An antimicrobial and phytochemical 

validation of southern African plants 

used as soap substitutes and 

formulation of herbal soap 
 

 

 

Nyiko Fortunate Mzimba 

 

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, 

Johannesburg, in fulfilment of the requirements for the degree of Master of Pharmacy 

 

March 2023 

 

Supervised by Professor Sandy van Vuuren 

Co-supervised by Professor Annah Moteetee 

 

 

 

 

 

 

 

 



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DECLARATION 

 

I, Nyiko Fortunate Mzimba, declare that this dissertation is my own work. It is being submitted 

for the Master of Pharmacy degree at the University of the Witwatersrand, Johannesburg. This 

work has not been submitted before for any other degree or examination at this or any other 

University. 

 

              

              N.F Mzimba                                                                                              Date 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

14/06/23 



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DEDICATION 

 

This dissertation is dedicated to my loving parents Vivian and Solani Mzimba and brothers 

Rifumo and Mahlori Mzimba. Thank you for your support and encouragement.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



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ACKNOWLEDGEMENTS 

 

• Firstly, I would like to thank the Almighty God for His grace, wisdom, strength, and peace 

which surpass all understanding. The Lord has been good, and His loving kindness endures 

forever (1 Chronicles 16:34).  

• I would like to thank my supervisor, Professor Sandy van Vuuren: Thank you for accepting 

me and believing that I can be a great addition to the research team. I have benefited from the 

knowledge, lessons, skills, and guidance that you shared during the course. I appreciate the 

positive attitude and confidence you had toward the project. It helped me become a better writer 

and researcher. It was an honour to be under your supervision.  

• To my co-supervisor, Professor Annah Moteetee: Thank you for the knowledge you have 

shared with me as a student throughout the years. Your guidance, patience, diligence, 

commitment, and constructive criticism are highly appreciated.  

• I extend my sincere gratitude to our laboratory manager and technician, Mrs Phumzile 

Moerane, for always making sure that we have a good working environment that is clean and 

work-friendly. I would not have completed my laboratory work without your assistance. Your 

assistance throughout this project is appreciated.  

• Special thanks to Mr Andrew Hankey, the specialist horticulturist, thank you for assisting in 

plant identification and authentication. 

• The financial assistance of the National Research Foundation (NRF), Postgraduate Merit 

Award (PMA), and Faculty Research Committee (FRC) towards this research project is hereby 

acknowledged.  

• A special thanks to the University of the Witwatersrand and the University of Johannesburg 

(Department of Chemistry) for giving me access to the resources and facilities I needed to 

complete this research project. 

• To my fellow students and colleagues, Sarhana Dinat and Salehah Moola, thank you for the 

support you provided with this project. It was always refreshing to work with you in the 

laboratory.  

• To Mpho Mohlakoana, I am truly grateful for your assistance and support in the laboratory.  



iv | P a g e  

 

• I also appreciate the love and care the Somakwabe family provided throughout this period, 

especially Gift and Vuyani, who were there with me when I spent late nights and weekends in 

the laboratory.  

• Last, but far from least, to my family (Vivian, Benneth, Progress, and Mahlori), partner 

(Blessing Rotondwa Sitabule), and mentors (Prophet Sibusiso Makamu, Prophetess Muhle 

Khoza and Mama Lydia Sitabule), thank you for the prayers, love, patience, emotional, and 

financial support. No words can express how grateful I am to have you in my life.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



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ABSTRACT 

 

A major proportion of the population in southern Africa relies on medicinal plants commonly 

known as soapy plants for bathing and washing. There is limited scientific research that assess the 

effectiveness of southern African soap plants. Therefore, this study investigated the 

phytochemistry, antimicrobial activity, and toxicity of plants used in southern Africa as soap 

substitutes. Thereafter, an effective antimicrobial herbal soap was formulated and assessed for its 

antimicrobial efficacy. 

 

A comprehensive literature review was conducted to gather information on plants used as soap 

substitutes in southern Africa. A total of 59 plant species were identified to be used for bathing 

and washing. A total of 26 plant species were collected based on availability at Walter Sisulu 

Botanical Garden, Random Harvest Indigenous Nursery and University of Johannesburg 

herbarium and University of the Witwatersrand storage. The organic and aqueous extracts were 

prepared and screened for the presence of alkaloids, terpenoids, and saponins. Methanol and 

acetone were the optimal solvents to extract alkaloids from 62.07% of plant extracts. Terpenoids 

were best extracted with ethanol (75.86% of plant extracts), followed by methanol (68.97% of 

plant extracts). Saponins were highly detectable using water (93.10% of plant extracts) and ethanol 

(82.76% of extracts). The qualitative evaluation of saponins using thin layer chromatography 

displayed a variety of saponins, including steroidal saponins that had Rf-values comparable to 

diosgenin (a steroidal aglycone used as a standard). Sideroxylon inerme subsp. inerme (16.3%) 

had a high percentage yield of saponins. Hermannia cuneifolia displayed the highest saponin 

content (262.41 ± 1.90 mg/g), followed by Sideroxylon inerme subsp. inerme (71.34 ± 1.01 

mg/ml), Acalypha glabrata (70.48 ± 2.05 mg/g), and Noltea africana (68.53 ± 2.43 mg/g). 

 

The organic and aqueous extracts of each of the selected soap plants were tested for their 

antimicrobial activity against skin-relevant pathogens. Pelargonium peltatum demonstrated the 

best antimicrobial activity against Brevibacterium linens and Cutibacterium acnes with an MIC 

value of 0.06 mg/ml. Calodendrum capense (leaves), Noltea africana (leaves), Olea europaea 

(leaves), Pelargonium peltatum (leaves), Plectranthus ciliatus (leaves), Ptaeroxylon obliquum 



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(bark), and S. inerme subsp. inerme (leaves) organic extracts displayed noteworthy antimicrobial 

activity against the pathogen C. acnes with an MIC value of 0.06 mg/ml. The plants that 

demonstrated notable broad-spectrum activity against most of the tested pathogens were 

Calodendrum capense (leaves), Pelargonium peltatum, Plectranthus ciliatus, and Ptaeroxylon 

obliquum (bark). 

 

The toxic profiles of the organic and aqueous extracts were evaluated to assess the safety of the 

plant species using brine-shrimp lethality assay (BSLA). Aqueous plant extracts were more toxic 

(65.52%) compared to organic plant extracts (62.07%). Acalypha glabrata (leaves), Aloe maculata 

(leaves), Bauhinia bowkeri (leaves), Deinbollia oblongifolia (leaves), Ledebouria luteola (bulb), 

Pouzolzia mixta (leaves), and Sideroxylon inerme subsp. inerme (leaves) organic and aqueous 

extracts demonstrated the lowest toxic effects at 24 and 48 h. Aristaloe aristata (leaves), 

Calodendrum capense (leaves) and P. obliquum (bark) organic extracts were non-toxic, and 

Hermannia cuneifolia (leaves), Plectranthus ciliatus (leaves), and Ptaeroxylon obliquum (leaves) 

aqueous extracts were non-toxic. Crinum bulbispermum (bulb), Haemanthus albiflos (bulb) and 

Ilex mitis (leaves) were highly toxic, with LC50 values > 250 µg/ml after 48 h. Pelargonium 

peltatum displayed low toxicity at a concentration of 125 µg/ml.  

 

The extracts of Calodendrum capense, Pelargonium peltatum, and Ptaeroxylon obliquum were 

then used for soap-making by the basic saponification reaction, and the physicochemical 

parameters and antimicrobial activity of the soaps were evaluated. The Calodendrum 

capense herbal soap had the lowest pH (10.79), moisture content (28%), and free caustic alkali 

(0.03%). Pelargonium peltatum and Calodendrum capense herbal soaps were categorized as first-

grade soaps (84 and 80%, respectively). The antimicrobial efficacy of the soaps was determined 

by inoculating selected skin micro-organisms on agar containing soap formulations using the 

multipoint inoculator.  Gram-positive micro-organisms were inhibited (MIC values of ≤ 1.57 

mg/ml). All the tested micro-organisms except for Enterobacter cloacae were inhibited at a 

concentration of 12.5 mg/ml, which is comparable to the control, Protex® commercial soap. The 

findings herein of the antimicrobial properties, phytochemistry, and toxicity contribute to the 

knowledge gaps that exist in the ethnobotanical literature of some southern African soap plants 

and provide evidence for their incorporation into soap formulation.  



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PRESENTATIONS ARISING FROM THIS STUDY 

 

NF Mzimba, A Moteetee, S van Vuuren. Antimicrobial and phytochemical validation of southern 

African plants used as soap substitutes. South Africa. 24th Indigenous Plant forum 2022. 4-7 July 

2022. University of Johannesburg. [online presentation, abstract Appendix A1]. 

 

NF Mzimba, A Moteetee, SF van Vuuren. Validating the antimicrobial activity, phytochemistry, 

and toxicity of southern African plants used as soap substitutes. South Africa. Faculty of Health 

Sciences Research Day, 15 September 2022, University of the Witwatersrand, Johannesburg. 

[poster presentation, abstract and poster Appendix A2]. 

 

 

 

 

 

 

 

 

 

 



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TABLE OF CONTENTS 

 

Declaration...................................................................................................................................... i 

Dedication ...................................................................................................................................... ii 

Acknowledgements ...................................................................................................................... iii 

Abstract .......................................................................................................................................... v 

Presentations arising from this study ........................................................................................ vii 

Table of contents ........................................................................................................................ viii 

List of tables................................................................................................................................. xii 

List of figures .............................................................................................................................. xiii 

List of equations ......................................................................................................................... xiv 

List of abbreviations ................................................................................................................... xv 

 

Chapter 1 ....................................................................................................................................... 1 

General Introduction .................................................................................................................... 1 

1.1 Overview of the human skin ..................................................................................................... 1 

1.1.1 Skin microbiota ...................................................................................................................... 1 

1.1.2 Pathogenesis of micro-organisms that usually form part of the skin microbiome ................ 2 

1.1.3 Bacterial skin infection: ......................................................................................................... 7 

1.1.3.1 Impetigo .............................................................................................................................. 7 

1.1.3.2 Folliculitis ........................................................................................................................... 7 

1.1.3.3 Cellulitis and erysipelas ...................................................................................................... 8 

1.1.4 Fungal (yeast) skin infections ................................................................................................ 8 

1.2 Skin hygiene.............................................................................................................................. 9 

1.3 Alternative: plants used as soap substitutes in southern Africa .............................................. 11 

1.4 Chemistry and toxicity of plants used as soap substitutes ...................................................... 12 

1.5 Antimicrobial activity of plants used as soap substitutes ....................................................... 21 

1.6 Formulation of herbal soap ..................................................................................................... 22 

1.7 Aim and objectives ................................................................................................................. 23 

 



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Chapter 2 ..................................................................................................................................... 24 

Phytochemical analysis of selected southern African plants used as soap substitutes ......... 24 

2.1 Introduction ............................................................................................................................. 24 

2.2 Methods and materials ............................................................................................................ 25 

2.2.1 Selection of plants ................................................................................................................ 25 

2.2.2 Collection of plants .............................................................................................................. 25 

2.2.3 Plant sample preparation ...................................................................................................... 27 

2.2.4 Preliminary phytochemical screening of alkaloids, terpenoids and saponins...................... 27 

2.2.4.1 Preparation of plant extracts ............................................................................................. 27 

2.2.4.2 Detection of alkaloids using Wagner’s test ...................................................................... 28 

2.2.4.3 Detection of terpenoids using Salkowski’s test ................................................................ 28 

2.2.4.4 Detection of saponins using the froth test ......................................................................... 28 

2.2.5 Qualitative evaluation of saponins ....................................................................................... 29 

2.2.5.1 Preparation of plant extracts ............................................................................................. 29 

2.2.5.2 Thin layer chromatography (TLC).................................................................................... 29 

2.2.6 Quantitative evaluation of saponins ..................................................................................... 30 

2.2.6.1 Sample and reagent preparation ........................................................................................ 30 

2.2.6.2 Preparation of standard curve ........................................................................................... 31 

2.2.6.3 Determining the absorbance of the samples ..................................................................... 32 

2.3 Results and discussion ............................................................................................................ 33 

2.3.1 The presence of alkaloids, terpenoids and saponins in soap plants ..................................... 33 

2.3.2 Qualitative analysis: thin layer chromatography (TLC) ...................................................... 34 

2.3.3 Quantitative analysis: vanillin-sulphuric acid assay ............................................................ 37 

2.4 Summary ................................................................................................................................. 41 

 

Chapter 3 ..................................................................................................................................... 42 

The antimicrobial activity of selected southern African plants used as soap substitutes .... 42 

3.1 Introduction ............................................................................................................................. 42 

3.2 Methods and materials ............................................................................................................ 43 

3.2.1 Preparation of organic and aqueous plant extracts .............................................................. 43 

3.2.2 Selection of test micro-organisms and preparation of cultures ............................................ 43 



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3.2.3 Minimum inhibition concentrations (MIC) assay ................................................................ 45 

3.3 Results and discussion ............................................................................................................ 46 

3.3.1 Antimicrobial activity of the organic extracts against Gram-positive bacteria ................... 52 

3.3.2 Antimicrobial activity of the organic extracts against Gram-negative bacteria .................. 54 

3.3.3 Antimicrobial activity of the organic plant extracts against C. albicans ............................. 55 

3.3.4 Overall antimicrobial effects of organic extracts on Gram-positive and Gram-negative 

micro-organisms ........................................................................................................................... 56 

3.3.5 Antimicrobial activity of plants that demonstrated broad-spectrum activity ...................... 57 

3.4. Summary ................................................................................................................................ 59 

 

Chapter 4 ..................................................................................................................................... 60 

Toxicity analysis of selected southern African plants used as soap substitutes .................... 60 

4.1 Introduction ............................................................................................................................. 60 

4.2 Methods and materials for Brine shrimp lethality bioassay.................................................... 61 

4.3 Results and discussion ............................................................................................................ 62 

4.3.1 The LC50 values of plant samples that possess a toxic effect on 50% of the                   

brine-shrimp……………………………………………………………………………………...64 

4.3.2 Dose-response of organic and aqueous extracts that demonstrated toxicity ....................... 70 

4.4 Summary ................................................................................................................................. 71 

 

Chapter 5 ..................................................................................................................................... 72 

Herbal soap formulation of three selected plants used as soap substitutes ........................... 72 

5.1 Introduction ............................................................................................................................. 72 

5.2 Methods and materials ............................................................................................................ 73 

5.2.1 Basic and herbal soap formulation ....................................................................................... 73 

5.2.2 Validation of the physicochemical properties of the soap ................................................... 73 

5.2.2.1 Determining foam height and foam retention ................................................................... 74 

5.2.2.2 Determining moisture content and total fatty matter content ........................................... 74 

5.2.2.3 Free caustic alkali and percentage chloride ...................................................................... 75 

5.2.3 Validation of the antimicrobial efficacy .............................................................................. 76 

5.2.3.1 Preparation of the soap-agar plates ................................................................................... 76 



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5.3 Results and discussion ............................................................................................................ 78 

5.3.1 Physicochemical properties of the formulated herbal soap ................................................. 78 

5.3.2 Antimicrobial efficacy of formulated herbal soaps ............................................................. 81 

5.4 Summary. ................................................................................................................................ 86 

 

Chapter 6 ..................................................................................................................................... 87 

Summary and conclusion ........................................................................................................... 87 

6.1 Overview of study ................................................................................................................... 87 

6.2 Limitations .............................................................................................................................. 93 

6.3 Recommendations ................................................................................................................... 93 

6.4 Conclusion .............................................................................................................................. 94 

 

References .................................................................................................................................... 96 

Appendix a: Presentations ....................................................................................................... 116 

A1: The 24th Indigenous Plant Use Forum (IPUF) conference 2022 – University of 

Johannesburg, South Africa. 4–7 July 2022. [online presentation]. ........................................... 116 

A2: Faculty of Health Sciences Research Day, 15 September 2022, University of the 

Witwatersrand, Johannesburg. .................................................................................................... 118 

Appendix B: Ethics waiver ....................................................................................................... 120 

Appendix C: TLC plates .......................................................................................................... 122 

C1: TLC plate sprayed with a vanillin-perchloric acid reagent. ................................................. 122 

C2: TLC plate sprayed with a vanillin-perchloric acid reagent. ................................................. 123 

C3: TLC plate sprayed with a 10% sulphuric acid reagent. ....................................................... 124 

C4: TLC plate sprayed with 10% sulphuric acid reagent. .......................................................... 125 

Appendix D: Dose-response graphs ........................................................................................ 126 

D1: The dose-response for organic extracts that were considered toxic after 48 h. ................... 126 

D2: The dose-response for aqueous extracts that were considered toxic after 48 h. .................. 127 

Originality report ...................................................................................................................... 128 

 



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LIST OF TABLES 

 
 

Table 1.1: The roles of selected pathogens in contaminating the skin, causing skin           

infection and bad body odour. ................................................................................................. 3 

Table 1.2: Plants used traditionally in southern Africa as soap substitutes. ................................ 13 

Table 2.1: List of plants that were used in this study, the parts used, voucher                   

numbers, supplier and when it was collected. ........................................................................ 26 

Table 2.2: The vanillin-sulphuric acid assay setup procedure for the reagent blank,          

standard, and sample. ............................................................................................................. 32 

Table 2.3: Preliminary phytochemical screening results and Rf values of 26                     

southern African soap plants. ................................................................................................. 35 

Table 2.4: The percentage yield of saponins and total saponin content of 26 plant               

species used as soap substitutes. ............................................................................................ 40 

Table 3.1: Pathogens and culturing conditions for antimicrobial screening. ............................... 43 

Table 3.2: The average MIC values of 26 plant species against Gram-positive                    

bacteria. .................................................................................................................................. 47 

Table 3.3: The average MIC values of 26 plant species against Gram-negative                   

bacteria and Candida albicans. .............................................................................................. 49 

Table 4.1: The average percentage mortality of 26 plant species extracts at 24                          

and 48 h. ................................................................................................................................. 63 

Table 4.2: The LC50 values of organic and aqueous plant extracts that displayed                    

toxic effects at 24 and 48 h. ................................................................................................... 65 

Table 5.1: Physicochemical properties of formulated herbal soaps............................................. 81 

Table 5.2: The antimicrobial activity of three formulated herbal soaps. ..................................... 85 

Table 6.1: Overall summary of the phytochemistry, antimicrobial activity and                    

toxicity of the three most ideal plants for soap formulation. ................................................. 90 

 

 

 

 



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LIST OF FIGURES 

 

Figure 2.1: The standard curve showing the absorbance of diosgenin at                                 

various concentrations. .......................................................................................................... 31 

Figure 3.1: A summary of the susceptibility of different micro-organisms                                   

(≤ 1.00 mg/ml). ...................................................................................................................... 57 

Figure 3.2: A summary of plant extracts that exhibited broad-spectrum activity. ...................... 59 

Figure 5.2: The three formulated herbal soaps. A - Calodendrum capense                            

herbal soap, B -Pelargonium peltatum herbal soap and C - Ptaeroxylon obliquum                

herbal soap. ............................................................................................................................ 78 

Figure 5.3: Indication of how the micro-organisms grew on the culture control                      

agar-soap plate. ...................................................................................................................... 82 

Figure 5.4: The herbal soaps inhibited all the tested micro-organisms at the               

concentration of 12.5 mg/ml except for E. cloacae which is the same for the control 

(Protex). ................................................................................................................................. 83 

 

 

 

 

 

 

 



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LIST OF EQUATIONS 

 

Equation 2.1: Saponin percentage yield (%) ............................................................................... 29 

Equation 2.2: Retention factor..................................................................................................... 30 

Equation 2.3: Regression line equation ....................................................................................... 32 

Equation 2.4: Total saponin content (mg/g) ................................................................................ 32 

Equation 4.1: Percentage mortality (%) ...................................................................................... 61 

Equation 5.1: Moisture content (%) ............................................................................................ 74 

Equation 5.2: Total fatty matter (%) ........................................................................................... 75 

Equation 5.3: Free caustic alkali (%) .......................................................................................... 75 

Equation 5.4: Percentage of chloride (%) ................................................................................... 76 

 

  

 

 

 

 

 

 

 



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LIST OF ABBREVIATIONS 

 

ATCC   American-type culture collection  

BSLA   Brine-shrimp lethality assay  

CC50   The 50% cytotoxic concentration 

CI   Confidence intervals 

COVID-19  Coronavirus disease 

CV   Crystal violet  

DCM   Dichloromethane 

DMSO  Dimethyl sulfoxide 

EICA    Encyclopaedia of industrial chemical analysis  

FCA   Free caustic alkali  

HPLC   High-performance liquid chromatography 

IC50   Half maximal inhibitory concentration  

INT   p-iodonitrotetrazolium violet 

ISO   International standard organisation  

JRAU   University of Johannesburg herbarium 

LC50   Median lethal concentration 

MDCK  Madin-Darby canine kidney cells 

MIC   Minimum inhibitory concentration  

MTT   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide 

NADH  Nicotinamide adenine dinucleotide 

NCSS   Number cruncher statistical systems 

pH   Potential hydrogen  

Rf   Retention factor  

RHIN   Random harvest indigenous nursery  

TFM   Total fatty matter 

TLC   Thin layer chromatography  

TSA   Tryptone soya agar 

TSB   Tryptone soya broth  



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TSC   Total saponin content  

UPLC   Ultra-pressure liquid chromatography  

UV   Ultraviolet  

UV/VIS  Ultraviolet-visible 

WHO   World Health Organisation  

WSBG  Walter Sisulu Botanical Gardens 

 



1 

 

CHAPTER 1 

GENERAL INTRODUCTION 

 

1.1 Overview of the human skin 

1.1.1 Skin microbiota 

 

The largest organ in the human body is the skin which regulates temperature and maintains fluids 

through eccrine glands (Grice and Segre, 2011). The skin contains sebaceous glands which secrete 

lipid-rich substances called sebum which assists in preventing the desiccation of the skin and in 

sealing moisture within the skin. The sebaceous glands also contribute to the production of most 

lipidic compounds on the skin surface through the sebum's assistance (Hoover et al., 2022). The 

sebum moderately regulates the skin pH since the skin pH is dependent on the skin moisture, i.e., 

high moisture areas such as the axilla have a higher pH (Schmid-Wendtner and Korting, 2006). 

Therefore, the characteristics of sebum contribute to creating an ecosystem which accommodates 

symbiotic bacteria as well as viruses, fungi, and mites on the skin (Boxberger et al., 2021). A 

collection of these symbiotic micro-organisms colonises the skin to form the skin microbiota that 

protects the skin against pathogenic micro-organisms. The T-cells on the skin and immune cells 

interact with these symbiotic micro-organisms, then respond to pathogenic micro-organisms that 

cause skin infections. They reinforce and repair the external barrier formed by the skin (Grice and 

Segre, 2011; Eisenstein, 2020). According to Byrd et al. (2018), the human skin is predominantly 

colonised by Gram-positive bacteria such as Corynebacterium spp., Cutibacterium spp., 

Enhydrobacter spp., Micrococcus spp. and Veillonella spp. Although Gram-positive bacteria make 

up a large proportion of the skin microbiota, there are also other Gram-negative commensal 

bacteria included (Boxberger et al., 2021). These include Acinetobacter spp., Enterobacterium 

spp., Moraxella osloensis, Pantoea septica, Roseomonas mucosa, and Pseudomonas spp. (Myles 

et al., 2016). Other commensal micro-organisms include Cryptococcus spp., Debaryomyces spp., 

Demodex mites, and Malassezia spp. (Grice and Segre, 2011). 

 

The colonization of the skin by a symbiotic bacterial community is primarily dependent on the 

physiology of the skin site. The abundance of microbial communities is associated with dry, moist, 



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or sebaceous environments (Grice and Segre, 2011; Byrd et al., 2018). Corynebacterium spp., 

Cutibacterium spp., and Staphylococcus aureus colonize the moist sites of the human skin. These 

bacteria colonize bodily locations with hair follicles such as the axilla (underarm) and external 

genitalia, as well as other sites such as the bends of elbows and feet (Byrd et al., 2018; Rudden et 

al., 2020). Grice and Segre (2011) recorded that there is low diversity of bacteria in sebaceous 

sites (behind the ear, inside the nostrils, face, back, and chest), which shows what conditions are 

not favourable for most bacteria. However, lipophilic Cutibacterium acnes is dominant on these 

sites because C. acnes degrades the lipids of sebum, releasing free fatty acids that aid in the 

colonisation of the skin surface at a pH of 5 (Grice and Segre, 2011). In contrast to the bacterial 

community, the fungal community does not depend on physiological skin sites for colonization. 

Malassezia spp. colonizes the armpits, while a combination of Aspergillus spp., Cryptococcus spp., 

Epicoccum spp., and Rhodotorula spp. colonises the core body (Hrestak et al., 2022). 

 

1.1.2 Pathogenesis of micro-organisms that usually form part of the skin microbiome 

 

Although micro-organisms found in the skin microbiota are normally commensal, at times they 

can cause skin infections and/or disorders reflecting the pathogenicity of the relative micro-

organisms. Skin infections or disorders often occur when micro-organisms from the skin 

microbiome invade other sites that they do not normally colonize. Furthermore, skin infections 

occur when they manage to overcome the normal host defence mechanism (Chiller et al., 2001). 

When these micro-organisms invade other sites, the balance of the microbiota is disturbed, thus, 

resulting in the development of skin infections such as boils, erythrasma, folliculitis, and impetigo 

(Aly, 1996). On healthy human skin, pathogens such as coryneform bacteria, Staphylococcus 

aureus, and Streptococcus pyogenes are inhibited by an acidic pH on the skin surface, encouraging 

the growth of Corynebacteria. However, during a time when the skin is occluded, the pH is 

elevated, thus, the growth of pathogenic micro-organisms is favoured (Grice and Segre, 2011). 

Table 1.1 shows the micro-organisms that are part of the microbiome and also have potential 

pathogenesis on the skin. Pathogens that are not usually found on the skin can nonetheless cause 

skin infections and an unpleasant body odour. 

 



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Table 1.1: The roles of selected pathogens in contaminating the skin, causing skin infection and 

bad body odour. 

Pathogen  Classification Role  Pathogenesis 

Acinetobacter 

baumannii a 

Gram-negative Skin contaminant 

(Meneghetti et al., 

2018). 

It causes a severe type of 

cellulitis and other skin and 

soft tissue infections (Ali et 

al., 2014). 

Brevibacterium agri  Gram-positive 

  

Commensal micro-

organisms on the 

human skin (Van 

Vuuren et al., 

2019). 

Digests dead skin and causes 

foot odour (methane thiol). 

They are also the leading 

cause of bromodosis (Van 

Vuuren et al., 2019). 

Brevibacterium 

epidermidis  

Brevibacterium 

linens  

Candida albicans  Yeast  Resides on the 

skin surface as a 

commensal 

(Kühbacher et al., 

2017). 

It causes cutaneous 

candidiasis, skin thickening, 

erythema, diaper rash, and 

hyperkeratosis (Kühbacher et 

al., 2017; Palese et al., 2018). 

Corynebacterium 

minutissimum 

Gram-positive  Found on the 

uppermost layers 

of the epidermis 

(Forouzan and 

Cohen, 2020). 

It causes erythrasma or 

interdigital erythrasma. Found 

on the toe webs, co-infecting 

with C. albicans and/or 

Trichophyton spp. (Forouzan 

and Cohen, 2020). 

Corynebacterium 

xerosis  

Gram-positive A commensal 

micro-organism 

that is normally 

present on the skin 

and nasopharynx 

(Chiller et al., 

2001). 

It causes skin infections when 

associated with other 

Corynebacterium species 

(Hernández-León et al., 2016). 



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Pathogen  Classification Role  Pathogenesis 

Cutibacterium acnes  Gram-positive Part of the normal 

skin microbiome 

(Findley and 

Grice, 2014). 

It is part of the normal flora of 

the skin that forms biofilms 

within the body, causing 

intense body odour (Elston et 

al., 2019). It plays a role in the 

development of Acne vulgaris 

in teenagers (Mayslich et al., 

2021). 

Cutibacterium avidum Gram-positive A skin commensal 

micro-organism is 

found in regions 

containing eccrine 

sweat glands 

(Corvec, 2018). 

It is associated with intense 

malodour (Lam et al., 2018) 

and acts as either superficial or 

invasive infections such as 

breast infections and skin 

abscesses (Corvec, 2018). 

Enterobacter cloacae  Gram-negative An opportunistic 

skin pathogen and 

a skin contaminant 

(Meneghetti et al., 

2018). 

It causes wound infections and 

is also a secondary causative 

agent of bacterial folliculitis 

(Ni Riain, 2013; Buckle, 2015; 

Meneghetti et al., 2018). 

Escherichia coli  Gram-negative Skin contaminant 

(Afsar and 

Khanam, 2016). 

It causes cellulitis localized to 

the lower or upper (Petkovšek 

et al., 2009).  

Klebsiella 

pneumoniae  

Gram-negative Skin contaminant 

(Meneghetti et al., 

2018). 

It gains access to the skin 

through breaks in the skin 

leading to bacterial infection 

(Chang et al., 2008).  

Malassezia species  Yeast It is a fungus 

found on many 

It is an infectious agent 

causing atopic dermatitis tinea 



5 | P a g e  

 

Pathogen  Classification Role  Pathogenesis 

human skin sites 

(Findley and 

Grice, 2014). 

versicolor, dandruff, and to a 

lesser extent psoriasis (Findley 

and Grice, 2014). 

Pseudomonas 

aeruginosa  

Gram-negative  Skin contaminant   

(Afsar and 

Khanam, 2016). 

It infects damaged skin, such 

as burns, wounds, or sores, and 

causes bacterial folliculitis and 

toe web infections (Findley 

and Grice, 2014; Afsar and 

Khanam, 2016). 

Staphylococcus 

aureus  

Gram-positive It colonizes the 

moist site of the 

human skin (Byrd 

et al., 2018).  

It is the most common skin 

pathogen that causes 

superficial and deep dermal 

infections. (Van Vuuren et al., 

2014). It also causes infections 

such as impetigo, folliculitis, 

and cellulitis (Ni Riain, 2013). 

Staphylococcus 

capitis  

Gram-positive  It is a commensal 

micro-organism on 

the skin of the 

human scalp, 

forehead, and ears 

(Kamalakannan et 

al., 2004). 

It has been implicated in 

biofilm-related infections. 

Additionally, it causes skin 

and soft tissue infections such 

as folliculitis and the 

formation of cysts (Natsis and 

Cohen, 2018). 

Staphylococcus 

epidermidis  

Gram-positive It is part of the 

normal flora of the 

skin (Meneghetti 

et al., 2018). 

It degrades leucine which is 

present in perspiration and 

responsible for an unpleasant 

odour (Kanda et al., 1990). 

Cause skin and soft tissue 

infections such as abscesses, 



6 | P a g e  

 

Pathogen  Classification Role  Pathogenesis 

folliculitis, cellulitis, formation 

of boils, and cysts (Natsis and 

Cohen, 2018). 

Staphylococcus 

haemolyticus  

Gram-positive  It is part of the 

normal flora of the 

skin (Meneghetti 

et al., 2018). 

It causes skin and soft tissue 

infections called felon, which 

is an infection that occurs on 

the pulp of the fingertips 

(Natsis and Cohen, 2018). 

Staphylococcus 

hominis 

Gram-positive It colonises the 

skin for several 

weeks or months, 

found under the 

arms (Lam et al., 

2018). 

It is associated with malodour. 

It produces thioalcohol 

compounds that contribute to 

unpleasant body odours (Lam 

et al., 2018). 

Staphylococcus 

lugdunensis  

Gram-positive It is part of the 

normal flora of the 

skin (Meneghetti 

et al., 2018). 

It causes skin and soft tissue 

infections such as paronychia, 

felon, formation of cysts, 

abscesses, and boils (Natsis 

and Cohen, 2018). 

Streptococcus 

pyogenes 

Gram-positive It is part of the 

normal skin 

microbiome (Grice 

and Segre, 2011). 

It causes skin infections such 

as impetigo, cellulitis, and 

erysipelas (Grice and Segre, 

2011). 

Trichophyton 

mentagrophytes  

N/A It is part of the 

normal skin 

microbiome (Grice 

and Segre, 2011). 

It is responsible for infections 

affecting the body, face, and 

feet such as ringworm, 

onychomycosis, and tinea 



7 | P a g e  

 

Pathogen  Classification Role  Pathogenesis 

pedis (Findley and Grice, 

2014). 

Key – a micro-organisms in bold are micro-organisms selected for the study. 

 

1.1.3 Bacterial skin infection:  

1.1.3.1 Impetigo 

 

 Impetigo is a superficial epidermal infection which is common in children and caused by Gram-

positive bacteria Staphylococcus aureus or Streptococcus pyogenes (Chiller et al., 2001). There is 

primary impetigo, which occurs on healthy skin (skin without infection), and secondary impetigo, 

which occurs on skin that previously had an underlying infection or an injury (Motswaledi, 2011). 

It is highly contagious; however, practising personal hygiene by washing the injuries and sores of 

infected patients with water and soap can limit the spread to healthy people. It is classified as one 

of the skin conditions that can lead to life-threatening complications (Abrha et al., 2020). 

According to Abrha et al. (2020), approximately 2% of the world’s population gets affected by 

impetigo. This includes isolated communities in first-world and third-world countries or 

unprivileged tropical countries. This is due to poor hygiene practices and the lack of adequate 

housing (Abrha et al., 2020). 

 

1.1.3.2 Folliculitis  

 

Folliculitis is a skin condition that causes the skin to become abnormally red due to inflammation 

of the hair follicles. The inflammation is caused by bacterial and fungal species (Winters and 

Mitchell, 2022). Staphylococcus aureus commonly causes superficial bacterial folliculitis, which 

occurs on the upper portion of the hair follicle. This infection is frequently found in the axillae, 

beard, and buttocks. The bacterial infection can extend deeper into the hair follicle, resulting in 

furuncles, commonly called boils. This leads to swelling and perifollicular inflammation. Gram-

negative bacterial folliculitis is commonly associated with pseudomonal infections from 



8 | P a g e  

 

contaminated water. It is usually caused by P. aeruginosa; however, it can also be caused by 

Enterobacter and Klebsiella spp. (Ni Riain, 2013). A form of folliculitis that is caused by fungal 

(yeast) species is called Pityrosporum folliculitis. Malassezia species are the most common 

causative agents. It usually occurs in adolescent patients since there is increased activity in the 

sebaceous glands (Suzuki et al., 2016). According to Winters and Mitchell (2022), one of the 

recommended ways to manage folliculitis is to maintain good skin hygiene by bathing with 

antimicrobial soap and clean water. This is also important as it prevents the reoccurrence of 

infection.  

 

1.1.3.3 Cellulitis and erysipelas 

 

Cellulitis is an acute inflammatory skin infection that naturally spreads on the subcutaneous tissue 

and lower dermis. The primary causative agents are S. aureus and Streptococci (S. pyogenes) 

which enter the dermis through a break in the skin such as wounds, injuries, bites, and burns 

(Maitre, 2006; Phoenix et al., 2012). A rash develops on the skin (most likely on the leg), resulting 

in swelling around the affected area. If not treated with antibiotics, the rash can expand, and the 

infection will get worse (Han et al., 2020). Unlike cellulitis which affects the deeper skin tissues, 

erysipelas is the more superficial form of cellulitis that occurs on the upper layers of the skin. It 

can be distinguished from cellulitis by the well-developed margins and bright red colour of the 

affected area. Similar to cellulitis, this superficial bacterial infection occurs due to a crack in the 

skin as a result of an injury, a bite, a wound, or a burn (Michael and Shaukat, 2022). According to 

Henry et al. (2004), cellulitis and erysipelas can be prevented by practising good personal hygiene 

and using antiseptics or antimicrobial soap adequately when there are breaks on the skin. 

 

1.1.4 Fungal (yeast) skin infections  

 

The skin microbiota includes fungi such as Candida, Cryptococcus, Malassezia, and Rhodotorula 

species; however, some of these species are pathogenic. According to Havlickova et al. (2008), an 

estimated 20-25% of the world’s population is affected by fungal skin infections. Candida albicans 

is often responsible for symptomatic skin infections among the Candida species, and the common 

symptoms include redness of the skin and increased thickness of the outer layer of the skin. Skin 



9 | P a g e  

 

infections caused by Candida species occur in closed regions where there is constant friction, 

accumulation of carbon dioxide, and humidity (Kühbacher et al., 2017). Fungal skin pathogens 

are classified into two classes, namely, dermatophytes and yeast infections. Dermatophytes cause 

a group of skin infections broadly referred to as dermatomycoses. This group of fungal infections 

is caused by the Trichophyton species. One of the infections is tinea corporis which is commonly 

known as ringworm. It is characterised by circular lesions with clear skin in the centre, scaling, 

and redness of the skin. It usually occurs on the legs, scalp, neck, and arms (O'Dell, 1998; Yee and 

Al Aboud, 2022). Candidal intertrigo is one of the skin infections caused by Candida species. It is 

an inflammatory skin condition that develops on the skin due to reduced air circulation, increased 

friction, and humidity.  It is characterised by the development of satellite lesions, crusts, exudative 

erosions, and fissures after presenting itself as mild erythematous papillae (Kalra et al., 2014). The 

inflammation of the skin is usually caused by treating the skin with harsh ointments, harsh 

antimicrobial soaps, or topical steroids. Preventative measures that can be taken to avoid recurrent 

infections include maintaining good hygiene by keeping the infected areas clean, dry, and cool 

(Metin et al., 2018).  

 

1.2 Skin hygiene  

 

Good hygiene is a vital principle that must be exercised to prevent skin infections and malodour 

(Afsar and Khanam, 2016). Odourless precursors of sweat, such as fatty acids, glycerol, and lactic 

acid are naturally secreted on the skin’s surface by apocrine glands, then decomposed by bacteria 

into human body odour (Lam et al., 2018; Rudden et al., 2020). Body odour is normal; however, 

can lead to discomfort and negative outcomes. Good skin hygiene can be achieved by bathing with 

commercial soaps (Felhaber and Mayeng, 1997). Soap is a chemical mixture of potassium or 

sodium salt with fatty acids that results in a saponification reaction (Mak-Mensah and Firempong, 

2011; Nchimbi, 2020).  

 

One of the principles for maintaining good hygiene is handwashing because hands are a critical 

vector that transmits micro-organisms faster between objects and/or individuals. Cross-

transmission of pathogens happens when hands are not washed effectively or often enough 

(Edmonds-Wilson et al., 2015). Recently, there has been a global effort to prevent the spread of 



10 | P a g e  

 

COVID-19, and one of the measures that have been strongly recommended is handwashing with 

soap and water for 20 seconds. The reason is that soap dissolves the lipid bilayer of the self-

assembled virus, causing it to disassemble (Türsen et al., 2020). Other alternatives such as alcohol-

based hand sanitisers are used when soap and water are not available (Alzyood et al., 2020). The 

COVID-19 pandemic revealed that it is important to maintain personal hygiene; however, 

according to the World Health Organisation (WHO), approximately 43% of the world’s population 

does not have access to water and soap (Unicef, 2020). 

 

The frequent use of antiseptic soaps can damage the skin by changing the composition of the skin 

flora (Weaver, 2005). This allows Staphylococci and other pathogenic Gram-negative bacteria to 

colonise the skin and cause skin conditions such as cellulitis, erysipelas, and impetigo (Stulberg et 

al., 2002). Furthermore, the overuse of alcohol-based sanitisers results in antimicrobial resistance 

as micro-organisms tend to mutate through the natural process due to repeated exposure to 

disinfectants (Mahmood et al., 2020). A study by Hayat and Munnawar (2016) reported that almost 

all Gram-negative bacteria are resistant to commercial hand sanitisers. Furthermore, 48% of 

Escherichia coli and 64% of Pseudomonas aeruginosa were resistant to all the sanitisers on the 

Pakistani market (Hayat and Munnawar, 2016). Therefore, a more naturally based approach should 

be considered.  

 

Plants have been used across the globe as soap substitutes by indigenous people for centuries. In 

southern Africa most of the population uses medicinal plants for bathing and washing. However, 

their use has only gained importance in recent years as the cornerstone of non-pharmaceutical 

interventions in combatting skin infections and COVID-19 (Sindhu et al., 2019; Kunatsa and 

Katerere, 2021). According to Solanki (2011), herbal soaps have about 60-80% antimicrobial 

activity; thus, they can inhibit micro-organisms from causing skin infections. Given that soap 

plants are natural, utilising them might be a promising route for achieving good health with fewer 

side effects and they are also more cost-friendly (Sindhu et al., 2019). However, the toxicity of 

some of the soap plants must be evaluated to ensure that the plants are safe to use (Mohlakoana, 

2020). 

 



11 | P a g e  

 

1.3 Alternative: plants used as soap substitutes in southern Africa 

 

In the past, indigenous people used plants as soap substitutes because when agitated in an aqueous 

solution, there is a formation of natural lather and foam (Levey, 1954; Akuaden et al., 2019; 

Mohlakoana, 2020). According to Akuaden et al. (2019), lather and foam are formed due to a 

group of naturally occurring plant steroids or triterpenoid glycosides called saponins. Saponins are 

characterised by their strong foam-forming properties in water; hence, plants containing 

substantial amounts of saponins have rich lather and are regarded as a natural soap base (Man et 

al., 2010). They are also recognised for their ability to rupture erythrocytes (Kunatsa and Katerere, 

2021). Apart from saponins, lather-forming plants are rich in phytochemicals such as alkaloids, 

tannins, flavonoids, terpenoids, and phenolic compounds which give the plants the ability to exert 

antimicrobial activity against skin pathogens. Furthermore, they have medicinal properties such as 

relieving pain and promoting wound healing (Van Wyk, 2008; Hulley and Van Wyk, 2019). Most 

soapy plants originate from the genus Saponaria L. since they are saponin-rich. Families such as 

Agavaceae, Dioscoreaceae, and Liliaceae (monocotyledons) are major sources of steroidal 

saponins, while families such as Fabaceae, Araliaceae, and Caryophyllaceae (dicotyledons) are 

major sources of triterpenoid saponins (Oleszek and Hamed, 2010; Rai et al., 2021). According to 

Kunatsa and Katerere (2021), families such as Aceraceae, Aloaceae, Fabaceae, Hippocastanaceae, 

Lamiaceae, Malvaceae, Pedaliaceae, Rhamnaceae, Sapindaceae, and Tiliaceae have saponin-rich 

species that are used in Southern Africa as soap substitutes. 

 

Herbal soap substitutes have been used globally for centuries. An example of such a plant is Yucca 

schidigera Roezl ex Ortgies used by Native Americans as a soap substitute and is currently used 

in a commercial soap (Oleszek and Hamed, 2010). The roots and rhizomes of Saponaria officinalis 

L. commonly known as soapwort, are also used as a soap substitute or as a detergent in Europe 

and Asia due to their cleaning abilities (Kregiel et al., 2017). Besides being a natural emulsifier 

and detergent, soapwort also has high biological activities (Jurek et al., 2019). Other soap plants 

with substantial amounts of saponins used internationally include Chlorogalum pomeridianum 

(DC.) Kunth, Sapindus saponaria L., Polygala L. and Primula L. spp. (Kregiel et al., 2017). Soap 

plants are also popular in Africa. In Angola, before the production of commercial soaps was 

introduced, plants were used as soap to cleanse the body (Bossard, 1993). In Nigeria, an open 



12 | P a g e  

 

market manufactures soap locally by mixing several plants to make lye. Some of the plants include 

Ageratum conyzoides Linn., Aloe vera (L.) Burm.f. and Theobroma cacao L. These soaps are used 

to treat wounds, ringworm, and body odours (Moody et al., 2004; Igbeneghu, 2013). In southern 

Africa, plants used as soap include Aloe maculata All., Artemisia afra Jacq. ex Willd and Ilex mitis 

(L.) Radlk. var. mitis. A list of southern African plants used as soap substitutes can be found in 

Table 1.2.  

 

1.4 Chemistry and toxicity of plants used as soap substitutes 

 

The two distinctive moieties of saponins after hydrolysis are glycan and aglycone. Glycan is water-

soluble and made up of sugar molecules, namely hexoses and pentoses. Aglycone has two types 

of carbohydrate moieties, namely triterpenoidal and steroidal aglycones which bring about 

diversity in the structure of saponins (Desai et al., 2009; Kunatsa and Katerere, 2021). Aglycone 

has a hydrophobic backbone that makes it non-soluble in water; thus, the hydrophilic glycan and 

hydrophobic aglycone give saponins their emulsifying and lathering properties. Saponins can be 

classified based on the carbon skeleton (–H, –COOH, or –CH3) on the aglycone as either 

triterpenes, steroids, or steroidal alkaloids (Oleszek and Hamed, 2010; Sandeep, 2020). 

Triterpenoid saponins are widely distributed in the plant kingdom, hence they are further 

subcategorized into dammarane, oleanane and ursolic acid saponins (Böttcher and Drusch, 2017). 

Steroidal saponins are subcategorized into furostanol and spirostanol. Alkaloidal saponins have a 

steroid-like structure; however, instead of having a pyranose ring in its structure, it has a piperidine. 

They usually occur in plant families such as Solanaceae (El Aziz et al., 2019; Rai et al., 2021). 

Besides saponins, there are also other secondary metabolites found in soap plants, namely alkaloids 

and terpenoids. In the past, alkalis used in soap-making were obtained from plants or ash (Levey, 

1954). Such plants produce a soap-like substance due to the presence of alkaloids; however, most 

of them have low amounts of saponins (Mohlakoana, 2020). Psilocaulon absimile N.E.Br and 

Sceletium tortuosum (L.) N.E.Br. are some of the plants with low saponin content; nonetheless, 

they have soap-like sap due to alkaloids (Watt and Breyer-Brandwijk, 1962).  

 

.



13 

 

Table 1.2: Plants used traditionally in southern Africa as soap substitutes. 

Medicinal plants Common name Family Use Part used Reference 

Acalypha glabrata 

Thunb.a 

Umthombothi (X), 

forest false nettle 

(Eng.) 

Euphorbiaceae Used to clean the skin  Bark (Afolayan et al., 

2014) 

Agathosma capensis 

(L.) Dümmer 

Spicy buchu (Eng.) Rutaceae Used to wash the body or as 

lotion  

Not 

specified  

(Hulley and Van 

Wyk, 2019) 

Albizia versicolor 

Welw. Ex Oliv. 

Muvhambangoma 

(V), large-leaved 

albizia (Eng.) 

Fabaceae Used as a soap substitute Bark, root, 

and leaves  

(Magwede et al., 

2019) 

Aloe ferox Mill. Inhlaba (Z), bitter 

aloe (Eng.) 

Asphodelaceae Used for cleansing the skin  The juice 

gel and the 

leaves 

(Afolayan et al., 

2014) 

Aloe maculata All. Soap aloe (Eng.), 

lekhala (S) 

Asphodelaceae Used as a soap substitute Leaves  (Felhaber and 

Mayeng, 1997; 

Ngalo, 2010) 

Aristaloe aristata 

(Haw.) Boatwr. & 

J.C. Manning 

Umathithibala (X), 

lace aloe (Eng.) 

Asphodelaceae The juice from the leaves is 

mixed with water to wash 

the body  

Leaves  (Watt and Breyer-

Brandwijk, 1962) 

Artemisia afra 

Jacq. ex Willd 

Umhlonyane (X), 

African wormwood 

(Eng.) 

Asteraceae Used as a body wash Leaves  (Hutchings et al., 

1996; Rabe and Van 

Staden, 1997) 

Bauhinia bowkeri 

Harv. 

Umdlandlovu (X), 

kei white bauhinia 

(Eng.) 

Fabaceae Used for steaming and 

bathing  

Leaves and 

bark  

(Ndawonde et al., 

2007) 



14 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Caesalpinia 

decapetala (Roth) 

Alston 

Luanakha (V), 

Mauritius-thorn 

(Eng.) 

Fabaceae Used as a soap substitute Fruit sap or 

seeds  

(Magwede et al., 

2019) 

Calodendrum 

capense (L.f.) 

Thunb. 

 

Umemezi 

omhlophe (Z), cape 

chestnut (Eng.) 

Rutaceae Used to clean the skin; used 

in soap; externally used to 

lighten skin, as a 

moisturizer, and to treat 

pimples. 

Fruits or 

seeds and 

Bark 

(Philander, 2011; 

Afolayan et al., 

2014) 

Carica papaya L. Pawpaw (Eng.) Caricaceae Used as a soap substitute  Leaves (Watt and Breyer-

Brandwijk, 1962) 

Citrus assamensis 

R.M Dutta & 

Bhattacharya 

Tshikavhavhe (V) Rutaceae Used as a facial wash  Fruit sap  (Ndhlovu et al., 

2019) 

Crinum 

bulbispermum 

(Burm.f) Milne-

Redh. & Schweick 

Orange river lily 

(Eng.), umnduze 

(Z) 

Amaryllidaceae Used as a soap substitute Bulb (Felhaber and 

Mayeng, 1997; 

Mohlakoana and 

Moteetee, 2021) 

Cussonia 

paniculata Eckl. & 

Zeyh. 

Umsengembuzi (Z), 

mountain cabbage 

tree (Eng.) 

Araliaceae Tree sap is mixed with 

water for bathing  

Tree sap  (Mogale et al., 2019) 

Cyathula cylindrica 

Moq. 

Bohome-bo-boholo 

(S) 

Amaranthaceae Used as a soap substitute Roots  (Moffett, 2010) 

Cyathula 

uncinulata 

(Schrad.) Schinz 

Bohome-ba-lipoli 

(S) 

Amaranthaceae Used as a soap substitute Roots  (Watt and Breyer-

Brandwijk, 1962; 

Moffett, 2010) 



15 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Deinbollia 

oblongifolia (E. 

Mey. ex Arn.) 

Radlk. 

Dune soapberry 

(Eng.), 

umbangabanga (X) 

Sapindaceae Used as a soap substitute Seed (Naidoo, 2009; 

Würger, 2010) 

Dianthus crenatus 

Thunb.  

Iningizimu (Z), 

wild pink (Eng.) 

Caryophyllaceae With Tephrosia lurida 

Sond. as a facial face wash 

(froth is produced) 

Roots  (Watt and Breyer-

Brandwijk, 1962) 

Dianthus thunbergii 

(Thunb.) S.S. 

Hooper  

Ungcane (X), wild 

pink (Eng.) 

Caryophyllaceae Used to remove body odour  Leaves  (Afolayan et al., 

2014) 

Dicerocaryum 

eriocarpum (Decne.) 

Abels 

Devil’s thorn 

(Eng.), intekelane 

(N) 

Pedaliaceae Used as soap and shampoo Leaves and 

flowers  

(Watt and Breyer-

Brandwijk, 1962; 

Hoveka, 2017) 

Dicerocaryum 

senecioides 

(Klotzsch) Abels 

Museto/dinda (V) Pedaliaceae Applied topically as a 

substitute for soap 

Leaves  (Ndhlovu et al., 

2019) 

Dicerocaryum 

zanguebaricum 

Merrill 

Museto (V) Pedaliaceae Used as a soap substitute Leaves and 

flowers  

(Watt and Breyer-

Brandwijk, 1962) 

Dissotis princeps 

Triana 

Purple dissotis 

(Eng.), 

kalwerbossie (Afr.) 

Melastomataceae Used as a soap substitute  Roots  (Watt and Breyer-

Brandwijk, 1962) 

Dysphania 

ambrosioides (L.) 

Imboya (X), 

Mexican tea (Eng.) 

Amaranthaceae Used to clean the skin  Leaf lotion  (Afolayan et al., 

2014) 



16 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Mosyakin & 

Clements  

Entada phaseoloides 

Merr. 

Box bean (Eng.) Fabaceae Used as a soap substitute Bark (Watt and Breyer-

Brandwijk, 1962) 

Foeniculum vulgare 

Mill. 

Imbozisa (X), 

fennel (Eng.) 

Apiaceae Used as a body wash  Not 

specified  

(Mhlongo and Van 

Wyk, 2019) 

Haemanthus 

albiflos Jacq. 

Umathunga (X), 

white paintbrush 

(Eng.) 

Amaryllidaceae Remove body odour  Leaf 

decoction  

(Afolayan et al., 

2014) 

Helinus integrifolius 

(Lam.) Kuntze 

Mupupuma (V) Rhamnaceae Applied topically as a 

substitute for soap 

Whole herb  (Ndhlovu et al., 

2019) 

Hermannia 

cuneifolia Jacq. 

Doll's roses (Eng.), 

poprosie, 

moederkappie 

(Afr.) 

Malvaceae Used for external wash  Not 

specified  

(Hulley and Van 

Wyk, 2019) 

Ilex mitis (L.) 

Radlk. var. mitis 

Mollo-oa-phofu 

(S), African holly 

(Eng.) 

Aquifoliaceae Produces a soap-like lather 

used to wash the body 

Leaves  (Phillips, 1917; 

Watt, 1927) 

Ipomoea simplex 

Thunb. 

Igontsi (X), 

seakhoe (S) 

Convolvulaceae Used to clean the skin  Leaves  (Afolayan et al., 

2014) 

Kedrostis capensis 

(Sond.) A. Meeuse 

Sesepa-sa-linoha 

(S) 

Cucurbitaceae Used as soap  Not 

specified  

(Phillips, 1917; Watt 

and Breyer-

Brandwijk, 1962; 

Moffett, 2010) 



17 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Ledebouria sp. 

[Ledebouria 

apertiflora (Baker) 

Jessop] 

Umathunga (Z), 

Sekanama (S), 

giant African 

hyacinth (Eng.)  

Hyacinthaceae Wash wounds, rubbed on 

painful parts of the body, 

mixed with bathing water, 

or used as soap 

Bulb (Komoreng et al., 

2017; Mogale et al., 

2019) 

Merwilla plumbea 

(Lindl.) 

Wild squill (Eng.) Hyacinthaceae It is considered a soap plant Bulb (Street and Prinsloo, 

2012; Mohlakoana, 

2020) 

Mesembryanthemum 

crystallinum L. 

Crystalline ice plant 

(Eng.), brakslaai 

(Afr.) 

Aizoaceae Used as a soap substitute  Leaves  (Watt and Breyer-

Brandwijk, 1962) 

Morella serrata 

(Lam.) Killick 

Malokela (S), 

lance-leaf waxberry 

(Eng.) 

Myricaceae Used as a soap substitute Branches  (Guillarmod, 1971; 

Hutchings and Van 

Staden, 1994) 

Noltea africana (L.) 

Endl. 

Soap glossy-leaf 

(Eng.), 

umkhuthuhla (X) 

Rhamnaceae Used as a soap substitute  Leaves and 

twigs 

(Watt and Breyer-

Brandwijk, 1962) 

Olea europaea L. 

subsp. africana 

(Mill.) P.S. Green 

Umquma (X), wild 

olive (Eng.) 

Oleaceae Used to remove body odour  Leaves  (Afolayan et al., 

2014) 

Pelargonium 

peltatum (L.) 

L'Hér.  

Ityolo (X), ivy-

leaved pelargonium 

(Eng.) 

Geraniaceae Used to clean the skin  Tubers  (Afolayan et al., 

2014) 

Piliostigma 

thonningii 

Camel's foot (Eng.) Fabaceae Used in soap making  Unripe 

pods  

(Ayisere et al., 2009; 

Ibrahim et al., 2019) 



18 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

(Schumach.) Milne- 

Redh. 

Plectranthus 

ciliatus E. Mey. 

Lephelephele (S), 

speckled spur-

flower (Eng.) 

Lamiaceae Used as a substitute for soap Leaves  (Phillips, 1917; Watt 

and Breyer-

Brandwijk, 1962; 

Guillarmod, 1971 ) 

Pouzolzia mixta 

Sohm 

Soap-nettle (Eng.), 

murovhadembe (V) 

Urticaceae Used as a soap substitute to 

wash hands and clothes 

Leaves (Masupa, 2013) 

Psilocaulon sp.cf. 

coriarium (Burch. 

ex N.E.Br.) N.E.Br. 

Asbos (H.) Mesembryanthemaceae The ash is placed in water, 

the solution is used to wash 

the hair; used to make soap 

Stems (De Beer and Van 

Wyk, 2011) 

Ptaeroxylon 

obliquum (Thunb.) 

Radlk. 

Umthathi (X), 

sneezewood (Eng.) 

Rutaceae Remove body odour Bark (Afolayan et al., 

2014) 

Pterocarpus 

angolensis DC. 

Mutondo (V) Leguminosae Wash or clean the skin  Stems  (Ndhlovu et al., 

2019) 

Rhynchosia 

caribaea (Jacq.) DC. 

Monya-mali (S), 

snoutbean (Eng.) 

Fabaceae Soap substitute Leaves  (Guillarmod, 1971; 

Hutchings and Van 

Staden, 1994; 

Moffett, 2010) 

Salsola aphylla L. f. Ganna bush (Eng.) Chenopodiaceae Used to supply the alkali for 

soap-making 

Leaves  (Watt and Breyer-

Brandwijk, 1962) 

Senna obtusifolia 

(L.) H.S. Irwin & 

Barneby. 

Mutsheketsheke 

(V) 

Leguminosae Soap substitute leaves  (Ndhlovu et al., 

2019) 



19 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Sesbania sesban (L.) 

Merr. subsp. sesban 

Mugunwa (V), 

frother (Eng.) 

Fabaceae Soap substitute Leaves (Magwede et al., 

2019) 

Sida rhombifolia L. Pretoria bossie 

(Afr.), arrow leaf 

sida (Eng.) 

Malvaceae Bathing and shampooing Shoot bark (Mehta and Bhatt, 

2007; Kunatsa and 

Katerere, 2021) 

Sideroxylon inerme 

L. subsp. inerme 

Unqwashu (X), 

white milkwood 

(Eng.) 

Sapotaceae Remove body odour  Leaves  (Afolayan et al., 

2014) 

Solanum 

aculeastrum Dunal 

subsp. aculeastrum 

Bitter apple (Eng.),  Solanaceae Used as a soap substitute  Fruits  (Watt and Breyer-

Brandwijk, 1962; 

Welman, 2004) 

Solanum 

tomentosum L. 

Slangbessiebos 

(Afr.) 

Solanaceae Wash body  Leaves  (De Beer and Van 

Wyk, 2011) 

Stoebe capitatum 

P.J. Bergius 

Groen slangbos 

(Afr.) 

Asteraceae Douche and a base for soap Foliage (Philander, 2011) 

Stoebe plumosa (L.) 

Thunb. 

Grys slangbos 

(Afr.) 

Asteraceae  As a base for soap  Foliage (Philander, 2011) 

Talinum caffrum 

(Thunb.) Eckl. & 

Zeyh. 

 Porcupine root 

(Eng.), impunyu 

(Z) 

Talinaceae Used as soap  Leaves  (Watt and Breyer-

Brandwijk, 1962) 

Taraxacum 

officinale F.H. Wigg 

Ikhokhoyi (X), 

common dandelion 

(Eng) 

Asteraceae  Remove body odour  Leaves  (Afolayan et al., 

2014) 



20 | P a g e  

 

Medicinal plants Common name Family Use Part used Reference 

Withania somnifera 

(L.) Dunal 

Geneesbos (Afr.), 

winter cherry 

(Eng.) 

Solanaceae Used as a soap wash and 

douche  

Foliage 

 
 

(Philander, 2011) 

Key – Afr. (Afrikaans); Eng. (English); H. (Hantam); S (Southern Sotho); V (Venda); X (Xhosa); Z (Zulu); a species names in bold = species selected for this 

study.



21 

 

Although these phytochemicals exhibit antimicrobial activity and other medicinal properties, they 

can also be toxic to humans and animals. Merwilla plumbea (Lindl.) Speta is one of the soap plants 

found in southern Africa that has been reported to have elevated levels of toxicity (Fennell et al., 

2004). According to Notten (2001), it causes skin irritation and itching. However, it is used 

traditionally as soap and in an ointment for wounds (Street and Prinsloo, 2012). Carica papaya L. 

is traditionally used as a soap substitute and skin-lightening agent due to the presence of papain 

and chymopapain. The saponins found in Carica papaya can heal wounds by boosting collagen 

production, and carpaine alkaloids serve as a detox agent within the body (Nugrahaningsih et al., 

2019). According to Aravind et al. (2013), latex causes skin irritation, and if the leaves are 

ingested, they can cause severe gastritis. The phytochemical analysis of Crinum L. spp. has yielded 

more than 150 Amaryllidaceae alkaloids, which are highly toxic. Crinum bulbispermum (Burm.f) 

Milne-Redh. & Schweick is traditionally used as a soap substitute (Felhaber and Mayeng, 1997); 

however, it has been reported to be highly lethal due to the crinamine and isoquinoline alkaloids 

found in the plant (Van Wyk et al., 2002; Maroyi, 2016). Haemanthus albiflos has elevated toxicity 

due to the presence of toxic alkaloids called homolycorine and albomaculine, thus it is important 

to assess potential soap plants for toxicity (Crouch et al., 2005). 

 

1.5 Antimicrobial activity of plants used as soap substitutes 

 

Saponins have a similar chemical structure as soaps and detergents, with hydrophilic heads and 

hydrophobic tails on both ends (Rai et al., 2021). The hydrophobic tails interact with the lipids of 

bacterial envelopes, causing them to rupture and release their contents. In the same way that soaps 

form micelles around dirt, proteins from damaged envelopes are enveloped in saponin molecules. 

In fungi, saponin contact with cell membranes induces cell content leakage, which leads to 

disintegration (Dong et al., 2020). Medicago arabica (L.) Huds. is one of the plants used as a soap 

substitute, and it was reported to have good antimicrobial activity against Gram-positive bacteria. 

This is due to phytochemicals such as alkaloids, saponins, and terpenoids (Avato et al., 2006). 

Saponins extracted from Sorghum bicolor L. Moench are active against Gram-positive bacteria 

(Soetan et al., 2006). 

  



22 | P a g e  

 

1.6 Formulation of herbal soap 

 

Historical studies show that the evidence for soap formulation dates back more than 6000 years, 

and it was mostly utilised by the Egyptians and Babylonians (Levey, 1954). According to Watt 

(1946), the Gauls were the original inventors of soap making by combining goats’ fat with the 

ashes of beech trees. This was found to produce crude soap that cleaned and washed grease 

effectively. According to Babayemi et al. (2010), when wood or plant material is burned by fire 

into ash, there are compounds (potassium hydroxide and sodium hydroxide) that are conserved 

into potash (potassium carbonate) and soda ash (sodium carbonate). These major alkali 

components (combined with oil or animal fats for the effect of saponification) were used for 

washing and cleaning (Levey, 1954).  Potash and soda are two remarkably similar chemicals; 

hence, the difference was only recognised in the nineteenth century by Lewkowitsch (1894).  

Lewkowitsch (1894) was able to differentiate between the two alkalis and that when using caustic 

soda, a hard soap will be produced, while when using caustic potash, a soft or soluble soap will be 

produced. This still applies where caustic potash (from wood) is used to make liquid soap and 

caustic soda, or lye is used to make hard bar soaps. Fatty acids (usually vegetable oils) are added 

to an alkaline (sodium or potassium salt) solution, resulting in the saponification reaction where 

glycerine and soap are produced (Akuaden et al., 2019). 

 

Excellent quality soap must produce lather; hence, foam retention and height are some 

phytochemical parameters to assess after the formulation. The formulated soap must have the 

moisturising ability and be compatible with the skin (Mak-Mensah and Firempong, 2011; Sindhu 

et al., 2019).  This is assessed by determining the strength and purity of the alkali and the moisture 

content in the soap. Free caustic alkali in soap prevents it from being oily; however, excess free 

alkali (caused by incomplete saponification) causes clothes to wear out and the skin to itch. 

Formulated soap must have a good fragrance, colour, and storage stability (Sindhu et al., 2019). 

The percentage of chlorine is vital since excess amounts can cause the soap to crack. It is also 

important to assess the pH of the soap since healthy skin has a pH range of 5.4-5.9 (Mak-Mensah 

and Firempong, 2011). Skin products are expected to have a pH close to this range to reduce skin 

irritation (Akuaden et al., 2019). Finally, yet importantly, the soap should have antimicrobial 

activity against skin pathogens (Igbeneghu, 2013). 



23 | P a g e  

 

1.7 Aim and objectives 

 

The study aimed to investigate the antimicrobial activity, phytochemistry, and toxicity of southern 

African plants used as soap substitutes; thereafter, an effective antimicrobial herbal soap was 

formulated and assessed for efficacy. The following objectives were implemented to align with 

the respective research chapters which are four in total: 

 

• To do undertake preliminary phytochemical screening, a qualitative screening (thin 

layer chromatography (TLC)) and quantitative screening to determine the presence of 

saponins.  

• To determine the antimicrobial activity of the selected plants using the minimum 

inhibitory concentration assay (MIC). 

• To determine the toxicity of the selected soap plants using the brine-shrimp lethality 

assay.  

• To formulate herbal soaps by the saponification process and assess the physicochemical 

parameters of the formulations by determining the percentage of free caustic alkali, 

moisture content, pH, foam retention, and total fatty matter, as well as determining the 

antimicrobial efficacy. 

 

 



24 

 

CHAPTER 2 

PHYTOCHEMICAL ANALYSIS OF SELECTED 

SOUTHERN AFRICAN PLANTS USED AS SOAP 

SUBSTITUTES 

 

2.1 Introduction  

 

A review conducted on the ethnobotanical literature (Chapter 1; Table 1.2) revealed that 59 plant 

species are used in southern Africa as soap substitutes. Although the phytochemistry of several 

plants listed in the review has been previously studied, there is still limited scientific data to assess 

the presence and quantity of saponins in selected plant species. Therefore, investigating the 

phytochemical properties of selected southern African soap plants may provide some evidence for 

the traditional use of the plants as soap substitutes. Terpenoids are the largest and most diversified 

class of secondary metabolites, produced from the five-carbon molecule isoprene, and subdivided 

according to the number of isoprene units present in the plant (Adefegha et al., 2022). Triterpenes 

are one of the subgroups classified in the terpenoid class. Triterpenes have active glycosylation 

sites used to convert the subgroup into triterpene glycoside, one of the major groups of saponins 

(Perveen, 2018; El Aziz et al., 2019). Historical studies show evidence that the ashes of plants 

were used as a detergent because they became soapy when mixed with fat or vegetable oil (Watt, 

1946). According to Kurek (2019), the alkalinity of alkaloids is caused by the nitrogen atoms in 

their structures. Hence, the plants that are used to make detergents have alkali-like properties. 

Thus, detecting alkaloids, terpenoids, and saponins in soap plants could help distinguish true soap 

plants from those with high alkaline content that act as surfactants. This chapter aimed to determine 

the presence of alkaloids, terpenoids, and saponins in selected plants. Furthermore, the quantity of 

saponins in the plants was determined. The following objectives were designed: 

 

• To carry out a preliminary phytochemical screening for alkaloids, terpenoids, and saponins. 

• To determine the presence of saponins using thin layer chromatography.  

• To determine the quantity of saponins using the vanillin-sulphuric acid assay. 

 



25 | P a g e  

 

2.2 Methods and materials  

2.2.1 Selection of plants 

  

The ethnobotanical information about plants used as soap substitutes was gathered from various 

sources, such as medicinal plant-based books (Watt and Breyer-Brandwijk, 1962; Felhaber and 

Mayeng, 1997), journal articles, dissertations, and theses. Databases such as EBSCOHost, JSTOR, 

PubMed, ProQuest Central, SABINET Online, Science Direct, Scopus, SpringerLink, Taylor & 

Francis Journals, Wiley Online Library, and Web of Science were utilised to search for 

ethnobotanical literature. Keywords such as “Ethnobotanical survey,” “Medicinal plant 

inventory,” “Indigenous knowledge,” “soapy plants,” “saponins,” “skin wash,” “herbal soap,” and 

“medicinal plants used for skin ailments” were used to search for ethnobotanical literature. The 

generated list of plant names (Chapter 1, Table 1.2) was sent to Mr Andrew Hankey, the chief 

horticulturist at Walter Sisulu Botanical Gardens (WSBG) who indicated which plants on the list 

were available for collection. The list was also sent to Random Harvest Indigenous Nursery 

(RHIN) and the University of Johannesburg Herbarium (JRAU). The plants that were available at 

these locations were selected for the study. 

 

2.2.2 Collection of plants  

 

Selected plants were collected from various locations, as indicated in Table 2.1. Mr Andrew 

Hankey, the chief horticulturist, granted permission and assisted in plant identification and 

collection from the Walter Sisulu Botanical Gardens (WSBG). Professor Sandy van Vuuren signed 

the documents to transfer plant material to the University of the Witwatersrand for research 

purposes. The two plants (Aloe ferox and Crinum bulbispermum) that were not collected from 

WSBG, were purchased from the Random Harvest Indigenous Nursery (RHIN), and two other 

plants (Cyathula uncinulata and Deinbollia oblongifolia) were obtained from the University of 

Johannesburg. The co-supervisor and herbarium curator at the University of Johannesburg 

(JRAU), Professor Annah Moteetee, assisted in plant identification and confirmation. The plant 

samples were dried and housed at the Department of Pharmacy and Pharmacology, University of 

the Witwatersrand. 



26 | P a g e  

 

Table 2.1: List of plants that were used in this study, the parts used, voucher numbers, supplier 

and when it was collected. 

 

Plant name Plant part collected Place obtained Voucher number 

Acalypha glabrata Bark and leaves WSBGa SVV249 

Albizia versicolor Bark WSBG SVV240 

Aloe ferox Leaves RHINb SVV264 

Aloe maculata Leaves WSBG SVV251 

Aristaloe aristata Leaves WSBG SVV250 

Artemisia afra Leaves WSBG SVV172 

Bauhinia bowkeri Leaves WSBG SVV252 

Calodendrum capense Leaves and bark WSBG SVV253 

Carica papaya Leaves WSBG SVV239 

Crinum bulbispermum Bulb RHIN SVV265 

Cussonia paniculata Leaves WSBG SVV271 

Cyathula uncinulata Roots JRAUc MRM4 

Deinbollia oblongifolia Leaves JRAU MRM5 

Haemanthus albiflos Bulb WSBG SVV256 

Hermannia cuneifolia Leaves De Rust SVV983 

Ilex mitis Leaves WSBG SVV145 

Ledebouria luteola Bulb WSBG SVV255 

Ledebouria zebrina Bulb WSBG SVV257 

Merwilla plumbea Bulb  WSBG SVV254 

Noltea africana Leaves  JRAU MRM8 

Olea europaea Leaves WSBG SVV238 

Pelargonium peltatum Leaves WSBG SVV258 

Plectranthus ciliatus Leaves WSBG SVV259 

Pouzolzia mixta Bark WSBG SVV260 

Ptaeroxylon obliquum Bark and leaves WSBG SVV261 



27 | P a g e  

 

Plant name Plant part collected Place obtained Voucher number 

Sideroxylon inerme subsp. 

inerme 

Leaves WSBG SVV262 

Key – a Walter Sisulu Botanical Gardens; b Random Harvest Indigenous Nursery; c University of Johannesburg 

herbarium. 

 

 

2.2.3 Plant sample preparation  

 

The plants were stored separately based on which part of the plant is traditionally used as a soap 

substitute. Leaf, root, and bark were left to dry at room temperature for 7–14 days, whereas the 

bulb and succulent leaves were left to dry in a warm air oven (Memmert, Germany) at 37℃ for 7–

14 days. The dried leaves, roots, and bulbs were ground into a fine powder using an electric grinder 

(Mellerware, Johannesburg), whereas the bark was crushed manually using a pounder (purchased 

at Faraday supermarket, Johannesburg). 

 

2.2.4 Preliminary phytochemical screening of alkaloids, terpenoids and saponins  

 

A qualitative phytochemical analysis was undertaken to identify the presence of secondary 

phytochemical constituents found in abundance in particular plant species that either have toxic or 

beneficial effects on a living system. Standard detection methods are used to test for the presence 

of phytochemicals such as saponins, alkaloids, and terpenoids (Harborne, 1984). According to 

Tiwari et al. (2011), the type of solvent used for extraction is vital in determining the biologically 

active compounds in the plant material. Hence, acetone, ethanol, methanol, and distilled water 

were used in the current study to extract and determine the presence of alkaloids, terpenoids, and 

saponins. All phytochemical screening tests were performed following methods by Tiwari et al. 

(2011) and Solomon et al. (2013). 

 

2.2.4.1  Preparation of plant extracts 

 

The plant material was extracted using four different solvents, namely distilled water, acetone 

(Associated Chemical Enterprise, South Africa), 70% ethanol (Univar, USA), and methanol 



28 | P a g e  

 

(Sigma-Aldrich, Germany). The extraction was done using a solvent-to-sample ratio of 10:1 (v/w). 

The solutions were left to stand at room temperature for a extraction 24 h. Thereafter, the solution 

was filtered twice using the Whatman No.1 filter paper. Thereafter, the filtrate was used for 

phytochemical screening of alkaloids, terpenoids, and saponins following Wagner’s reagent test, 

Salkowski’s test, and the froth test, respectively.  

 

2.2.4.2  Detection of alkaloids using Wagner’s test 

 

Wagner’s reagent was prepared by making up 1.27 g of iodine (BDH Chemicals Ltd, Poole 

England) and 2 g of potassium iodide (BDH Chemicals Ltd, Poole England) in 100 ml of distilled 

water. Five drops of Wagner’s reagent were used to treat a small fraction of each plant filtrate. The 

formation of a brown-reddish precipitate indicated the presence of alkaloids. The potassium metal 

ion from Wagner’s reagent covalently bonds with nitrogen on an alkaloid compound to form a 

potassium-alkaloid complex, thus the formation of the brown-reddish precipitate (Parbuntari et al., 

2018). 

 

2.2.4.3  Detection of terpenoids using Salkowski’s test 

 

Salkowski’s test detects sterols and terpenoids by yielding a reddish-brown precipitate on the 

bottom layer when treated with chloroform and concentrated sulphuric acid. Two millilitres of 

each extract were added to test tubes, altogether with 1 ml of chloroform (Associated Chemical 

Enterprise, South Africa) and a few drops of sulphuric acid (Rochelle Chemicals, South Africa). 

The formation of a reddish-brown precipitate indicated the presence of terpenoids. 

 

2.2.4.4  Detection of saponins using the froth test 

 

In test tubes containing 2 ml of plant filtrates each, 18 ml of distilled water was added. The test 

tubes were then shaken vigorously for 15 mins. The formation of a 1 cm layer of foam indicated 

the presence of saponins.  

 

 



29 | P a g e  

 

2.2.5 Qualitative evaluation of saponins  

2.2.5.1 Preparation of plant extracts  

 

A method by Makkar et al. (2007) was followed with modifications. Ten grams of ground plant 

material was defatted using 100 ml of hexane (Associated Chemical Enterprise, South Africa) and 

left overnight. The solvent was filtrated, and the residue was left to dry under a fume hood. After 

the residue was dry, 100 ml of 50% aqueous methanol was added, and the solution was left on a 

magnetic stirrer overnight at room temperature. The contents were centrifuged at 3000 xg for 10 

min, and the supernatant was collected. The extraction process was repeated using the same solvent 

by stirring on a magnetic stirrer overnight and then centrifuging. The first and second supernatants 

were combined and filtrated. Utilizing a rotary evaporator (Buchi, Switzerland), methanol was 

evaporated from the solution under a vacuum at approximately 42℃. Water-insoluble components 

were removed from the aqueous phase by centrifuging at 3000 xg for 10 mins. The aqueous phase 

was transferred to a separating funnel and extracted three times with an equal volume of 

chloroform (Associated Chemical Enterprise, South Africa) to remove pigments. Thereafter, 

concentrated saponins were extracted from the aqueous solution (twice) with an equal volume of 

n-butanol (Rochelle Chemicals, South Africa) using a separating funnel. The solvent (n-butanol) 

was evaporated under a vacuum at a temperature not exceeding 45℃. The dried fraction containing 

saponins was dissolved in 40 ml of distilled water, and the solution was transferred into a pre-

weighed container. The fraction was then lyophilized for 4-7 days using a freeze-dryer (Virtis, 

California), and the percentage recovery of saponins was calculated using Equation 2.1. 

% yield of saponins = 
W1- W2

W3

 × 100 

 Equation 2.1  

 

W1 = weight of the vessel, W2 = weight of the vessel and freeze-dried saponins and W3 = weight 

of plant material. 

 

2.2.5.2 Thin layer chromatography (TLC) 

 

A mixture of chloroform, methanol (Associated Chemical Enterprise, South Africa), and distilled 

water (65:35:10, v/v/v) was prepared as the developing solution. Thereafter, 120 ml of the 



30 | P a g e  

 

developing solution was poured into a chromatographic tank, and the tank was saturated overnight. 

The sample was prepared by dissolving 5 mg of freeze-dried crude saponin residue in 1 ml of 50% 

aqueous methanol. The TLC plates were marked with a pencil, 2.5 cm from the bottom of the plate 

(20 cm × 20 cm, silica gel 60; Merck catalogue No. 1.05721, South Africa). Each sample was 

applied on the plate using capillary tubes and allowed to dry. After the spots were dry, the TLC 

plates were inserted into the saturated chromatographic tank containing the developing solution. 

When the developing solution had reached 1 cm below the top of the TLC plate, the plate was 

carefully removed and allowed to dry at room temperature. The vanillin-perchloric acid reagent 

was prepared by making up 1% vanillin in ethanol (w/v) and 2% perchloric acid in ethanol in 

separate bottles. Thereafter, equal volumes of 1% vanillin (Merck, South Africa) and 2% 

perchloric acid (Merck, South Africa) were combined. The vanillin-perchloric acid reagent was 

sprayed on the plates. A different set of plates were also sprayed with 10% sulphuric acid 

(Associated Chemical Enterprise, South Africa). The plate was heated at 100℃ for 5 mins. Violet- 

or blue-coloured spots were visually located on the plates as saponins (Oleszek, 2002). The 

retention factor (Rf) values were calculated using Equation 2.2.  

 

Rf = 
Distance moved by the solute/compound (mm)

Distance moved by the solvent front (mm)
 

Equation 2.2  

 

2.2.6 Quantitative evaluation of saponins  

2.2.6.1 Sample and reagent preparation 

 

The vanillin-sulphuric acid assay developed by Hiai et al. (1976) was used to determine the total 

saponin content (TSC). The reaction of oxidised triterpene saponins with vanillin is the basic 

principle of the assay (Cheok et al., 2014). A review by Kunatsa and Katerere (2021) recorded that 

plants with a saponin concentration of 40 mg/g and above were considered to be saponin-rich. In 

this study, the same benchmark was used. The freeze-dried saponin residues (Section 2.2.5.1) were 

prepared to a concentration of 5 mg/ml using 80% aqueous methanol. The standard saponin 

solution was prepared by dissolving 10 mg of diosgenin in 80% methanol. The final concentration 

of diosgenin in the solution was 0.5 mg/ml. The vanillin reagent was prepared by dissolving 800 



31 | P a g e  

 

mg in 10 ml of 99.5% ethanol. Thereafter, 72% (v/v) sulphuric acid was made up by carefully 

adding 72 ml of sulphuric acid to 28 ml of distilled water.  

 

2.2.6.2 Preparation of standard curve 

 

Diosgenin was used as a standard compound, and the total saponin content was expressed as mg/g 

diosgenin equivalents. Different volumes (0, 50, 100, 150, 200, and 250 μl) of diosgenin standard 

solution were made up to 0.25 ml with 80% aqueous methanol. The vanillin reagent was added at 

a volume of 2.5 ml. Thereafter, 2.5 ml of 72% sulphuric acid was slowly added to oxidize triterpene 

saponins (Cheok et al., 2014). The samples were mixed well using a vortex (Benchmixer V2) and 

transferred to a water bath (Labcon (Pty) Ltd, South Africa) set at 60℃ for 10 mins. Thereafter, 

the tubes were placed in ice-cold water for 3-4 min to cool. The distinctive purple colour indicates 

indicated that the oxidation reaction was complete (Cheok et al., 2014). The absorbance was 

measured using a UV/VIS spectrophotometer (Biochrom WPA Lightwave II) at 544 nm against a 

reagent blank containing 0 µl of diosgenin standard solution. A standard curve was generated. The 

linear relationship between the concentration of the standard (diosgenin) and the absorbance value 

is demonstrated in Figure 2.1. 

  

 

Figure 2.1: The standard curve showing the absorbance of diosgenin at various concentrations. 

 

0,282

0,384

0,641

0,74

0,972

y = 0,0017x + 0,083

R² = 0,9805

0

0,2

0,4

0,6

0,8

1

1,2

0 100 200 300 400 500 600

A
b

so
rb

an
ce

 a
t 

5
4
4
 n

m

Concentration (µg/ml)



32 | P a g e  

 

2.2.6.3 Determining the absorbance of the samples  

 

A mixture of 0.25 ml of sample extracts and reagent blank, together with 0.25 ml of 8% vanillin 

reagent and 2.50 ml of 72% sulphuric acid, was incubated for 10 min at 60℃ in a water bath, with 

the reagent blank made up of the solvent used for extracting the plant samples (methanol) (Table 

2.1). After cooling in ice water for 3-4 min, the absorbance of the extracts was measured at 544 

nm. This was done in triplicate.  

 

Table 2.2: The vanillin-sulphuric acid assay setup procedure for the reagent blank, standard, and 

sample. 

Component Reagent blank (ml) Standard (ml) Sample (ml) 

Extraction solvent (80% methanol) 0.25 - - 

Diosgenin in 80% methanol - 0.25 - 

Sample in 80% methanol - - 0.25 

8% vanillin in ethanol  0.25 0.25 0.25 

72% sulphuric acid 2.50 2.50 2.50 

 

The unknown concentration of the samples was calculated from the regression equation (Equation 

2.3) from the calibration curve of diosgenin. 

A = 0,0017C + 0,083 

Where A = absorbance of sample, C = unknown sample concentration (µg/ml)  

Equation 2.3 

 

Thereafter, the TSC was calculated using Equation 2.4, where C = concentration of the sample 

(µl/ml), V = volume of extract in the test tube (ml), the mass of extract (g)  

 

TSC (mg g) = ⁄ C×10
-3

×
V

W
 

Equation 2.4 

 



33 | P a g e  

 

2.3 Results and discussion 

2.3.1 The presence of alkaloids, terpenoids and saponins in soap plants 

 

The preliminary phytochemical screening results of 26 plant species are presented in Table 2.3. 

The results revealed that most of the alcoholic and aqueous extracts contained terpenoids and 

saponins. Saponins were detected in high concentrations in aqueous extracts (93.10% of plant 

extracts), followed by ethanol extracts (82.76% of plant extracts). Saponins are naturally occurring 

amphiphilic glycosides that contain a glycosidic linkage between a lipophilic non-polar aglycone 

and a hydrophobic polar sugar chain (glycone moieties). Therefore, the surfactant properties 

observed when saponins are agitated in water are attributed to the amphiphilic structure (El Aziz 

et al., 2019; Rai et al., 2021). According to Oleszek and Hamed (2010), saponins with one sugar 

chain (monodesmoside) have the best foaming properties, while those with two or three sugar 

chains (bidesmoside and tridesmoside) have decreased foaming abilities. Some saponins have no 

foaming properties in water solutions; however, they are still considered saponins due to their 

chemical structure (Oleszek and Hamed, 2010). Thus, this explains the high presence of saponins 

in aqueous extracts. Furthermore, the high concentration of saponins in the aqueous extracts 

validates the traditional use of 93.10% of the selected plants as soap substitutes. 

 

Methanol and acetone were the optimal solvents to extract alkaloids from 62.07% of plant extracts. 

Terpenoids were best extracted with ethanol (75.86% of plant extracts), followed by methanol 

(68.97% of plant extracts), and distilled water (65.52% of plant extracts). A review by Tiwari et 

al. (2011) highlights that methanol and distilled water are the best solvents for the extraction of 

terpenoids and saponins, while ethanol is recorded as a good solvent for extracting terpenoids and 

alkaloids. Acetone is a good extractant for phenols and flavanols; hence, terpenoids and saponins 

were not detected in most acetone plant extracts. The results observed in this study are comparable 

to those obtained by Truong et al. (2019), where alcoholic solvents and distilled water proved to 

be the best extraction solvents for saponins and terpenoids.  

 

A few previous studies conducted preliminary phytochemical screening for some of the plants 

investigated in this study. In a study by Kane et al. (2019), the ethanol extract of Artemisia afra 

contained terpenoids and alkaloids, while the aqueous extract displayed the presence of saponins. 



34 | P a g e  

 

These results corroborate those of the current study. The presence of saponins in ethanol and 

aqueous Carica papaya extracts and alkaloids in the ethanol extract is consistent with findings by 

Singh et al. (2018) and Nduche et al. (2019). The presence of alkaloids and saponins in Aloe ferox 

was reported by Wintola and Afolayan (2011). Choi et al. (2015) did a preliminary screening of 

Aloe maculata leaf and reported that the extract displayed the presence of terpenoids and saponins. 

These results are comparable with those in the current study. The presence of terpenoids and 

saponins in Crinum bulbispermum, Cyathula uncinulata, Deinbollia oblongifolia, Ilex mitis, 

Merwilla plumbea, Noltea africana, and Plectranthus ciliatus was previously reported by 

Mohlakoana and Moteetee (2021), and the results correlate with those in the current study. A 

previous study (Nahal et al., 2012) showed that Olea europaea does have saponins. 

 

2.3.2 Qualitative analysis: thin layer chromatography (TLC)  

 

The retention factor (Rf) values for 29 extracts are presented in Table 2.3. The vanillin-perchloric 

acid and 10% sulphuric acid reagent were used as visualisation reagents. The saponin compounds 

in the plant samples displayed similar mobilities. The extracts in this study were not hydrolysed, 

hence the similar mobilities. When the plates were sprayed with vanillin-perchloric acid reagents 

(Appendix C1 and C2), the Rf values ranged from 0.10 to 0.97, whereas when sprayed with 10% 

sulphuric acid (Appendix C3 and C4) the Rf values ranged from 0.06 to 0.98. The compounds 

found in most of the extracts were considered less polar since the Rf values were higher due to a 

wider migration distance. Hermannia cuneifolia displayed a total of six different bands. Out of the 

six bands, three were present on plates sprayed with vanillin-perchloric acid (Rf values = 0.23; 

0.40; 0.60), and three were on plates sprayed with 10% sulphuric acid (Rf values = 0.76; 0.85; 

0.95). Out of the six bands, three were present on plates sprayed with vanillin-perchloric acid (Rf 

values = 0.23; 0.40; 0.60) and three were on plates sprayed with 10% sulphuric acid (Rf values = 

0.76; 0.85; 0.95). Acalypha glabrata (bark), Aloe ferox (leaves), Calodendrum capense (leaves), 

and Sideroxylon inerme subsp. inerme (leaves) extracts displayed two bands on each plate (Table 

2.2).  

 

 



35 

 

Table 2.3: Preliminary phytochemical screening results and Rf values of 26 southern African soap plants.  

Plant sample  Extraction solvents Vanillin-

perchloric acid 

10% Sulphuric 

acid 
Acetone Ethanol Methanol Aqueous 

A T S A T S A T S A T S Rf values  

Acalypha glabrata (leaves) + - + + + + + + + - + + 0.76; 0.98 0.93 

Acalypha glabrata (bark) - - + - + ++ - + - - - + 0.35; 0.60 0.79; 0.95 

Albizia versicolor  + - - - + + + - - - + - 0.97 0.79 

Aloe ferox ++ + - - - ++ ++ ++ - ++ ++ ++ 0.73; 0.95 - 

Aloe maculata  - + - - ++ + ++ ++ - - ++ + - 0.88 

Aristaloe aristata - - - - - + - - - - + + 0.33 0.92 

Artemisia afra  + + - ++ + + - + - + ++ + 0.33; 0.91 0.79; 0.88 

Bauhinia bowkeri  ++ - - - + ++ - + - - + + 0.26 0.07; 0.82 

Calodendrum capense (leaves) + - - + + + - + - - - ++ 0.18; 0.51 0.14; 0.93 

Calodendrum capense (bark) - - - + - + + + - - - ++ 0.77; 0.90 0.84 

Carica papaya + - - - + ++ + - + - - + 0.19; 0.42 0.90 

Crinum bulbispermum - + + - - - + + - - - + 0.27 0.06; 0.69 

Cussonia paniculata  + + - - - + + + - - - + 0.10 0.95 

Cyathula uncinulata  - - - - + ++ - - + - + ++ 0.24 0.98 

Deinbollia oblongifolia   + - - - - ++ - + + - - + 0.82; 0.93 0.82 

Haemanthus albiflos  + - - + + + - + - + + ++ 0.79 0.95 

Hermannia cuneifolia  + - - - + ++ - + - + + + 0.23; 0.40; 0.60 0.76; 0.85; 0.95 

Ilex mitis - - - - + ++ + + + ++ + + 0.90 - 



36 | P a g e  

 

Plant sample  Extraction solvents Vanillin-

perchloric acid 

10% Sulphuric 

acid 
Acetone Ethanol Methanol Aqueous 

A T S A T S A T S A T S Rf values  

Ledebouria luteola  + - - - - ++ + - - ++ ++ + 0.84 0.70; 0.84 

Ledebouria zebrina - - - - ++ ++ - - - + - + 0.30 0.93 

Merwilla plumbea  - - - - + - ++ + ++ + + ++ 0.19 0.82 

Noltea africana  ++ - - - + ++ + ++ - ++ + ++ 0.35; 0.64 0.61 

Olea europaea - + ++ - + ++ ++ - ++ - - ++ 0.97 - 

Pelargonium peltatum  ++ - - - + + + - - - - ++ 0.92 0.79 

Plectranthus ciliatus  + - - - + + + + + - ++ + - 0.70 

Pouzolzia mixta  - + - - + - - + - ++ + - 0.91 0.52 

Ptaeroxylon obliquum (leaves) + - + - + + ++ ++ - - + ++ 0.94 0.85 

Ptaeroxylon obliquum (bark) + + - - + - + + - - + ++ 0.91 0.82 

Sideroxylon inerme subsp. 

inerme 

++ - - - + ++ + ++ + ++ ++ ++ 0.13; 0.73 0.73; 0.76 

Diosgenin (standard saponin)  ++  ++  ++  ++ 0.21; 0.61; 0.76 0.18; 0.79 

Key – A: alkaloids, T: terpenoids, S: saponins; highly present: ++ (as determined by the intensity of brown-reddish precipitate for the presence of alkaloids, reddish-

brown precipitate for the presence of terpenoids and formation of a foam layer greater than 1 cm for the presence of saponins), slightly present/present: +, not 

present: -



37 

 

Compounds separated from extracts of Aristaloe aristata (Rf value = 0.33), Artemisia afra (Rf 

value = 0.33), Calodendrum capense (leaves) (Rf value = 0.18), Carica papaya (Rf value = 0.19; 

0.42), Crinum bulbispermum (Rf values = 0.06; 0.27), Cussonia paniculata (Rf value = 0.10), 

Cyathula uncinulata (Rf value = 0.24), Hermannia cuneifolia (Rf values = 0.23; 0.40), Noltea 

africana (Rf value = 0.35) and Sideroxylon inerme subsp. inerme (Rf value = 0.13) were 

considered polar since they had lower Rf values. According to Oleszek et al. (2008), the number 

of sugars in an extract determines the polarity of the compounds, which can range widely.  

 

Diosgenin showed three violet-coloured bands on the vanillin-perchloric acid plate, with Rf values 

of 0.21, 0.61, and 0.76, respectively. Two bands with Rf values of 0.18 and 0.79 were observed 

on a TL