Antibacterial, antioxidant activities and cytotoxicity of plants against Propionibacterium acnes

The use of plants to treat skin ailments has strong support in the current trend of drug discovery. Propionibacterium acnes, an anaerobic pathogen, plays an important role in the occurrence of acne. The present study was conducted to evaluate the antimicrobial and antioxidant activities against P. acnes and cytotoxic effects of 48 medicinal plants grown in South Africa. The broth dilution and DPPH radical scavenging methods were used to determine antibacterial and antioxidant activities, respectively. Cytotoxicity was determined on mouse melanocytes (B16-F10). The ethanolic bark extract of Acacia galpinii Burtt Davy. (Leguminosae) exhibited the lowest minimum inhibitory concentration of 62.5 μg/mL. Excellent antioxidant activity was shown by Aspalathus linearis (Burm.f.) R.Dahlgren (Leguminosae), Combretum apiculatum Sond. (Combretaceae), Harpephyllum caffrum Bernh. ex Krauss (Anacardiaceae) and Sclerocarya birrea Hochst. (Anacardiaceae), with 50% radical scavenging activity (EC50) at concentrations ranging from 1.6 μg/mL to 3.5 μg/mL. Greyia sutherlandii Hook. & Harv. (Greyiaceae) also exhibited good antioxidant activity with an EC50 value of 7.9±0.23 μg/mL. A. linearis, G. sutherlandii and S. birrea showed low toxicity with 50% viability of cells (EC50) at concentrations of 125.09±0.71 μg/mL, 107.85±1.53 μg/mL and 92.07±0.09 μg/mL, respectively. The extracts of A. linearis, G. sutherlandii and S. birrea showed good antibacterial and antioxidant activities and low toxicity. Therefore, these plants can be considered as possible anti-acne agents and warrant further investigation.


Introduction
Acne, one of the most common disorders of the skin, is a polymorphic disease with non-inflammatory (blackhead or whitehead) and inflammatory (papules, pustules, or nodules) aspects and a wide spectrum of severity.Acne can have a significant impact on the psychosocial and physical aspects of life.It affects up to 85% of adolescents to some extent but is less common among infants.[3][4] Propionibacterium acnes, a Gram-positive anaerobic bacterium, is a normal component of the microbiota of human skin.P. acnes causes an increase in the secretion of sebum from sebaceous glands, which is accompanied by the thickening of the epidermis at the outlet to the pilosebaceous follicles.As a result, there is an obstruction to the flow of sebum outwards, and a comedone develops.Colonisation of the follicles with P. acnes and the host's inflammatory response play a pivotal role in the development of typical inflammatory papulopustular lesions. 5In an anaerobic environment, the bacteria secretes nucleases, nuraminidases, hyaluronidases, acid phosphatises, lecithinases and other lipases.As a result of the action of these enzymes, the sebum content changes and reactive oxygen species may be released from the damaged follicular walls.Reactive oxygen species may also be the reason for the progression of inflammation in the pathogenesis of disease. 6nventional drugs commonly used in acne treatment -such as tetracycline, erythromycin, mynocycline and metronidazole -act as antioxidants and antibacterials.Benzoyl peroxide, a topical agent for the treatment of acne, shows the ability to induce an inflammatory reaction mediated by reactive oxygen species in addition to its antibacterial activity. 6These drugs also have various known side effects.The topical antibiotics can lead to dryness, redness and irritation of the skin, as well as hypopigmentation while oral antibiotics have age restrictions, can cause gastrointestinal disorders and increase the risk of venous thromboembolism. 5rbal medicines are an important part of African tradition and also have very deep roots in the treatment of dermatological ailments.Ethnobotanical studies have documented the use of plants by traditional healers for the treatment of various skin ailments. 7Different plant parts commonly used as cosmetics or face masks, known as umemezis, are widely used in southern Africa for skin problems like inflammation, wounds, burns, eczema and puberty acne. 8cause many skin disorders like atopic dermatitis and acne are associated with inflammation and the release of free radicals, which lead to oxidative and cellular damage and bacterial infections such as P. acnes, the presence of antioxidant and antimicrobial agents can explain the effectiveness of plants in the treatment of skin infections.In order to develop the therapeutic and drug potential of these plants, it is important to know whether they have any cytotoxic effects.Therefore, ethanol extracts of selected plants were evaluated for their antibacterial and antioxidant activities and cytotoxicity.
Limitations in the usage of some drugs and the prevailing side effects of the various chemically derived compounds have led to the search for alternative herbal agents to treat acne.The aim of this study was to test the effect of selected plant extracts on the pathogenic bacteria P. acnes, and to identify which plant extracts could be considered as possible anti-acne agents.

Preparation of plant extracts
Different plant parts (leaves, roots, bark and twigs) were collected from the Botanical Garden of the University of Pretoria (Pretoria, South Africa).The plants were identified by a taxonomist, Professor A.E. (Braam) van Wyk, at the H.G.W.J. Schweickerdt Herbarium of the University of Pretoria.The shade-dried plant material (80 g) was ground with a mechanical grinder, then soaked in 300 mL of ethanol and left on a shaker for 3 days.The plant material was then filtered and the solvent was evaporated under vacuum (Buchi Rotavapor, Labotech, Switzerland) to yield dry extracts.The plants were selected based on their medicinal usage as summarised in Table 1.

Antibacterial bioassay
The minimal inhibitory concentration (MIC) of the ethanolic extracts of the 48 selected plants was determined by a microdilution assay.This assay was done using the method described by Mapunya et al. 36 , with slight modifications.For this purpose, P. acnes (ATCC 11827) was cultured from a Kwik-Stick on nutrient agar and incubated at 37 °C for 72 h under anaerobic conditions.The ethanolic extracts were dissolved in 10% dimethyl sulphoxide (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; 100 µL of the plant stock samples and positive control were added to the first row of wells in triplicate.Twofold serial dilutions were made in broth over a range to give concentrations of 3.9-500 µg/mL and 0.3-50 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.Then 100 µL of this bacterial inoculum with 10 5 -10 6 CFU/mL was 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 (defined as the lowest concentration that showed no bacterial growth) was determined by observing the colour change in the wells after the addition of p-iodonitrotetrazolium salt.

Antioxidant assay
The antioxidant activity of selected plant extracts was investigated using the DPPH radical scavenging method as previously described by Du Toit et al. 37 , with slight modifications.DPPH is a free radical, which is stable at room temperature and produces a violet solution in ethanol.When reduced in the presence of an antioxidant molecule, it gives rise to a colourless solution.DPPH was dissolved in ethanol to obtain a solution of 0.04% w/v.The selected plant samples and the positive control (vitamin C) stock solutions (2 mg/mL) were serially diluted to final concentrations ranging from 0.78 µg/mL to 100 µg/mL.Ethanol and DPPH without any plant material were used as blanks while plant samples diluted in distilled water were used as controls.DPPH solution (90 µg/mL) was then added to all the wells except for the controls and allowed to react at room temperature.After 30 min, the absorbance values were measured at 515 nm using a Biotek Power-wave XS multiwell reader (A.D.P., Johannesburg, South Africa).The values were converted into the percentage antioxidant activity (AA) using the formula given below.The 50% inhibitory concentration (EC 50 ) values were then calculated by linear regression of the plots using GraphPad Prism version 4. AA% = {Abs blank (Abs sample -Abs control ) / Abs blank }*100

Mouse melanocyte cytotoxicity assay
The cytotoxicity of selected plant extracts was determined following a previously described method. 36Briefly, mouse melanocyte (B16-F10) cells were plated in complete Roswell Park Memorial Institute medium (10% foetal bovine serum and 1% gentamycin) directly in the wells of a 96-well plate (10 5 cells per well).After an overnight incubation at 37 °C in 5% CO 2 and a humidified atmosphere, extract samples and the positive control (actinomycin D) were added to the cells to give the final concentrations of plant extract and positive control of 3.13-400 µg/mL and 0.03x10 -2 -0.05 µg/mL, respectively.Plates were incubated at 37 °C in 5% CO 2 in a humidified atmosphere for 3 days.The toxicity effects of the extracts on the B16-F10 cells were assayed using the sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro ben zene sulphonic acid hydrate (XTT) cytotoxicity assay.Thereafter, 50 µL of XTT reagent (1 mg/mL XTT with 0.383 mg/ mL penazine methosulphate) was added to the wells and incubated for 1 h.The optical densities of the wells were measured at 450 nm with background subtraction at 690 nm.Cell survival was assessed by comparison with the controls (medium with DMSO).The EC 50 value, which represents the concentration of plant extract that causes death in 50% of the cells, was analysed using GraphPad Prism version 4.

Statistical analysis
All the assays were performed in triplicate with three independent studies for each assay.EC 50 values for antioxidant and cytotoxicity tests were derived from a non-linear regression model (curve fit) based on a sigmoidal dose response curve (variable) and computed using GraphPad Prism version 4 (GraphPad, San Diego, CA, USA).

Antibacterial activity of ethanolic extracts
The antibacterial activity of the selected plants against P. acnes is summarised in Table 2.After the addition of p-iodonitrotetrazolium salt, the MIC value of the positive drug control (tetracycline) was determined to be 3. Chiov.(Cancellaceae) -exhibited MIC values of 125 µg/mL.Another 28 extracts inhibited the growth of bacteria at MIC values ranging from 250 µg/mL to 500 µg/mL, whereas the remaining 10 extracts did not show any antibacterial activity, even at the highest concentration (500 µg/mL) tested.A threshold MIC value of ~100 µg/mL is suggested for rating plant extracts as having significant antimicrobial activity. 38herefore, the plant extracts exhibiting MIC values ranging from 62.5 µg/ mL to 125 µg/mL were selected for the evaluation of antioxidant activity.
Treatment of dermatological diseases and ophthalmic disorders 14 Arbutus unedo L.
Treatment of diarrhoea 22 Dahlia imperialis Roezl Treatment of skin ailments like rashes, grazes, infected scratches 23 Datura stramonium L.
Treatment of body pains, elephantiasis, sores and skin ailments, toothache 7,12 Diospyros lycioides Desf.Chewed and used as a toothbrush, and to ease body pains 7 Dodonaea viscosa Jacq.
As chewing sticks for toothache, headaches; as a purgative; bark infusion is used to enhance appetite 7 Euclea natalensis A.DC. Treatment of bronchitis, chronic asthmas, pleurisy, toothache, urinary tract infections 7 Galenia africana L. Treatment of asthma, coughs, skin diseases, eye inflammation, venereal sores, wounds 24 Gomphocarpus fruticosus R.Br.Treatment of headache and tuberculosis and to relieve stomach pain and general aches in the body 7,12 Greyia flanaganii Bolus.

Cytotoxicity of selected extracts
Cytotoxicity was assessed on the plant extracts which demonstrated EC 50 values of ≤10 µg/mL for radical scavenging activity.The plant extracts of A. linearis, G. sutherlandii and S. birrea showed low toxicity with 50% viability of cells (EC 50 ) at concentrations of 125.09±0.71µg/mL, 107.85±1.53µg/mL and 92.07±0.09µg/mL, respectively (Figure 2).During a previous study by our research group, the leaf extract of H. caffrum showed toxicity to B16-F10 cells at a concentration of 100 µg/mL. 39The plant extract of C. molle showed moderate toxicity with an EC 50 value of 48.83±0.21µg/mL, whereas C. apiculatum was found to be the most toxic with an EC 50 value of 12.15±0.03µg/mL and was found to be lethal to almost all cells at the highest concentration of 400 µg/mL.Actinomycin D, the positive control, showed an EC 50 value of 4.5x10 -3 ± 0.5x10 -3 µg/mL (Figure 2).
(Eucommiaceae) and Ilex paraguariensis A. St.-Hil.(Aquifoliaceae) inhibited the growth of P. acnes at MIC values of 2000 µg/mL, 500 µg/mL and 1000 µg/mL, respectively.To the best of our knowledge, the present study is the first scientific report of the antibacterial activity of all the selected plants against P. acnes.However, some of the plants used in this study have been previously reported to be active against other pathogens.In another study, leaf extracts of A. linearis showed zones of inhibition against Bacillus cereus, Micrococcus luteus and Candida albicans of 7.0 mm, 6.4 mm and 8.5 mm, respectively. 42e antibacterial activity of C. apiculatum against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was reported by Serage 43 .The acetone extract of the stem bark of C. molle showed antimicrobial activity against E. coli and Shigella spp. at an MIC of 50 000 µg/mL.The extract also showed inhibitory effects on the fungus C. albicans with complete inhibition at a concentration of 400 µg/mL. 44In a study done by Lining et al. 45 , the crude methanolic extract of Diospyros lycioides Desf.(Ebenaceae) showed activity against Streptococcus mutans and Prevotella intermedia at an MIC of 1250 µg/mL.In contrast, our results showed no activity of the ethanolic extract of D. lycioides against P. acnes.In another study conducted by Mativandlela et al. 46 , the ethanolic extract of G. africana showed antimycobacterial activity against Mycobacterium tuberculosis and Mycobacterium smegmatis at MIC values of 780 µg/mL and 1200 µg/mL, respectively.The ethanolic extract of H. caffrum was reported to be active against four bacterial species, namely Bacillus subtilis, E. coli, Klebsiella pneumoniae and S. aureus, while an aqueous extract showed activity against C. albicans. 47he acetone extracts of the bark and leaves of S. birrea were reported to be active against S. aureus, P. aeruginosa, E. coli and Enterococcus faecalis at MIC values ranging from 150 µg/mL to 3000 µg/mL. 48In a study done by Motsei et al. 49 , the leaf extracts of W. salutaris inhibited growth of C. albicans at MIC values ranging from 12 500 µg/mL to 25 000 µg/mL and the bark extracts showed growth of inhibition against S. aureus, Staphylococcus epidermis, B. subtilis and E. coli. 50o reports regarding the antimicrobial activity of G. sutherlandii and R. repens were found in the literature.However, in the present study, both of these plants showed growth inhibitory activity against P. acnes at an MIC of 125 µg/mL.In a study conducted by Eloff and Katerere 51 , the acetone and chloroform leaf extracts of A. galpinii inhibited the growth of S. aureus and E. coli.Similar to our findings, the ethanol bark extract of  Acne is associated with the production of free radicals along with the infection of P. acnes.Reactive oxygen species are produced as a result of the action of hydrolytic enzymes released from bacteria on the follicular walls of pilosebaceous units.Therefore, the plant extracts were evaluated for antioxidant activity along with antibacterial activity.In our study, the ethanol extracts of A. linearis, C. apiculatum, H. caffrum, S. birrea, C. molle and G. sutherlandii exhibited significant antioxidant activity with EC 50 values of ≤10 µg/mL.Our results are in agreement with other researchers.During a previous study by Joubert et al. 52 , the DPPH radical scavenging activity of A. linearis and its constituents were confirmed.The polar fractions of C. apiculatum showed antioxidant activity with an EC 50 value of 3.91 µg/mL. 53The DPPH radical scavenging activity of H. caffrum and S. birrea was confirmed by Moyo et al. 54 with EC 50 values of 6.8 µg/mL and 5.02 µg/mL, respectively.In another study, acetone and dichloromethane extracts of C. molle displayed antioxidant activity after spraying with DPPH. 55It has been reported that DPPH free radicals abstract the phenolic hydrogen of the electron-donating molecule, which could be the general mechanism for the scavenging action of flavonoids. 56Based on the mechanism of reduction of the DPPH molecule that is correlated with the presence of hydroxyl groups on the antioxidant molecule, the antioxidant activity of the polar plant extracts in the present study can be explained as a result of the presence of their phytoconstituents (phenolics or flavonoids) which are radical scavengers with an available hydroxyl group and are known to occur abundantly in plant species.
In order to evaluate the therapeutic potential of the plants, the cytotoxicity of selected samples was tested on B16-F10 cells.To the best of our knowledge, the cytotoxicity of the extracts described in the present study is reported for the first time.However, previous researchers have documented similar cytotoxic effects on different cell lines.In a study by McGaw et al. 57 , A. linearis showed low toxicity on vero cells and brine shrimp larvae with LD 50 values of >1000 µg/mL.S. birrea showed low cytotoxicity on vero cells with an IC 50 value of 361.24 µg/mL. 58According to previous studies by Fyhrquist et al. 59 on the cytotoxicity of C. molle, the extract showed IC 50 values of 27.7 µg/mL, 72.6 µg/mL and 42.6 µg/ mL on T24 (bladder carcinoma), HeLa (cervical carcinoma) and MCF-7 (breast carcinoma) cells, respectively, while the C. apiculatum extract showed IC 50 values of 65.0 µg/mL and 40.1 µg/mL for T24 and MCF-7 cells, respectively.No records of cell cytotoxicity for G. sutherlandii were found in the literature.
The results shown in this study prove the capability of medicinal plants as anti-acne agents, although the mode of action and in vivo studies are required to give conclusive results.

Conclusions
Based on the results obtained, it can be concluded that the ethanol bark extract of A. galpinii demonstrated the best activity against P. acnes with acceptable antioxidant activity.This plant might have other attributes that were not investigated in the present study which could be useful in the treatment of P. acnes.Although the plant extracts of H. caffrum, C. apiculatum and C. molle showed good antibacterial and excellent antioxidant activity, these samples also showed moderate toxicity to mouse melanocyte cells.The plant extracts of A. linearis, S. birrea and G. sutherlandii also exhibited good antibacterial and antioxidant activity but had low toxicity to the mouse melanocytes; these extracts therefore have potential as anti-acne agents, either alone or in combination.

Table 2 :
Minimum inhibitory concentrations (MICs) for antibacterial activity of extracts against Propionibacterium acnes determined by microdilution assay †Na, not active at the highest concentration (500 µg/mL) tested.
. galpinii exhibited good inhibitory effect on P. acnes (MIC 62.5 µg/mL).However, no reports on the antimicrobial activity of bark extracts of A. galpinii were found in the literature.