. The results were expressed as µM Trolox equivalent/g sample in dry mass using a trolox standard curve (R² = 0.9976).

4.7 Antimicrobial Assays

4.7.1 Disc Diffusion Assay

The antimicrobial activity with the disc diffusion assay was done by measuring bacterial growth inhibition zone, as described by Mahmoudi et al. (2016) with some modifications. Mueller- Hinton agar (Oxoid) medium was seeded with 100 µL of freshly prepared bacterial suspensions (108 CFU/mL). Inoculum was spread uniformly with a sterile cotton swab on the surface of the petri plates (Merck, South Africa). Leaves extracts (0.2 mg) were dissolved in 2 mL 10 % DMSO. Thereafter, 20 µL samples of each extracts were loaded onto sterile filter paper discs (6 mm in diameter, Whatman #1) which were subsequently placed on top of the agar plates. The plates were left for 30 min at room temperature (25 °C) and then incubated at 37 °C for 24 hrs. Antimicrobial activity was evaluated by measuring the inhibition zone (mm) against the studied microorganisms, including disc diameter. Negative control treatments consisted of discs impregnated with 10 % DMSO (20 µL). Streptomycin (0.032 g/mL) was used as positive control.

4.7.2 Minimum inhibitory concentration (MIC)

The minimum inhibition concentration (MIC) was determined via micro dilution according to the method outlined elsewhere (e.g. Andrews et al., 2001). The experiment was done with Mueller Hinton (MH) broth using 96-well micro titer plates. The extracts were dissolved in sterile 10 % DMSO and serial dilutions were made with MH broth, maintaining a constant volume of 1000 µL in each tube. At that stage, the samples (100 µL) were tested at varied concentration (16 mg/mL to 0.03125 mg/mL). The outer wells of the plate were filled with sterile distilled water. An inoculum of 100 µL of the standardized (106 CFU/mL) overnight bacterial cultures were added into each well (horizontally and vertically) in five repeats for each bacterium. Thereafter, 100 µL of the diluted samples were added and the plates were covered and incubated at 37 °C for 18 h. After that, 10 µL of 0.02 % (w/v), resazurin sodium salt solution was added to the wells followed by further incubation for two hours. The wells were then visually inspected for colour changes. Blue coloured wells indicated no bacterial growth while pink coloured wells indicated bacterial growth. The minimal inhibitory concentration value was observed as the lowest concentration where no viability was observed in the 96- microwell plates after incubation.

4.7.3 Statistical Analysis: A one way analysis of variance (ANOVA) was performed to determine the differences between individual extraction conditions using SPSS statistical software version 23 (SPSS/IBM, Chicago, Illinois). The results were expressed as mean values ± standard deviation values and statistical significance was set at (p < 0.05). Means and standard deviations from the TPC, TFC, TC, DPPH, ABTS and FRAP are results of experiments performed in triplicate. The Pearson’s correlation test was used to determine the relationship between TPC, TFC, TC and antioxidant activity.

4.8 Results and Discussion

4.8.1 Total phenolic, flavonoid and tannin content

Plants are generally reported to contain substantial amounts of polyphenols, which include flavonoids, tannins, phenolics and tocopherols (Lizcano et al., 2010; Hygreeva et al., 2014). In this study, the total phenolic content (TPC), flavonoid content (FC) and tannin content (TC) of S. retroflexum leaves were determined in aqueous methanol extracts (40 %, 60 % and 80 %). This is because polyphenols are reportedly extracted better in more polar solvents such as aqueous methanol compared to absolute methanol (Alothman et al., 2009; Calabrone et al., 2015). In general, the TPC, TFC and TC levels increased with an increase in the concentration of methanol (Table 4.1).

With TPC, extraction with 80 % aqueous methanol yielded significantly (p<0.05) higher phenolic content (4.57 mg GAE /g) than extraction with 60 % (3.90 mg GAE /g) and 40 % (2.4 mg GAE/g) aqueous methanol. This suggests that S. retroflexum leaves may contain less polar polyphenols than polar compounds. Contrarily, other workers have reported that phenolics are extracted in higher amounts with more polar solvents such as aqueous methanol than with absolute methanol (Sultana et al., 2009). The discrepancy could be attributed to differences in plant species and composition of polar compounds in each plant. The total polyphenol content in S. nigrum leaves extracted with methanol at 80 °C was reported to be 13.2 mg/100g of leaves (Akubugwo et al., 2007). These levels are approx. ten times lower than the TPC reported here. However, apart from the differences in assays, their study did not indicate the standard used for quantification, which makes it difficult to compare their results with those of the current study. Elsewhere, Zarzecka et al., (2017) reported a polyphenol content of between 274.1 – 304.1 GAE mg/Kg fresh matter, in selected cultivars of Solanum tuberosum L. (potato) leaves extracted with 80 % methanol. Although their results are expressed on, as is basis, they are substantially lower than the total phenol content found in S. retroflexum leaves here.

Table 4.1: Observations of total phenolic, flavonoid and tannin content of Solanum retroflexum leaf extract at different concentrations of methanol in water.

Methanol TPC (mg GAE/ g)* TFC (mg QE/ g)* TC (mg CE/ mg)* Composition

40 % 2.40 ± 0.10a 1.30 ± 0.02a 0.03 ± 0.05a

60 % 3.90 ± 0b 2.47 ± 0.14b 1.04 ± 1.0a

80 % 4.57 ± 0.58c 3.03 ± 0.24c 2.90 ± 1.09b

*Results expressed in dry matter weight (dw). TPC – Total Phenolic Content, TFC – Total Flavonoid Content, TC – Tannin Content, GAE – Gallic Acid Equivalent, QE –Quercetin Equivalent, CE – Catechin Equivalent. All data reported as mean ± SD (n=3). Alphabets in superscripts within a column show significant differences among the extracts at different percentages.

Similar to the TPC, a significantly (p<0.05) higher TFC (3.03 mg QE/g) was observed with 80 % aqueous methanol than with the 60 % and 40 % extractions (Table 4.1). The total flavonoid content in two tomato varieties were reported to be 33.03 mg QE/g (Pitenza) and 61.96 (Floradale) mg QE/g (Silva-Beltrán et al., 2015). These levels are substantially higher than values found in S. retroflexum here, because of differences in plant species and extraction assays. In their study, extraction was done with acid-ethanol (5:95) and via sonication. Elsewhere, Akubugwo et al. (2007) reported flavonoid content of 0.81 mg/ 100 g leaves in S. nigrum. Although these levels are lower than the TFC reported in S. retroflexum here, their results cannot be compared to those of the current study due to the afore mentioned reasons.

A significantly (p<0.05) higher tannin content (2.90 mg CE/ mg) was obtained with the 80 % aqueous methanol extracts than with the 60 % (1.04 mg CE/ mg) and 40 % (0.03 mg CE/ mg) methanol extracts. Modilal et al., 2015 screened S. nigrum leaves for the presence of tannins in ethanolic, aqueous and petroleum ether extracts. All the extracts tested positive for the presence

of tannins. Similarly, Kumar et al., 2016 reported the presence of tannins in S. nigrum L. leaves after conducting qualitative phytochemical tests on hexane, chloroform, acetone, ethanol and water extracts. A tannin content of 65.9 mg/g was also reported in Solanum torvum leaves extracted with methanol (Kusirisin et al., 2009). Similar to what was obtained with TFC, the tannin content reported by these workers is more than ten times higher than the levels reported in the current study. The possible reason for the discrepancies in results has been previously mentioned.

No results have been documented on the polyphenol content of S retroflexum leaves. Herein, S. retroflexum has been shown to contain a TPC that is lower than that of other Solanum plants. It is worth noting though that the level of polyphenols in plants might differ due to availability of extractable components, resulting from the varied chemical matrix composition and plant environment (Naczk and Shahidi, 2006). Polyphenol content in plants can also be detected at different concentration ratios because the amount and composition of secondary metabolites are not constant and their concentration depends on the tissue type and age of the plant (Silva- Beltrán et al., 2015). In addition, the extraction yield of polyphenols from plant material may also be influenced by the ratio of solvent-to-sample (Naczk and Shahidi, 2006).

4.8.2 Antioxidant Activity

In this study, the antioxidant activity of S. retroflexum leaf extracts was determined with the DPPH, ABTS and FRAPS assays. Previous research has reported that methods for determining antioxidants activity give different results because of their different mechanistic principles (Floegel et al., 2011). The DPPH and ABTS are colometric assays and they measure the relative antioxidant potential of natural extracts to scavenge free radicals (Shalaby and Shanab, 2013). Due to excellent reproducibility under strict conditions, both assays are the most and commonly utilised antioxidant methods despite differences in their response to antioxidants. The FRAP assay, on the other hand, is used to measure the capacity of reductants in a sample (Bontempo et al., 2013).

Here, the extracts showed strong scavenging activities against DPPH and ABTS radicals (Table 4.2). The scavenging of the ABTS radical by the extracts was found to be much higher than that of DPPH radical. Similarly, other researchers have reported such a phenomenon in other plants

(Adedapo et al., 2008; Bhoyar et al., 2011; Teow et al., 2007). This has been attributed to the fact that some compounds which have ABTS scavenging activity do not exhibit DPPH scavenging activity (Bhoyar et al., 2011). The scavenging activity on DPPH is one of the quickest methods to evaluate antioxidant activity. In this study, the scavenging abilities of the aqueous methanol (40 %, 60 % and 80 %) extracts against DPPH radical did not differ significantly (p<0.05) (Table 4.2). Overall, effective use of the DPPH results show that all three solvent concentrations yield hydrogen donating capabilities and can equally be used to determine antioxidant activity from the plant.

ABTS is one of the radicals generally used for testing preliminary radical scavenging activity of a compound or plant extract. The ABTS+, generated from oxidation of ABTS by potassium persulfate, is an exceptional tool for determining the antioxidant activity of hydrogen donating antioxidants and chain-breaking antioxidants (Le Grandois et al., 2017). The aqueous methanolic extracts of S. retroflexum leaves were effective scavengers of the ABTS radical (Table 4.2). The three aqueous methanolic extracts gave similar values (approx. 60 µM TE/ g) for quenching free radicals despite the 40 % aqueous methanol giving a slightly less but statistically significant value.

Table 4.2: Antioxidant activity of Solanum retroflexum methanol leaf extract.

Methanol DPPH (µM TE/ g) FRAP (µM Fe (II) E/ g) ABTS ( µM TE/ g) Composition 40 % 23.93 ± 0.58a 16.57 ± 0.58a 59.87 ± 0.58a

60 % 23.93 ± 0.58a 16.90 ± 0.0b 60.13 ± 0.12b

80 % 24.07 ± 0.06a 17.20 ± 0.0c 60.17 ± 0.58b

*Result expressed in dry matter weight. DPPH – 2, 2-diphenly-1-picrylhydrazl assay, FRAP – Ferric Reducing Antioxidant assay, ABTS – 2, 2-azinobis (3- ethyl- benzothiazoline - 6 - sulfonic acid), TE – Trolox Equivalent, Fe (II) E – Iron (II) Equivalent. All data reported as means ± SD (n=3). Alphabets in superscripts within a column are significantly different (p≤0.05).

The FRAP assay measures the antioxidant effect of any substance in the reaction medium as reducing ability (Bontempo et al., 2013). As shown in Table 4.2, the FRAP assay gave a significantly higher value with the 80 % aqueous methanol extracts compared to the other extracts (60 % and 40 % aqueous methanol). This trend was similar to what was observed with the DPPH and ABTS assays.

The observed antioxidant activity of S. retroflexum was expected since other Solanum plants have shown such activities. For example, Abdennacer et al., (2015) reported antioxidant activities of 0.31 TEAC (mg ET mg DW), 0.44 TEAC (mg ETmg D`W) and an RC50 of 555.5 (µg/mL) with the DPPH, ABTS and FRAP assays, respectively, in Lycium intricatum (Solanaceous shrub) leaves extracted with n-hexane, petroleum ether, chloroform and finally

MeOH/H2O (80:20 v/v). Loganayaki et al., (2010) reported antioxidants activities in S. nigrum and S. torvum leaves and fruit extracts with the DPPH and ABTS assays. DPPH radical scavenging activities (up to 43.4 % in 150 µL extract) were also reported by Muthuvel et al.,

(2014) in S. nigrum leaf aqueous extracts. Elsewhere, ethanol extracts of Solanum lycocarpum (tomato) leaves were capable of scavenging DPPH radicals in a concentration dependent manner (da Costa et al., 2015). Poongothai et al., (2011) reported that the antioxidant activity of S. xanthocarpum (herb plant) leaves methanol extracts might synergistically aid in treating diabetes mellitus. Their study was carried on diabetic induced rats.

According to recent reports, a highly positive relationship between total phenols and antioxidant activity appears to be the trend in many plant species (Ghasemzadeh et al., 2010; Fazio et al., 2013; Babu et al., 2013). Yousaf et al. (2013) reported that in vitro antioxidant activity was highly dependent on total phenolic content. The antioxidant activity of phenolic compounds such as flavonoids has been reported to be more effective in protecting cells from free radical damage than vitamin C and E (Vinson et al., 1995; Wiseman et al., 1997). It is known that the free radical scavenging activity of plants extracts is mainly due to phenolic compounds Wiseman et al., 1997). In general, the highest methanol extraction here, which contained the highest amount of total phenols, did not show substantially high scavenging activity compared to the 60 % and 40 % aqueous methanol extractions. This could be attributed to the presence of non-phenolic antioxidants with increase in methanol concentration. Further, the composition (types) of phenolic may vary at different solvent concentrations.

The antioxidant activity of the S. retroflexum leaf extracts is most likely attributed to the presence of phenolic compounds. These compounds have redox properties, hence they can act as reducing agents, hydrogen donors and single oxygen quenchers (Abdennacer et al., 2015). In addition, the identified hydroxycinnamic acids and their derivatives in S. retroflexum leaves could have contributed to the observed antioxidant activity (Askun et al., 2009). Most flavonoids possess antioxidant activities and they may be used as sweeteners and flavouring agents in food (Rauha et al., 2000).

The presence of antioxidant activity in S. retroflexum leaves is important because natural antioxidants are known to minimise the adverse effects of free radicals in living systems (Rahal et al., 2014). In addition, dietary antioxidant can reduce the risk of cardiovascular disease (Colarusso et al., 2016; Liu et al., 2003), while some phytochemicals with antioxidant properties are associated with reduced systematic inflammation (Devaraj et al., 2007) and reduced

oxidative stress (Dragsted et al., 2004). This means that leaves from the plant may potentially be used as a source of natural antioxidants for various food applications. The antioxidants in food materials can reduce spoilage of food through lipid oxidation.

4.8.3 Correlation between Total phenolic, Flavonoid Tannin Content and Radical Scavenging Activity

To determine if the phenolic compounds in Solanum retroflexum were responsible for its antioxidant activity, the correlation between TPC, TFC, TC and antioxidant activity were investigated. The Pearson’s correlation coefficient revealed that there was a significant positive linear relationship between Total Phenolic, Total Flavonoid, Tannin Content and DPPH, FRAP and ABTS assays (p < 0.01) (Table 4.3). Other studies did not find any correlation between phenolic compounds and antioxidant activity (Ka et al., 1999). Interestingly, in this study there is correlation between phenolic compounds and antioxidant activity.

Table 4.3: Pearson’s Correlation coefficient (r) between Radical Scavenging assay with total phenolic, flavonoid and tannin content.

DPPH ABTS FRAP

TPC 0.568 0.865 0.975 TFC 0.569 0.862 0.965 TC 0.682 0.719 0.841 Significant correlation at P < 0.01

4.9 Antimicrobial activity of Solanum retroflexum leaf extracts

The antibacterial activity of methanol leaf extracts (40 %, 60 % and 80 %) of S. retroflexum was investigated against 14 microorganisms that included 6 Gram-positive and 8 Gram-negative bacteria. In general, the methanolic extracts of S. retroflexum exhibited moderate antibacterial activity against only 6 of the investigated microorganisms (Table 4.4). These were Bacillus cereus, Mycobacterium smegmatis, Staphylococcus aureus, Staphylococcus epidermidis,

Klebsiella pneumonia and Proteus vulgaris. Although some of these microorganisms are found in humans as normal flora, they may become pathogenic once the host is immuno-compromised. The S. retroflexum leaf extracts showed no inhibitory effect on the growth of E. aerogenes, E. cloacae, E.coli, K. oxytoca, P. mirabilis and P. aeruginosa. Overall, the extent of inhibition was dependent on solvent concentration with the highest concentration (80 % methanol) showing better inhibition. As expected, Gram-positive microorganisms were more susceptible to the extracts with only K. pneumonia and P. vulgaris showing susceptibility among Gram-negative microorganisms. Interestingly, Mycobacterium smegmatis showed a higher (10 mm) zone of inhibition at 80 % methanol than the positive control (8 mm).

As shown in Table 4.5, the MIC values for the S. retroflexum methanolic leaf extracts against susceptible bacteria ranged from 4 mg to 16 mg/mL. The lowest MIC, which could inhibit microbial growth, was recorded for M. smegmatis and S. epidermidis.

Table 4.4: Assessment of Disc diffusion assay of extracts isolated from Solanum retroflexum.

Bacteria species Gram Streptomycin Disc Diffusion Result

Reaction Zone of Inhibition (mm)

40 % 60 % 80 %

B. cereus (BC) + 12 mm 8 ±0.2 9 ±0.3 11 ±0.0 B. subtilis (BS) + NA NA NA E. faecalis (EF) + NA NA NA M. smegmatis (MS) + 8 mm 6 ±0.3 8 ±0.4 10 ±0.2 S. aureus (SA) + 16 mm 8 ±0.1 9 ±0.0 10 ±0.4 S. epidermidis (SE) + 10 mm 7 ±0.2 8 ±0.3 8 ±0.1 E. aerogenes (EA) - NA NA NA E. cloacae (EC) - NA NA NA E. coli (ECOLI) - NA NA NA K. oxytoca (KO) - NA NA NA K. pneumoniae (KP) - 12 mm 8 ±0.3 9 ±0.2 10 ±0.1 P. mirabilis (PM) - NA NA NA P. vulgaris (PV) - 11 mm 7 ±0.3 8 ±0.2 8 ±0.3 P. aeruginosa (PA) - NA NA NA

NA – No activity

This was followed by B. cereus, S. aureus, K. pneumoniae and P. vulgaris with all showing 8 mg/mL MIC. Similar to what occurred with the disc diffusion method, the MIC value was dependent on methanol concentration. Further, the MIC results were in agreement with those of the disc diffusion assay in terms of susceptible microorganisms.

Table 4.5: Assessment of Minimum Inhibitory Concentration of extracts isolated from Solanum retroflexum.

Bacteria species Gram Minimum Inhibitory Concentration on (mg/mL) Reaction 40 % 60 % 80 %

B. cereus + 16 mg/mL 16 mg/mL 8 mg/ mL B. subtilis + >32 mg/mL >32 mg/mL >32 mg/ mL E. faecalis + >32 mg/mL >32 mg/mL >32 mg/ mL M. smegmatis + >32 mg/mL 8 mg/ML 4 mg/ mL

S. aureus + 16 mg/mL 8 mg/mL 8 mg/mL S. epidermidis + 8 mg/mL 4 mg/mL 4 mg/mL E. aerogenes - >32 mg/mL >32 mg/mL >32 mg/mL E. cloacae - >32 mg/mL >32 mg/mL >32 mg/mL E. coli - >32 mg/mL >32 mg/mL >32 mg/mL K. oxytoca - >32 mg/mL >32 mg/mL >32 mg/mL K. pneumoniae - 16 mg/mL 16 mg/mL 8 mg/mL P. mirabilis - >32 mg/mL >32 mg/mL >32 mg/mL P. vulgaris - 16 mg/mL >32 mg/mL 8 mg/mL P. aeruginosa - >32 mg/mL >32 mg/mL >32 mg/mL NOTE: No inhibition is written as >32 mg/mL

Generally, it is known that Gram-negative bacteria are usually more resistant to antibacterial agents than their Gram-positive counterparts due to morphological differences between these microorganisms (Fair and Tor, 2014; Eliopoulos et al., 2009). In this regard, S. retroflexum extracts showed moderate antibacterial activity in the disc diffusion test on Gram-positive bacteria except for B. subtilis and E. faecalis while most Gram-negative bacteria were not inhibited. The antimicrobial inhibition of S. aureus and Proteus spp was expected since it has been observed in other Solanum plants. For example, methanolic leaf extracts of S. nigrum showed antimicrobial activity of 14 mm and 12 mm, respectively, against the two microorganisms. The sensitivity of S. aeurus and P. vulgaris is important since the two

microorganisms reportedly show resistance to antibiotics. The former shows resistance to a number of antimicrobial agents leading to therapeutic problems such as methicillin-resistant S. aureus (Goldstein et al., 2007), while the latter is resistant to ampicillin and first generation cephalosporins. This means that people who consume S. retroflexum leaves could potentially be safe from diseases caused by these two opportunistic pathogens.

Contrary to the findings of this study, E. coli showed susceptibility to methanolic leaf extracts from S. nigrum. The difference in the findings could be attributed to strain variation and extracts concentration. Elsewhere, E. coli was not inhibited by methanol leaf extracts of the same plant (Das et al., 2015). In S. macranthum leaf extracts, E. coli was the least sensitive at a high concentration (2 g/mL), while a high antimicrobial activity against S. aureus and P. aeruginosa was reported elsewhere (Olayemi et al., 2005). Bacillus cereus is a well-known foodborne pathogen, but because it causes mild symptoms, its cases are infrequently reported (Dierick et al., 2005). The inhibition (11 mm) that occurred with S. retroflexum leaf extract against this pathogen means that extracts from the plant could potentially be used in the control of the B. cereus food poisoning such as the emetic and diarrheal types.

Methanolic extracts from leaves and fruit have been described as potentially good sources of antimicrobial agents (Hossain et al., 2011; Kuete et al., 2007). These in-vitro results collectively support the ethnobotanical use of Solanum retroflexum. The antimicrobial activity of the plant extracts may be partly due to the presence of phytochemicals, as previously reported (Bontempo et al., 2013; Eugenia et al., 2017). Many flavonoids are known to possess both antimicrobial and antioxidant activities (Manqin et al., 2017). For example, Kaempferol identified in S. retroflexum here has been shown to possess activity against Gram-positive and Gram-negative bacteria (Salem et al., 2010). The antibacterial activity of flavonoids is thought to be due to their ability to inactivate microbial adhesion and cell envelope (Plaper et al., 2003; Naoumkina et al., 2010). Moreover, fat-soluble flavonoids may disrupt microbial membranes, alter their fluidity and negatively affect the respiratory chain (Haraguchi et al., 1998; Mishra and Jha, 2009). The presence of alkaloids in the leaf extracts could have also contributed to the antimicrobial activity of S. refloflexum. Alkaloids play a crucial role in the defence mechanisms of plants, including protection against invading microorganism (Patel et al., 2013). Similar to other compounds, the antimicrobial effect of alkaloids depends on the type of alkaloid and concentration. For example,

it was found that Aconitum (herbal plant) alkaloids extracted with water did not substantially inhibit the growth of E. coli, while S. aureus was inhibited at higher concentration (Shi et al., 2015).

The current study showed that 4 Gram-positive and 2 Gram-negative organisms were sensitive to S. retroflexum leaf extract and the MIC ranged from 16-4 mg/mL. Further, as far as the bacterial species are concerned, our results are in agreement with reports in the literature with regard to the antimicrobial activity of such plants against bacterial species. Research into the effects of local medicinal plants is expected to boost the use of plants in the control of foodborne diseases in food as an alternative to chemical preservatives. Based on these results, it can be concluded that the leaves of S. retroflexum exhibit moderate antibacterial activity against a number of bacteria that can potentially cause food poisoning.

4.10 Conclusion

This study is the first report characterising the phytochemical composition, antioxidant and antimicrobial activity of S. retroflexum leaves. The results demonstrate a higher total phenolic content compared to other plants within the Solanum genus such as S. nigrum and S. tuberosum. However, flavonoid and tannin content in S. retroflexum leaves were found to be substantially lower than in other Solanum plants. The highest concentration of methanol yielded substantially higher polyphenols than lower concentrations of the solvent. This suggests that the leaves of S. retroflexum may contain more non-polar compounds. The leaves of S. retroflexum also exhibited antioxidant activity in vitro when investigated with the DPPH, ABTS and FRAP assays. With respect to antimicrobial activity, S. retroflexum aqueous methanolic leaf extracts exhibited moderate antibacterial activity against 6 out of the 14 investigated microorganisms. This study provides baseline information for the possible utilisation of S. retroflexum aqueous methanolic leaf extracts in food applications and in the control of foodborne infectious diseases.

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CHAPTER FIVE

5.0 GENERAL DISCUSSION AND CONCLUSIONS

5.1 Methodological considerations

In this study, the phytochemical composition of S. retroflexum leaves was investigated. The leaves of the plant are consumed in some parts of the Limpopo province of South Africa. Solanum plants have a long history of medicinal usage, probably due to the fact that they are rich in phytochemicals. There is however, paucity of knowledge on the phytochemical composition and antimicrobial properties of S. retroflexum leaves. It was expected that the study on S. retroflexum would provide knowledge about the bioactive composition of this poorly exploited plant to promote consumption of the plant or its use as a food addictive.

Two extraction solvents, water and aqueous methanol, were used to extract phytochemicals in this study. The use of organic solvents such as methanol is known to produce good extraction yields. In addition, aqueous methanol has been reported to extract higher amounts of phenolic compounds compared to absolute methanol (Alothman Bhat and Karim, 2009; Calabrone et al., 2015). Extracting with water, on the other hand, is considered a green technique. In this work, pressurised hot water extraction (PHWE) was used to extract compounds from S. retroflexum leaves. Pressurised hot water extraction is an environmentally-friendly technique that generates small volumes of waste with reduced costs and time (Andreu et al., 2016; Vazquez-roig and Picó, 2015). The technique uses elevated temperature and pressure to extract bioactive compounds. It provides better selectivity as its solvation power can be manipulated over a wide spectrum of polarities. Currently, the PHWE is the most utilised extraction technique with potential to overcome the setbacks associated with organic solvents. It has been accepted as an official US Environmental Protection Agency (USEPA) method (Aguilar et al., 2014). The problem associated with PHWE is thermal degradation of some target analytes due to the high temperatures (Khoza et al., 2015). In addition to this, high temperature and pressure attributed to PHWE i.e. critical temperature and pressure of water could pose risk of reactiveness of water, this simply means, water could oxidise or catalyse hydrolysis of some compounds (Teo et al., 2010). Interestingly, cosolvent or surfactant could assist in eliminating the problem associated

with thermal degradation or reactivity of water at elevated temperature, as satisfactory recovery could be attained even at mild extraction conditions.

The separation of the analytes was done with High Performance Liquid Chromatography (HPLC) and the chromatographic elution was monitored using an electrospray ionisation (ESI) quadruple time of flight mass spectrometer (ES1-qTOF –MS) detector. The mass spectrometer was operated in both positive and negative ionisation modes to scan the range of 100-1000 Da with a scan time of 0.2 s. Although analysis of phenolic compounds is normally done with ESI ionisation in the negative mode, isoflavonoids are usually analysed in the positive mode (Churchwell et al., 2005; Prokudina et al., 2012). The use of HPLC with reversed-phase columns in the separation of phenolic compounds is a standard method, while gas chromatography is a powerful tool for analysis of volatile compounds. Coupling the HPLC with MS does not only provide excellent sensitivity and selectivity but also allows for determination of unknown and known phytochemical compounds (Zhao et al., 2015; Wu et al., 2015). Quadruple Time-of- Flight Mass Spectrometry (QTOF-MS) fingerprinting method is one of the improved methods that has been proved to generate stable and reproducible results, particularly in the identification of chlorogenic acid (Ncube et al., 2014). The technique has been demonstrated to rapidly differentiate components and potential chemical markers for confirmatory purposes (Guo et al., 2008; Lu et al., 2013; Pihlava et al., 2014). In a Q-TOF system, ions held in an ion accumulator are first selected by the quadruple (first analyser) and then released in a gush into the TOF flight tube (second analyser) (Motilva, Serra and Macià, 2013). The latter can analyse larger masses and combining the two analysers provides more accurate mass measurements.

The antioxidant activity of the extracts was determined using three assays, namely DPPH, ABTS and FRAP. The scavenging activity on DPPH is one of the quick methods to evaluate antioxidant activity of foods. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a free radical that is stable at room temperature and accepts an electron or hydrogen radical to become a diamagnetic molecule (Pisoschi Cheregi and Danet, 2009). After reacting, the reduced DPPH results in a loss of the violet colour and reduction in absorbance (Thaipong et al., 2006). ABTS is one of the radicals generally used for testing the preliminary radical scavenging activity of a compound or plant extract. The ABTS+, generated from oxidation of ABTS by potassium persulfate, is presented as an exceptional tool for determining the antioxidant activity of hydrogen donating antioxidants

and chain-breaking antioxidants (Le Grandois et al., 2017). The ABTS cation radical is formed by the loss of an electron by the nitrogen atom of ABTS (2, 2’-azino-bis (3-ethylbenzthiazole-6- sulphonic acid). In the presence of a hydrogen donating agent or trolox, decolorisation occurs due to the nitrogen atom quenching the hydrogen atom (Marc et al., 2004). The change in colour is measured using a spectrophotometer. The FRAP (ferric reducing antioxidant power) assay measures the antioxidant effect of any substance in the reaction medium as reducing ability. A very intense navy blue colour is created by the binding of Fe2+ to the ligand (Thaipong et al., 2006). The change in absorbance measures the amount of iron reduced and this can be linked to the amount of antioxidant in the sample (Pellegrini et al., 2003). Therefore, the inactivation of oxidants by reductants can be described as a redox reaction in which one reaction specie (oxidant) is reduced at the expense of the oxidation of another antioxidant.

DPPH is one of the few stable and commercially available organic nitrogen radicals (Shah and Modi, 2015). In terms of preparation of chemicals and also in terms of performing the assay, DPPH is not a very tedious assay and hence can be utilized for its operational simplicity. The only limitations of this assay is that it is not very cost effective and it is not suitable for measuring the antioxidant capacity of plasma, because proteins are precipitated in alcoholic reaction medium (Boligon et al., 2014). The ABTS assay uses ABTS radicals performed by oxidation of ABTS with potassium persulphate. Thus, this assay becomes time consuming in terms of waiting for the ABTS radicals to be generated, unlike DPPH assay where one does not have to wait for it to be generated. However once the radicals are generated, it is a very simple assay in term of performing the assay. The ABTS radical is soluble in water and organic solvents enabling the determination of antioxidant capacity of both hydrophilic and lipophilic compounds/ samples (Apak et al., 2007). The major limitation of this assay is that the radicals formed are not very stable and the results are not reproducible. Nevertheless, this assay is also widely reported for the measurement of antioxidant activity. The FRAP assay is more tedious and time consuming in terms of preparing the chemicals of the working solution. It is a simple and inexpensive method and does not require the use of any exclusive chemicals. The results obtained in FRAP are found to be reproducible for all the concentrations. Hence, FRAP is a suitable method for the determination of antioxidant activity.

The antimicrobial activity of the S. retroflexum leaf extracts was determined using the disc diffusion assay and the dilution assay. The paper disc diffusion assay is a standard method that is widely used for screening antimicrobial activities of plant extracts. The assay is, however, not good for non-polar samples and samples that will have a poor diffusion into the agar (Cos et al., 2006). The disc size used is normally 6-8 mm and the inoculum concentration is normally between 104 – 108 cfu/ mL (Eldeen Elgorashi and Staden, 2005; Pyun and Shin, 2006). In the dilution assay, the test extracts are mixed with suitable media, such as broth, followed by inoculation with test microorganisms (Jayanta Kumar Patra and Mohanta, 2014). The growth inhibition is expressed as minimal inhibition concentration which is defined as the lowest concentration of test extract that inhibits any visible microbial growth.

5.1.1 Phytochemical composition of S. retroflexum leaf extracts

In this study, the phytochemical composition of S. retroflexum leaves extracted with aqueous methanol and pressurised hot water was investigated with UPLC-qTOF-MS. The total phenolic, flavonoid and tannin content were also determined. Later, the antioxidant and antimicrobial activities of the methanol extracts were determined. The overall aim of the project was to first identify secondary metabolites in S. retroflexum leaves extracts and also to investigate the effect of extraction method on the yield of the metabolites. Secondly, the potential of the extracted metabolites to inhibit bacterial growth, particularly pathogenic bacteria was investigated.

The first experiments involved investigating the effect of solvent concentration (40 %, 60 % and 80 % v/v, methanol/water) on the phytochemical composition of S. retroflexum leaves. It was hypothesised that altering the solvent concentration would allow for extraction of polar and non- polar compounds. With UPLC-qTOF-MS, the results revealed that the concentration of solvent did not affect the type of compounds extracted. However, the total phenolic, flavonoid and tannin content increased with an increase in the concentration of methanol. Contrary to the current findings, Calabrone et al., (2015) reported that a higher yield of phenolic compounds was achieved with a more polar solvent (70/30, v/v, methanol/water) compared to methanol/water (50/50, v/v). The results of the current study suggest that S. retroflexum leaves contain less polar compounds, hence the difference in results compared to other plants. This was further confirmed by the increase in the total phenolic content when the solvent contained more methanol. A total of 30 phytochemicals including alkaloids, flavonoids and cinnamic acids derivatives, were

identified from the methanolic leaf extracts. The identified compounds were from different classes namely, phenolics (22), flavonoids (6) and alkaloids (2).

As expected, chlorogenic acids were identified in S. retroflexum leaf extracts here. The presence of chlorogenic acids has been previously reported in other Solanum plants such as Solanum corymbiflorum leaves but not in S. retroflexum (Abdennacer et al., 2015). Importantly, both trans- and cis- isomers were identified in this study. The presence of the cis- isomers was thought to be due to exposure of the plant to hot environmental conditions. Such isomers enhance the biological activities possessed by plants. These include antioxidant, antimicrobial, antiviral and antidiabetic activity (Plaza et al., 2014; Willems et al., 2016). Therefore, S. retroflexum leaves may potentially confer health benefits associated with chlorogenic acids. A total of 6 flavonoids were detected and characterised in S. retroflexum here. Among them was quercetin which is the main dietary flavonoid associated with various properties that suppress certain chronic diseases (Suganthy et al., 2016; Mascaraque et al., 2014). The presence of quercetin and other flavonoids, such as kaemferol suggests that S. retroflexum leaf extracts may possess antioxidant and antibacterial properties as observed in other plants with such compounds (Toku Ünal and Yildirim, 2003; Govindaraju et al., 2010).

Alkaloids are the largest group of secondary metabolites made largely of ammonium compounds (Zia et al., 2011). They function in the defence of plants against herbivores and pathogens hence they are known to be intensely bitter and poisonous due to their potent biological activities. In nature, alkaloids exist in large proportions in the seeds and roots of plants and often in combination with plant organic (Ukachukwu Effiom and Uchechukwu, 2015). Two alkaloids (Solasonine and Solamargine) were identified in S. retroflexum leaves in this study with methanol (Table 3.1). Similar alkaloids have been identified in other Solanum plants such as S. nigrum. The presence of alkaloids in S. retroflexum leaves suggest that the plant could potentially be toxic to humans since high doses are associated with acute toxicity and more severe symptoms such as neurological disorder and low blood pressure (Ruprich et al., 2009). However, in this study the concentration of alkaloids was not quantified hence their effect on human health cannot be estimated based on the current findings.

Here, PHWE was used at different temperatures (50, 100, 150, and 250 °C) to extract metabolites from S. retroflexum leaves. As expected, optimum extraction occurred at the highest temperature (250 °C). This was thought to be due to enhanced solubility of compounds at high temperatures, which allows breakage of the matrix-analyte interactions and achieves high diffusion rates (Liu et al., 2009; Vazquez-roig et al., 2010). Unlike what occurred with aqueous methanol, a total of 6 health promoting metabolites were extracted with PHWE. These included Quercetin-3-rutinoside, Kaempferol-3-0-rutinoside, Kaempferol-3-0-glucoside, 3-Caffeoylquinic acid, 5-Caffeoylquinic acid and 3, 4-di-Caffeoylquinic acid (Table 3.1). The substantially low yield at high temperatures with PHWE could be due to degradation of compounds during extraction. Other workers have used low extraction temperatures (40 °C) to avoid degradation of compounds such as alkyl phenols (Font Juan-garc and Pic, 2007). The reason for the poor extraction with low temperatures in this study warrants further investigation. Particularly because other studies concluded that PHWE give results that are better than those of conventional extraction methods (Liau et al., 2017).

Quercetin rutinoside was identified at all temperatures with PHWE, while alkaloids were absent at all extraction temperatures. The absence of alkaloids in the current study was thought to be due to the fact that alkaloids dissolve better in alcohol. Other workers have observed degradation of alkaloids (hydrastine and berberine) at high temperatures of up to 140 °C (Mokgadi Turner and Torto, 2013) but this could have been unlikely in the current study since low temperature extractions did not yield alkaloids. The absence of alkaloids with PHWE may further justify why the S. retroflexum leaves are not toxic when prepared as food.

With total phenolic, flavonoid and tannin contents, the extraction yield increased with increase in the concentration of methanol. This indicates that the majority of metabolites in S. retroflexum may be somewhat non polar. When compared to other Solanum plants such as S. nigrum and S. tuberosum, the total phenolic content of S. retroflexum is substantially higher. Conversely, the total flavonoid found here was approx. ten times lower than what was reported in tomato varieties (Silva-Beltrán et al., 2015). Such inconsistencies in the results could be attributed to differences in plant species, extraction techniques and varying solvents.

5.1.2 Antioxidant and antimicrobial activity of S. retroflexum leaf extracts

There is a growing interest nowadays on the antioxidant activity of medicinal plants probably due to their health promoting properties such as prevention of chronic diseases, DNA damage, ageing, cardiac and cerebral ischema (Aslani and Ghobadi, 2016; Oloyede et al., 2016). In addition, available synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), tertiary butylated hydroquinone and gallic esters have been associated with negative health and environmental effects (Antonopoulou et al., 2016). In this study, S. retroflexum aqueous methanol leaf extracts were found possessing strong scavenging activities against DPPH and ABTS radical (Table 4.2). Similarly, the FRAP assay showed some antioxidant activity from the plant leaf extracts. The concentration of methanol did not substantially affect the antioxidant activity with all the assays. As previously mentioned, the observed antioxidant activity of S. retroflexum was expected since other Solanum plants leaf extracts have shown such activities. Generally, plant extracts with high phenolic content yield the highest antioxidant activity. However, in the current study, the highest amount of total phenols did not show high antioxidant properties. The discrepancy was thought to be due to the presence of non-phenolic antioxidants with increase in methanol concentration. The antioxidant activity of S. retroflexum leaves extract was also attributed to the identified hydroxycinnamic acids and their derivatives (Askun et al., 2009).

It is worth noting that there are several limitations when accessing antioxidant activity in food extracts. According to Floegel et al., 2011, the ABTS and DPPH assays are in vitro models and do not assess all of the antioxidant activities in food plants. Another study reported that antioxidant capacity depends on the solvent used as well as extraction method (Pérez-Jiménez and Saura-Calixto, 2006; Radhi Addai et al., 2013). Elsewhere, it was reported that the antioxidant capacity of foods may differ depending on the growing season, geographic origin and agricultural practices (Chun et al., 2005).

The antimicrobial activity of S. retroflexum aqueous methanolic leaf extracts was investigated with the disc diffusion and minimal inhibitory concentration assays. About 14 microorganisms constituting of 6 Gram-negative and 8 Gram-positive bacteria were used in the study. In general, the S. retroflexum leaf extracts showed moderate inhibition against 6 (B. cereus, M. smegmatis, S. aureus, S. epidermis, K. pneumonia and P. vulgaris) of the investigated microorganisms

(Table 4.5). As expected, Gram-positive bacteria were more susceptible to the extracts than Gram-negative bacteria as reported by other authors (Mahmoudi et al., 2016). This was thought to be due to differences in morphology between the two bacterial groups. Gram-negative bacteria have an outer membrane with peptidoglycan and lipopolysaccharide layers that can exert strong hydrophilicity and act as permeability barrier (Mann et al., 1997). Better inhibition was also observed in samples extracted with the highest methanol concentration. This was thought to be due to the high total phenol, flavonoid and tannin content that occurred at the highest (80 %) methanol concentration compared to the 60 and 40 % aqueous methanol extractions. Flavonoids and tannins have been reported to possess antimicrobial activities (Mishra et al., 2009; Mohanta et al., 2007) The inhibition mechanism of the former has been reported to be due to inactivation of microbial adhesion and cell envelope transport proteins (Plaper et al., 2003; Naoumkina et al., 2010). As previously mentioned, alkaloids which were extracted with aqueous methanol could have also contributed to the antimicrobial activity of S. retroflexum leaf extracts.

Among the Gram-positive microorganisms, B. cereus was the most susceptible bacteria to the extracts followed by S. aureus, K. pneumonia and M. smegmatis (Table 4.5). Elsewhere, S. aureus and B. cereus were the most sensitive bacteria to methanolic leaf extracts from fig tree (Mahmoudi et al., 2016). Li et al., 2014 studied the mode of action of chlorogenic acid (CA) on S. aureus. These workers reported, among others, the destruction of the cell membrane, decreased intracellular ATP concentrations and a significant release of cell constituents after CA treatment. Since CA was identified in S. retroflexum leaves here, similar negative effects on the cell membrane of susceptible bacteria could have occurred.

There have been a major interest in phytochemicals from medicinal plants, due to numerous evidence that links them to various protective health benefits (Doughari et al., 2009). The fundamental objective of this vital step was to adopt fast and reliable technique that offers better sensitivity and shifts towards green chemistry. In this study, methanolic extracts have been a well favoured extraction technique but the presence of health threatening phytochemicals in methanol extracts warrant further investigation using pressurised hot water extraction in combination with UPLC-qTOF-MS which further reiterates the assertion that S. retroflexum is a rich source of phytochemicals and also a safe food source. The difference in extractability of metabolites suggests that these metabolites have different physicochemical properties such as polarity.

5.2 Conclusions and Recommendation

A total of 30 phytochemical compounds that include alkaloids, flavonoids and cinnamic acid derivatives were identified in S. retroflexum aqueous methanolic leaf extracts using the UPLC- qTOF-MS technique. The concentration of methanol did not affect the type of metabolites identified, hence similar metabolites were identified at 40 %, 60 % and 80 % aqueous methanol (methanol/water, v/v). With PHWE, the extraction yield increased with increase in temperature. The highest extraction temperature (250 °C) resulted in qualitative yields of molecules, such as Quercetin-3-rutinoside, Kaempferol-3-0-rutinoside, Kaempferol-3-0-glucoside, 3-Caffeoylquinic acid, 5-Caffeoylquinic acid and 3, 4-di- Caffeoylquinic acid. Glycoalkaloids were identified in the methanol extracts but not in the PHWE. The absence of glycoalkaloids in the PHWE may potentially justify the use of S. retroflexum leaves as food.

The total phenolic content (TPC) of S. retroflexum aqueous methanol extracts was found to be approx. 4.57 mg GAE/g. This level is substantially higher, by more than tenfold, than that reported in other Solanum plants such as S. nigrum and S. tuberosum. The discrepancy is attributed to differences in plant species growth environment and extraction methods. The total flavonoid (TFC) and tannin content (TC) of S. retroflexum were found to be approx. 3.03 mg QE/g and 2.90 mg CE/mg, respectively. The extract yield of TPC, TFC and TC was affected by the concentration of solvent with more compounds extracted at the highest level of methanol. With regard to antioxidant activity, S. retroflexum leaf extracts exhibited some antioxidant activity in vitro when investigated with the DPPH, ABTS and FRAP assays. In general, solvent concentration did not affect the antioxidant activity, this can help in cost reduction of the extraction process. The aqueous methanol extracts of S. retroflexum showed moderate antimicrobial activity against 6 out of the 14 microorganisms investigated in this study. The susceptible microorganisms included B. cereus, M. smegmatis, S. aureus, S. epidermis, K. pneumonia and P. vulgaris. The minimum inhibitory concentration (MIC) as determined by micro dilution method was found to be 4-16 mg/mL.

This study shows that S. retroflexum leaves contain phytochemical compounds that are, to some extent, similar to those found in other Solanum plants. The extraction solvent affects the extract yield with aqueous methanol yielding more compounds than PHWE. The concentration of methanol does not affect the type of metabolites extracted, however, the extract yield is affected

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by the solvent concentration used. Understanding the type of compounds found in S. retroflexum will be useful in promoting the use of the plant as food and also its application in other foods to control foodborne bacteria. Further work is needed to firstly, quantify the metabolites found in S. retroflexum leaves with techniques such as the HPLC. This will help indicate the type of compounds that are abundant in S. retroflexum. In addition, quantification of compounds is useful in food application to know toxicity levels of compounds such as alkaloids. Secondly, further work is needed to optimise the PHWE technique on S. retroflexum. This is because the technique is environmentally friendly and less costly compared to conventional methods.

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