The potential of Leucosidea sericea against Propionibacterium acnes

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Possible potential of plants for treating acne

The plant kingdom is known to contain many novel biologically active compounds, many of which could potentially have a higher medicinal value when compared to some of the current medications. So there arises a need for search of new effective bioactive compounds to overcome this. A well-known plant extract studied for acne treatment, Tea tree oil or Melaleuca oil, originating from the Australian medicinal plant Melaleuca alternifolia, has been used in a clinical trial study to determine its effectiveness against acne (Carson et al., 2006). A crude drug extract called Kushen that is made from the fried roots of Sophora flavescens (Leguminosae) contained prenylflavanone derivatives were shown to have antibacterial activity against P. acne (Kuroyanagi et al., 1995). During previous studies Hemidesmus indicus, Eclipta alba, Cucubito pepo, Euphorbia hirta showed MIC value of 0.05, 0.66, 1.25 and 1.55 mg/ml, respectively (Kumar et al., 2007). Rhinacanthins-rich Rhinacanthus nasutus extract exhibited potent bacterioststic activity against P. acnes with MIC value of 8-16 μg/ml (Puttarak et al., 2010). The antibacterial activity of pomegranate rind extract containing 13% w/w ellagic acid exhibited a bacteriostatic activity against P. acnes at MIC of 15.6 μg/ml (Panichayupakaranant et al., 2010). Methanolic extracts of Rosa damascene, Eucommia ulmoides and Ilex paraguariensis were found to inhibit the growth of P. acnes with MICs of 2, 0.5 and 1 mg/ml, respectively (Tsai et al., 2010).
South Africa has remarkable biodiversity and a rich, extant herbal medicine tradition with origins that probably reach back to Paleolithic times. It is estimated that there are at least 2,00,000 indigenous healers in South Africa. Medicinal plants are widely used in traditional therapeutics, and it is likely that at least 2500 species of plant are commonly used as medicines. A South African pharmaceutical company, Noristan Ltd, investigated South African medicinal plants over a period of almost 20 years. Noristan found that 80% of the local medicinal plants that they had tested exhibited pharmacological There are many South African plants which are used in herbal cosmetics. Rooibos (Aspalathus linearis) is rich in flavanoids, polyphenols, phenolic acids, oligosaccharides and polysaccharides (Dos et al., 2005). Rooibos proved to exhibit anti-inflammatory and anti-microbial properties and is used for cosmetic applications. Artemisia herba-alba is also popular for skin ailments. A poultice of leaf is applied to any glandular or skin inflammation (Dweck, 1995). The leaves and roots of Aloe ferox are applied topically, sometimes mixed with animal fat, or taken internally to treat conditions such as eczema, dermatitis and acne (Van Wyk et al., 1997).
Therefore, there is a wide scope to obtain new drugs from plants that can have potential as antibacterial, anti-inflammatory and antioxidant agents.

Chemicals, microbial strain and culture media 

Tetracycline, vitamin C, p-iodonitrotetrazolium salt and DPPH (2,2-diphenyl-1-picrylhydrazyl) were obtained from Sigma-Aldrich (Kempton Park, South Africa). Nutrient agar, nutrient broth, Anaerocult A and Gram stain were obtained from Merck SA (Pty) Ltd. P. acnes (ATCC 11827) was purchased from Anatech Company South Africa. The cell culture reagents and the equipment were purchased from Highveld Biological (Sandringham, South Africa), Labotech (Midrand, South Africa) and The Scientific Group (Midrand, South Africa). The B16-F10 mouse melanoma cell line was obtained from Highveld Biological (Sandringham, South Africa).

Gram stain for the determination of the identity of bacteria

The Gram stain test was performed for the identification of bacteria in pure cultures. A smear of P. acnes culture suspended in nutrient broth was heat fixed on a glass slide. Crystal violet was used as primary stain. Iodine resublime solution was subsequently added as mordant. A mixture of acetone and ethyl alcohol were then added as a decolourant. Finally, a counterstain Safranine O was applied to the smear (Gerhardt et al., 1981).
The cell wall structure of microorganism determines the ability to be stained differentially by Gram’s method. The cell wall of Gram positive bacteria possess a higher peptidoglycan and lower lipid content, in contrast, high lipids and a thinner peptidoglycan layer is found in Gram negative bacteria. Therefore, the purple colour of crystal violet is retained by Gram positive bacteria whereas the same is washed by alcohol in Gram negative bacteria which appears red due to the counter stain (Gerhardt et al., 1981).

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Determination of antibacterial activity

The ethanol extract of the plant samples were tested against P. acnes by determining the minimum inhibitory concentration (MIC) values obtained by a microdilution method. This assay was done using the methods as described by Mapunya et al. (2011), with slight modifications. For this purpose, P. acnes were cultured as explained in section 4.2.4.1. The ethanolic extracts were dissolved in 10% DMSO to obtain a stock solution of 2 mg/ml. The positive control (tetracycline) was dissolved in sterile distilled water to obtain a stock solution of 0.2 mg/ml. The 96 well plates were prepared by dispensing 100 μl of the nutrient broth into each well. Hundred micro litres (100 μl) of the plant stock samples and positive control were added to the first row of wells in triplicates. Twofold serial dilutions were made in broth over a range to give concentrations of 500 to 3.9 μg/ml and 50 to 0.3 μg/ml for the plant extracts and positive control, respectively. The 72 h culture of bacteria was dissolved in nutrient broth and the suspensions were adjusted to 0.5 McFarland standard turbidity at 550 nm. About 100 μl of this bacterial inoculum with 105—106 CFU/ml was then added to all the wells. The wells with 2.5% DMSO and bacterial suspension without samples served as the solvent and negative controls, respectively. The plates were then incubated at 37°C for 72 h under anaerobic conditions. The MIC value was determined by observing the colour change in the wells after the addition of p-iodonitrotetrazolium salt (INT) (defined as the lowest concentration that showed no bacterial growth).

Chapter 1 General Introduction
1.1 Background and motivation of the study
1.2 Objectives of the study
1.3 Structure of thesis
1.4 References
Chapter 2 Acne: a review on epidemiology, pathogenesis and treatment options
2.1 Introduction
2.2 Cosmeceuticals
2.3 Skin and acne
2.4 Epidemiology
2.5 Propionibacterium acnes, the causative agent
2.6 Pathogenesis of Propionibacterium acnes
2.7 Immunology of acne
2.8 Conventional drugs available for treating acne
2.9 Possible potential of plants for treating acne
2.10 References
Chapter 3 Plants selected for the present study
3.1 Introduction
3.2 Description of selected plants
3.3 References
Chapter 4 Antibacterial, antioxidant activities and cytotoxicity of plants against Propionibacterium acnes
4.1 Introduction
4.2 Materials and methods
4.3 Results and discussions
4.4 Conclusion
4.5 References
Chapter 5 Antibacterial and anti-inflammatory effects of Syzygium jambos (L.) Alston and isolated compounds on acne vulgaris
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussions
5.4 Conclusion
5.5 References
Chapter 6 The potential of Leucosidea sericea against Propionibacterium acnes
6.1 Introduction
6.2 Materials and methods
6.3 Results and discussions
6.4 Conclusion
6.5 References
Chapter 7 Synergistic activity of Syzygium jambos and Leucosidea sericea
7.1 Introduction
7.2 Materials and methods
7.3 Results and discussions
7.4 Conclusion
7.5 References
Chapter 8 Conclusions
Chapter 9 Appendix
Appendix A
9.1 1H-NMR and 13C-NMR spectra of isolated compounds from Syzygium jambos
9.2 1H-NMR and 13C-NMR spectra of isolated compounds from Leucosidea sericea
Appendix B 
9.3 Efficacy of Syzygium jambos and Leucosidea sericea in clinical studies

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