- Email: [email protected]
Antibacterial and antioxidant strategies for acne treatment through plant extracts
Accepted Manuscript Antibacterial and antioxidant strategies for acne treatment through plant extracts Jaykant Vora, Anshu Srivastava, Hashmukh Modi PII:
S2352-9148(17)30201-0
DOI:
10.1016/j.imu.2017.10.005
Reference:
IMU 70
To appear in:
Informatics in Medicine Unlocked
Received Date: 26 June 2017 Revised Date:
10 October 2017
Accepted Date: 18 October 2017
Please cite this article as: Vora J, Srivastava A, Modi H, Antibacterial and antioxidant strategies for acne treatment through plant extracts, Informatics in Medicine Unlocked (2017), doi: 10.1016/ j.imu.2017.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibacterial and Antioxidant Strategies for Acne Treatment through Plant Extracts Jaykant Voraa, Anshu Srivastavab , Hashmukh Modia*
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b
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Department of Life Science, School of Science, Gujarat University, Navrangpura, Ahmedabad-380009, Gujarat, India
B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Ahmedabad, Gujarat, India
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*Address for Correspondence Email: [email protected], [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Acne vulgaris is a chronic skin infection affecting the majority of Indian population. There are several internal and external factors responsible for this infection. The present study emphasizes on screening of medicinal plant extracts against acne causing bacteria and antioxidant activity of these plant extracts. Anti-bacterial activities of methanolic extracts of Rosmarinus officinalis, Pelargonium asperum, Ocimum tenuiflorum, Acacia nilotica, Azadirachta indica, Matricaria chamomilla and Calendula officinalis was carried out using agar well diffusion method against Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 2079), Kocuria sp. (NCBI: Kocuria KM24375), Bacillus subtilis (MTCC 736) and Propionibacterium acnes (ATCC 11827). The results revealed that different plant extracts showed noticeable activity against different test organisms. The plant extracts of R. officinalis, M. Chamomilla and A. nilotica showed significant activity against P. acnes with diameter of 8 mm, 6 mm and 4 mm inhibition zone respectively. The minimum inhibition concentration (MIC) was measured for these three potential plant extracts. The MIC values of extracts of R. officinalis, M. Chamomilla were comparable to standard reference drugs isotretinoin. Since antioxidants also play an important role in the pathophysiology of acne, anti-oxidant activity of plant extracts was also carried out using 1, 1-diphenyl-2-picryl hydrazyl (DPPH) method. Ascorbic acid was used as a standard in anti-oxidant activity assay. The extracts of R. officinalis, A. nilotica and A. indica also exhibited moderate antioxidant activity with IC50 value of 109.3, 136.9 and 108 µg/ml, respectively. The outcome of the study suggested that R. officinalis, M. chamomilla and A. nilotica plant extracts could be possible to use as the natural anti-acne formulations.
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Keywords: Acne, Antibacterial, Antioxidant, DPPH, MIC, Plant extracts, Propionibacterium acnes
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ACCEPTED MANUSCRIPT 1. Introduction Acne is one of the most common and chronic skin problem in most adolescents and young adults [1]. Pathophysiology of acne is attributed to different notable factors such as androgenmediated stimulation of sebaceous gland activity, follicular hyperkeratinization, hormonal imbalance, inflammation and external bacterial infection. Propionibacterium acnes and
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Staphylococcus epidermidis are the major bacteria found on skin causes acne [2, 3]. A number of topical and systematic therapies are available for acne; various antibiotics, comedolytic agents, and anti-inflammatory drugs are available as a topical therapy, whereas modern systematic cure includes antibiotics, hormones, zinc and laser treatment. Still, anti-
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acne drugs are being primarily used in the treatment for more than 40 years. Various topical and oral drugs are available in the market like Clindamycin, Salicylic acid, Isotretinoin, Erythromycin, Triclosan, Tetracycline, Minocycline, and Metronidazole for treatment of
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acne. However, an excessive use of these drugs over a long time can lead to the rising resistance of bacteria. These drugs have limitations with respect to toxicity and side effects also such as skin drying, headache, nausea etc. To overcome these limitations, there is an imperative need for the development of effective, safe and low-cost anti-acne drugs. Exploration of herbal resources may provide valuable leads that can be further developed as
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anti-acne drugs [4]. There has been reported that natural substances derived from plants are used for various types of acne treatments. Therefore, in this study, mostly aromatic plants were selected on the basis of their common antibacterial properties but not particularly for acne causing bacterial strain using literature survey.
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The skin bacteria such as P. acnes (Gram-positive, anaerobic bacterium) and S. epidermidis are common pus forming bacteria that produce inflammation in acne. P. acnes is involved in
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the development of inflammatory acne by activating complements and metabolizing sebaceous triglycerides into fatty acids that irritate the follicular wall and surrounding dermis [5].
It is known that oxidative stress is responsible directly or indirectly for causing many diseases including acne. Oxidative stress is initiated by reactive oxygen species (ROS). In acne, sebum is produced by damaged follicular walls of sebaceous glands, which contains ROS, namely hydroxyl, superoxide and nitrous oxide. These free radicals are responsible for the occurrence of irritation during the acne infection [6, 7]. The synthetic antioxidant compounds have an
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ACCEPTED MANUSCRIPT adverse side effect. Herbal sources are the substitute antioxidants which are beneficial in facilitating the repair of the damage caused by these free radicals [8]. There is an increased interest among the researchers to discover and develop a natural therapy for various diseases. The present investigation highlights the potential plant extracts that can
drug resistivity and side effects of current acne therapy. 2. Materials and methods 2.1. Extraction of plant material
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be explored for the treatment of acne and development of futuristic drugs to overcome the
The powder of leaves of Rosmarinus officinalis, Pelargonium asperum, Ocimum tenuiflorum,
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Acacia nilotica, Azadirachta indica, Matricaria chamomilla and Calendula officinalis were procured from Vivvan Herbals and Healthcare, Ahmedabad. Powdered plant materials (20
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gm) were immersed in 200 ml methanol and shaken vigorously for 30 min. They were further left for 48 hr at 25°C. The extracts were filtered through Whatman 1 filter paper. The methanol was allowed to evaporate from the extracts at room temperature resulting in dried extracts and used for antibacterial as well as anti-oxidant assay [9]. 2.2. Assay for Antibacterial activity by agar well diffusion method
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The test organisms used in this study were procured from National Collection of Industrial Microorganisms (NCIM), Pune. Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 2079), Kocuria sp. (NCBI: Kocuria KM24375), Bacillus subtilis (MTCC 736) and Propionibacterium acnes (ATCC 11827) was
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used for the study. Nutrient broth and Nutrient Agar Medium (Hi-media) were used for bacterial growth and antibacterial assay. The 24 hours cultured fresh bacterial inoculums (100
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µl) were uniformly spread using the sterile cotton swab on a sterile nutrient agar plate. 50 µl methanolic plant extracts were added in agar well (7 mm) under aseptic conditions. For negative control, an equal volume of methanol (50 µl) was added in one well. All the plates were incubated for 24-36 hour at 37°C. The experiment performed in triplicate and results were recorded as the average zone of inhibition [10]. 2.3. Minimum Inhibitory Concentration (MIC) The lowest concentration of the extracts which inhibited the growth of tested bacteria was measured by the MIC. A broth micro-dilution bioassay in 96-well polystyrene micro titre plate was used to determine the MIC of potential extracts. The well of each column (1-12) 4
ACCEPTED MANUSCRIPT was filled with 50 µl of sterilized nutrient broth (except first well of each column). 50 µl of plant extract having a concentration of 10 mg/ml dissolved in DMSO (0.1 %) was added to the first well of columns 4-12 (each extracts obtained in triplicates). Serial twofold dilutions were made from first well of columns 4-12 in the 7 consecutive wells of the columns (50 µl). Subsequently, 50 µl mixtures from the last well of each column were discarded. The
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concentration of the extracts ranged from 5 to 0.0781 mg/ml. Subsequently, 50 µl sterilized nutrient broth was added in all the well of the plate. Next, 50 µl of the bacterial inocula (109 CFU/ml) was added to each well so that the final volume of each well was 150 µl. The first column of the plate served as a positive control. Anti-acne drug Isotretinoin (1mg/ml) was
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used as the antibiotic control and was serially diluted in same way. The second column of the plate served as a negative control having 50 µl DMSO, 50 µl bacterial inocula, and 50 µl nutrient broth. The third column of the plate served as a sterility control which has only
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sterile nutrient broth medium (150 µl). Later on, the plates were incubated at 37°C for 24 hours. After incubation period INT indicator dye was added to each well. These dye indicated bacterial growth with the red-pink color solution and the growth inhibition was indicated by no change of color [11]. 2.4. Assay for antioxidant activity
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Antioxidant activities of extracts were measured spectophotometrically by using 1, 1diphenyl 2-picryl hydrazyl (DPPH). 0.1 mM DPPH solution was prepared in methanol. 1 ml of DPPH stock solution was mixed with 1 ml of plant extract solution of different concentrations (50, 100, 150 and 200 µg/ml). The mixer of 1 ml methanol and 1 ml DPPH
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stock solution was used as a control. Ascorbic acid was used as the standard reference compound with the same concentration. The reactions were carried out in triplicate and
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allowed to stand at room temperature for 30 min. Reduction in the absorbance was measured by UV-Vis spectrophotometer at 517 nm. The inhibition percentage was calculated using the following formula [12].
DPPH scavenging effect (%) = Abs Control- Abs Sample/Abs Control X 100 IC 50 values were calculated for plant extract has potent antioxidant activity. 2.5. Statistical analysis The statistical significance of differences between the values of selected plant extract samples and controls were determined by using Two-way ANOVA with Dunnett’s post-hoc test using 5
ACCEPTED MANUSCRIPT Graphpad Prism version 5.00 for windows (GraphPad Software, San Diego, USA). In each case P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Antibacterial activities of extracts In the present study, methanolic extracts of seven plants were analyzed for antimicrobial
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activity against test bacteria. All extracts showed activity against P. acnes and the range of inhibition zone was between 1-8 mm. Among these, extracts of R. officinalis showed highest inhibition zone against P. acnes at 8 mm. M. chamomilla and A. nilotica showed moderate inhibition zone against P. acnes at 6 mm and 4 mm respectively. None of the plant extracts
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showed the inhibition zone against P. aeruginosa and E. coli. The value of the zone of inhibition by different extracts against test bacteria is shown in Table 1. The zone of
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inhibition of potential plant extracts against test pathogens is shown in figure 1.
Table 1. Average inhibition zones of selected plant extracts against test organisms Average diameter of zone of inhibition (mm)
O. tenuiflorum A. nilotica A. indica
8
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1
-
2
4
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M. chamomilla C. officinalis
E. coli S. aureus
Kocuria sp.
B. subtilis
15
15
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4
10
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4
10
-
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-
10
20
3
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6
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-
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3
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P. asperum
P. aeruginosa
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R. officinalis
P. acnes
6
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Figure 1: Zone of the inhibition of plant extracts against test organisms 1 & 2 indicate effects of plant extracts against P. acnes (2: Ch- Chamomile, T- Tulsi, R- Rosemary, G- Geranium) 3 indicates effects of plant extracts against Kocuria sp.4 & 5 indicates effects of plant extracts against B. subtilis
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ACCEPTED MANUSCRIPT The R. officinalis is a good source of iron, calcium and vitamin B-6. This herb was traditionally used to help alleviate muscle pain, improve memory, boost the immune and promote hair growth. But, in this study, we described the role of this plant extract to protect against several common skin bacteria and acne causing bacteria. Issabeagloo et al. (2012) studied the activity of methanolic extract of R. officinalis against S. aureus and found good
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inhibition at 98.5 µg/ml concentration [13]. The present study also showed the potential activity of R. officinalis against S. aureus at lower concentration along with other pathogenic strains viz. Kocuria sp. and P. acnes (Table 2).
A. nilotica is a plant which has multipurpose medicinal properties and used from an ancient
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time. In earlier study on A. Nilotica, methanolic extract showed its effectiveness against E. coli, S. aureus, P. aeruginosa, K. Pneumonia, and S. typhi [14]. However, the present study
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indicates that A. nilotica is not effective against E. coli and S. aureus but very efficient in inhibiting Kocuria sp., B.subtilis and P. acnes which are responsible for acne. M. chamomilla has effective compounds with cytotoxic effects and antiproliferative as well as anti-inflammatory properties. Many cosmetic products contain the extracts of this plant. Therefore, in the present study, we obtained the antibacterial activity of this plant extracts against acne causing bacteria. The extract of M. chamomilla revealed diameter of zone of
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inhibition as 6 mm against acne strain P. acnes (Table 1). The graphical representation of zone of inhibition of selected plant extracts against test
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organisms with statistical analysis is shown in figure 2.
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Figure 2: Graphical representations are indicated zone of inhibition of selected plant extracts against test organisms. A Effect of R. officinalis plant extract B Effect of P.asperum plant extract C Effect of O. tenuiflorum plant extract D Effect of A. nilotica plant extract E Effect of A. indica plant extract F Effect of M. chamomilla plant extract G Effect of C. officinalis plant extract. These results are representative of three independent experiment in triplicates with mean ± SEM. Statistical significance against respective control *P<0.05, **P<0.01; ***P<0.001. (*** denotes significant value) 9
ACCEPTED MANUSCRIPT 3.2. Minimum Inhibitory Concentration (MIC) of potential plant extracts The MIC of potential three plant extracts which gave anti-bacterial activity against acne causing bacteria were determined using broth micro dilution method. This assay was performed only for those extracts which gave a significant zone of inhibition against acne causing strain along with other bacteria causes skin infection. The MIC values of the R.
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officinalis, M. chamomilla and A. nilotica extracts against the tested bacteria are shown in table 2. The minimum inhibitory concentration values of R. officinalis was near to reference standard isotretinoin against S. aureus and Kocuria sp. as 0.0781
and 0.0625 mg/ml
respectively. The MIC value of standard anti-acne drug (Isotretinoin) was 0.0625 mg/ml
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against acne causing strain P. acnes. The result showed that R. officinalis and M. chamomilla gave significant MIC against P. acnes. So, these to plant extract would be used for anti-acne
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topical formulation.
Table 2. Minimum inhibitory concentration (MIC) values of potential plant extracts against test organisms
Minimum Inhibitory Concentration (mg/ml)
R. officinalis
B. subtilis
0.0781
0.0781
ND
0.312
ND
0.0781
0.0781
0.156
ND
ND
ND
0.0625
0.0625
0.0625
0.0625
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Isotretinoin
Kocuria sp.
0.156
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A. nilotica M. chamomilla
S. aureus
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P. acnes
ND means not detected
3.3. Antioxidant assay by DPPH-Free radical scavenging activity DPPH is a rapid and sensitive method to evaluate the antioxidant capacity of plant extracts. It is based on the decolourization of DPPH in the presence of antioxidants in the tested sample. The color change from purple to yellow shows the strong antioxidant capacity of the sample. The percentage values of antioxidant activity of selected plant extracts at different concentration 50-200 µg/ml were shown in figure 3. In this assay, ascorbic acid gave 10
ACCEPTED MANUSCRIPT antioxidant activity with IC50 value of 35.45µg/ml. The results of the present investigation indicated that the extracts of R. officinalis, A. nilotica and A. indica exhibited noteworthy
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antioxidant activity with the IC50 value of 109.3, 136.9 and 108 µg/ml, respectively.
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Figure 3: Percentage of antioxidant activity of plant extracts These results are representative of the antioxidant experiment in triplicates with mean ± SEM. Statistical significance against Ascorbic acid as a control *P<0.05, **P<0.01; ***P<0.001. (*** denotes significant value)
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4. Conclusion
The results of this study indicate that extracts of R. officinalis, M. Chamomilla and A. nilotica
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show antibacterial activity with less MIC against acne causing bacteria. Additionally R. officinalis and M. Chamomilla also showed good antioxidant activity along with A. indica extract. As these are the natural extracts, so they have no or less side effects. These can also be an alternative and better option for resistant acne causing bacteria. The topical anti-acne formulations can be developed using these plant extracts individually or in combination. 5. Acknowledgement All authors are thankful to the Head of the Department, Life Science Department, Gujarat University, Ahmedabad, Gujarat, for providing all the facilities for this research work.
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ACCEPTED MANUSCRIPT References Sinha P, Srivastava S, Mishra N, Yadav NP. New perspectives on anti acne plant drugs: contribution to modern therapeutics. Biomed Res Int 2014; 2014:301–304.
[2]
Coenye T, Peeters E, Nelis HJ. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Micro biol 2007; 158:386–392.
[3]
Williams HC, Dellavalle RP, Garner S. Acne vulgaris. Lancet 2017; 379:361–372.
[4]
Kumar A, Baboota S, Agarwal S, Ali J, Ahuja A. Treatment of acne with special emphasis on herbal remedies. Expert Rev Dermatol 2008; 3:111–122.
[5]
Webster GF. Acne vulgaris. BMJ Br Med J 2002; 325:475–479.
[6]
Briganti S, Picardo M. Antioxidant activity, lipid peroxidation and skin diseases. What’s new? J Eur Acad Dermatology Venereol 2003; 17:663–669.
[7]
Nand P, Drabu S, Gupta K R. Screening for antioxidant and antibacterial potential of common medicinal plants in the treatment of acne. Int Journal of Drug Dev Res 2012; 4:65–71.
[8]
Alonso AM, Guillen DA, Barroso CG, Puertas B, Garcia A. Determination of antioxidant activity of wine by products and its correlation with polyphenolic content. J Agric Food Chem 2002;50:5832–5836.
[9]
Das K, Tiwari RKS, Shrivastava DK. Techniques for evaluation of medicinal plant products as antimicrobial agent : Current methods and future trends. J Med Plants Res 2010; 4:104–111.
[10]
Smania A, Monache FD, Smania E de FA, Cuneo RS. Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum Pat. (Aphyllophoromycetideae) fruit body. Int J Med Mushrooms 1999; 1:325–330.
[11]
Shah P, Modi HA, Shukla MD, Lahiri SK. Preliminary phytochemical analysis and antibacterial activity of Ganoderma lucidum collected from dang district of gujarat , india. Int J Curr Microbiol App Sci 2014; 3:246–255.
[12]
Veeru P, Kishor MP, Meenakshi M, Linn P, Linn S. Screening of medicinal plant extracts for antioxidant activity. J Med Plants Res 2009; 3:608–612.
[13]
[14]
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[1]
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6.
Issabeagloo E, Kermanizadeh P, Taghizadieh M, Forughi R. Antimicrobial effects of rosemary (Rosmarinus officinalis L .) essential oils against Staphylococcus spp. African J Microbiol Res 2012;6:5039–5042. Srivastava M, Kumar G, Mohan R, S M. Phytochemical studies and antimicrobial activity of babool seeds. J Sci Ind Res 2014; 73:724–728.
12
S2352-9148(17)30201-0
DOI:
10.1016/j.imu.2017.10.005
Reference:
IMU 70
To appear in:
Informatics in Medicine Unlocked
Received Date: 26 June 2017 Revised Date:
10 October 2017
Accepted Date: 18 October 2017
Please cite this article as: Vora J, Srivastava A, Modi H, Antibacterial and antioxidant strategies for acne treatment through plant extracts, Informatics in Medicine Unlocked (2017), doi: 10.1016/ j.imu.2017.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Antibacterial and Antioxidant Strategies for Acne Treatment through Plant Extracts Jaykant Voraa, Anshu Srivastavab , Hashmukh Modia*
a
b
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Department of Life Science, School of Science, Gujarat University, Navrangpura, Ahmedabad-380009, Gujarat, India
B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Ahmedabad, Gujarat, India
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*Address for Correspondence Email: [email protected], [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Acne vulgaris is a chronic skin infection affecting the majority of Indian population. There are several internal and external factors responsible for this infection. The present study emphasizes on screening of medicinal plant extracts against acne causing bacteria and antioxidant activity of these plant extracts. Anti-bacterial activities of methanolic extracts of Rosmarinus officinalis, Pelargonium asperum, Ocimum tenuiflorum, Acacia nilotica, Azadirachta indica, Matricaria chamomilla and Calendula officinalis was carried out using agar well diffusion method against Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 2079), Kocuria sp. (NCBI: Kocuria KM24375), Bacillus subtilis (MTCC 736) and Propionibacterium acnes (ATCC 11827). The results revealed that different plant extracts showed noticeable activity against different test organisms. The plant extracts of R. officinalis, M. Chamomilla and A. nilotica showed significant activity against P. acnes with diameter of 8 mm, 6 mm and 4 mm inhibition zone respectively. The minimum inhibition concentration (MIC) was measured for these three potential plant extracts. The MIC values of extracts of R. officinalis, M. Chamomilla were comparable to standard reference drugs isotretinoin. Since antioxidants also play an important role in the pathophysiology of acne, anti-oxidant activity of plant extracts was also carried out using 1, 1-diphenyl-2-picryl hydrazyl (DPPH) method. Ascorbic acid was used as a standard in anti-oxidant activity assay. The extracts of R. officinalis, A. nilotica and A. indica also exhibited moderate antioxidant activity with IC50 value of 109.3, 136.9 and 108 µg/ml, respectively. The outcome of the study suggested that R. officinalis, M. chamomilla and A. nilotica plant extracts could be possible to use as the natural anti-acne formulations.
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Keywords: Acne, Antibacterial, Antioxidant, DPPH, MIC, Plant extracts, Propionibacterium acnes
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ACCEPTED MANUSCRIPT 1. Introduction Acne is one of the most common and chronic skin problem in most adolescents and young adults [1]. Pathophysiology of acne is attributed to different notable factors such as androgenmediated stimulation of sebaceous gland activity, follicular hyperkeratinization, hormonal imbalance, inflammation and external bacterial infection. Propionibacterium acnes and
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Staphylococcus epidermidis are the major bacteria found on skin causes acne [2, 3]. A number of topical and systematic therapies are available for acne; various antibiotics, comedolytic agents, and anti-inflammatory drugs are available as a topical therapy, whereas modern systematic cure includes antibiotics, hormones, zinc and laser treatment. Still, anti-
SC
acne drugs are being primarily used in the treatment for more than 40 years. Various topical and oral drugs are available in the market like Clindamycin, Salicylic acid, Isotretinoin, Erythromycin, Triclosan, Tetracycline, Minocycline, and Metronidazole for treatment of
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acne. However, an excessive use of these drugs over a long time can lead to the rising resistance of bacteria. These drugs have limitations with respect to toxicity and side effects also such as skin drying, headache, nausea etc. To overcome these limitations, there is an imperative need for the development of effective, safe and low-cost anti-acne drugs. Exploration of herbal resources may provide valuable leads that can be further developed as
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anti-acne drugs [4]. There has been reported that natural substances derived from plants are used for various types of acne treatments. Therefore, in this study, mostly aromatic plants were selected on the basis of their common antibacterial properties but not particularly for acne causing bacterial strain using literature survey.
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The skin bacteria such as P. acnes (Gram-positive, anaerobic bacterium) and S. epidermidis are common pus forming bacteria that produce inflammation in acne. P. acnes is involved in
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the development of inflammatory acne by activating complements and metabolizing sebaceous triglycerides into fatty acids that irritate the follicular wall and surrounding dermis [5].
It is known that oxidative stress is responsible directly or indirectly for causing many diseases including acne. Oxidative stress is initiated by reactive oxygen species (ROS). In acne, sebum is produced by damaged follicular walls of sebaceous glands, which contains ROS, namely hydroxyl, superoxide and nitrous oxide. These free radicals are responsible for the occurrence of irritation during the acne infection [6, 7]. The synthetic antioxidant compounds have an
3
ACCEPTED MANUSCRIPT adverse side effect. Herbal sources are the substitute antioxidants which are beneficial in facilitating the repair of the damage caused by these free radicals [8]. There is an increased interest among the researchers to discover and develop a natural therapy for various diseases. The present investigation highlights the potential plant extracts that can
drug resistivity and side effects of current acne therapy. 2. Materials and methods 2.1. Extraction of plant material
RI PT
be explored for the treatment of acne and development of futuristic drugs to overcome the
The powder of leaves of Rosmarinus officinalis, Pelargonium asperum, Ocimum tenuiflorum,
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Acacia nilotica, Azadirachta indica, Matricaria chamomilla and Calendula officinalis were procured from Vivvan Herbals and Healthcare, Ahmedabad. Powdered plant materials (20
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gm) were immersed in 200 ml methanol and shaken vigorously for 30 min. They were further left for 48 hr at 25°C. The extracts were filtered through Whatman 1 filter paper. The methanol was allowed to evaporate from the extracts at room temperature resulting in dried extracts and used for antibacterial as well as anti-oxidant assay [9]. 2.2. Assay for Antibacterial activity by agar well diffusion method
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The test organisms used in this study were procured from National Collection of Industrial Microorganisms (NCIM), Pune. Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 2079), Kocuria sp. (NCBI: Kocuria KM24375), Bacillus subtilis (MTCC 736) and Propionibacterium acnes (ATCC 11827) was
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used for the study. Nutrient broth and Nutrient Agar Medium (Hi-media) were used for bacterial growth and antibacterial assay. The 24 hours cultured fresh bacterial inoculums (100
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µl) were uniformly spread using the sterile cotton swab on a sterile nutrient agar plate. 50 µl methanolic plant extracts were added in agar well (7 mm) under aseptic conditions. For negative control, an equal volume of methanol (50 µl) was added in one well. All the plates were incubated for 24-36 hour at 37°C. The experiment performed in triplicate and results were recorded as the average zone of inhibition [10]. 2.3. Minimum Inhibitory Concentration (MIC) The lowest concentration of the extracts which inhibited the growth of tested bacteria was measured by the MIC. A broth micro-dilution bioassay in 96-well polystyrene micro titre plate was used to determine the MIC of potential extracts. The well of each column (1-12) 4
ACCEPTED MANUSCRIPT was filled with 50 µl of sterilized nutrient broth (except first well of each column). 50 µl of plant extract having a concentration of 10 mg/ml dissolved in DMSO (0.1 %) was added to the first well of columns 4-12 (each extracts obtained in triplicates). Serial twofold dilutions were made from first well of columns 4-12 in the 7 consecutive wells of the columns (50 µl). Subsequently, 50 µl mixtures from the last well of each column were discarded. The
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concentration of the extracts ranged from 5 to 0.0781 mg/ml. Subsequently, 50 µl sterilized nutrient broth was added in all the well of the plate. Next, 50 µl of the bacterial inocula (109 CFU/ml) was added to each well so that the final volume of each well was 150 µl. The first column of the plate served as a positive control. Anti-acne drug Isotretinoin (1mg/ml) was
SC
used as the antibiotic control and was serially diluted in same way. The second column of the plate served as a negative control having 50 µl DMSO, 50 µl bacterial inocula, and 50 µl nutrient broth. The third column of the plate served as a sterility control which has only
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sterile nutrient broth medium (150 µl). Later on, the plates were incubated at 37°C for 24 hours. After incubation period INT indicator dye was added to each well. These dye indicated bacterial growth with the red-pink color solution and the growth inhibition was indicated by no change of color [11]. 2.4. Assay for antioxidant activity
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Antioxidant activities of extracts were measured spectophotometrically by using 1, 1diphenyl 2-picryl hydrazyl (DPPH). 0.1 mM DPPH solution was prepared in methanol. 1 ml of DPPH stock solution was mixed with 1 ml of plant extract solution of different concentrations (50, 100, 150 and 200 µg/ml). The mixer of 1 ml methanol and 1 ml DPPH
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stock solution was used as a control. Ascorbic acid was used as the standard reference compound with the same concentration. The reactions were carried out in triplicate and
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allowed to stand at room temperature for 30 min. Reduction in the absorbance was measured by UV-Vis spectrophotometer at 517 nm. The inhibition percentage was calculated using the following formula [12].
DPPH scavenging effect (%) = Abs Control- Abs Sample/Abs Control X 100 IC 50 values were calculated for plant extract has potent antioxidant activity. 2.5. Statistical analysis The statistical significance of differences between the values of selected plant extract samples and controls were determined by using Two-way ANOVA with Dunnett’s post-hoc test using 5
ACCEPTED MANUSCRIPT Graphpad Prism version 5.00 for windows (GraphPad Software, San Diego, USA). In each case P < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Antibacterial activities of extracts In the present study, methanolic extracts of seven plants were analyzed for antimicrobial
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activity against test bacteria. All extracts showed activity against P. acnes and the range of inhibition zone was between 1-8 mm. Among these, extracts of R. officinalis showed highest inhibition zone against P. acnes at 8 mm. M. chamomilla and A. nilotica showed moderate inhibition zone against P. acnes at 6 mm and 4 mm respectively. None of the plant extracts
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showed the inhibition zone against P. aeruginosa and E. coli. The value of the zone of inhibition by different extracts against test bacteria is shown in Table 1. The zone of
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inhibition of potential plant extracts against test pathogens is shown in figure 1.
Table 1. Average inhibition zones of selected plant extracts against test organisms Average diameter of zone of inhibition (mm)
O. tenuiflorum A. nilotica A. indica
8
-
-
1
-
2
4
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M. chamomilla C. officinalis
E. coli S. aureus
Kocuria sp.
B. subtilis
15
15
-
-
-
4
10
-
-
-
4
10
-
-
-
10
20
3
-
-
-
-
-
6
-
-
-
-
-
3
-
-
-
-
-
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P. asperum
P. aeruginosa
EP
R. officinalis
P. acnes
6
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SC
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EP
TE D
Figure 1: Zone of the inhibition of plant extracts against test organisms 1 & 2 indicate effects of plant extracts against P. acnes (2: Ch- Chamomile, T- Tulsi, R- Rosemary, G- Geranium) 3 indicates effects of plant extracts against Kocuria sp.4 & 5 indicates effects of plant extracts against B. subtilis
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ACCEPTED MANUSCRIPT The R. officinalis is a good source of iron, calcium and vitamin B-6. This herb was traditionally used to help alleviate muscle pain, improve memory, boost the immune and promote hair growth. But, in this study, we described the role of this plant extract to protect against several common skin bacteria and acne causing bacteria. Issabeagloo et al. (2012) studied the activity of methanolic extract of R. officinalis against S. aureus and found good
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inhibition at 98.5 µg/ml concentration [13]. The present study also showed the potential activity of R. officinalis against S. aureus at lower concentration along with other pathogenic strains viz. Kocuria sp. and P. acnes (Table 2).
A. nilotica is a plant which has multipurpose medicinal properties and used from an ancient
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time. In earlier study on A. Nilotica, methanolic extract showed its effectiveness against E. coli, S. aureus, P. aeruginosa, K. Pneumonia, and S. typhi [14]. However, the present study
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indicates that A. nilotica is not effective against E. coli and S. aureus but very efficient in inhibiting Kocuria sp., B.subtilis and P. acnes which are responsible for acne. M. chamomilla has effective compounds with cytotoxic effects and antiproliferative as well as anti-inflammatory properties. Many cosmetic products contain the extracts of this plant. Therefore, in the present study, we obtained the antibacterial activity of this plant extracts against acne causing bacteria. The extract of M. chamomilla revealed diameter of zone of
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inhibition as 6 mm against acne strain P. acnes (Table 1). The graphical representation of zone of inhibition of selected plant extracts against test
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organisms with statistical analysis is shown in figure 2.
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EP
TE D
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SC
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Figure 2: Graphical representations are indicated zone of inhibition of selected plant extracts against test organisms. A Effect of R. officinalis plant extract B Effect of P.asperum plant extract C Effect of O. tenuiflorum plant extract D Effect of A. nilotica plant extract E Effect of A. indica plant extract F Effect of M. chamomilla plant extract G Effect of C. officinalis plant extract. These results are representative of three independent experiment in triplicates with mean ± SEM. Statistical significance against respective control *P<0.05, **P<0.01; ***P<0.001. (*** denotes significant value) 9
ACCEPTED MANUSCRIPT 3.2. Minimum Inhibitory Concentration (MIC) of potential plant extracts The MIC of potential three plant extracts which gave anti-bacterial activity against acne causing bacteria were determined using broth micro dilution method. This assay was performed only for those extracts which gave a significant zone of inhibition against acne causing strain along with other bacteria causes skin infection. The MIC values of the R.
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officinalis, M. chamomilla and A. nilotica extracts against the tested bacteria are shown in table 2. The minimum inhibitory concentration values of R. officinalis was near to reference standard isotretinoin against S. aureus and Kocuria sp. as 0.0781
and 0.0625 mg/ml
respectively. The MIC value of standard anti-acne drug (Isotretinoin) was 0.0625 mg/ml
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against acne causing strain P. acnes. The result showed that R. officinalis and M. chamomilla gave significant MIC against P. acnes. So, these to plant extract would be used for anti-acne
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topical formulation.
Table 2. Minimum inhibitory concentration (MIC) values of potential plant extracts against test organisms
Minimum Inhibitory Concentration (mg/ml)
R. officinalis
B. subtilis
0.0781
0.0781
ND
0.312
ND
0.0781
0.0781
0.156
ND
ND
ND
0.0625
0.0625
0.0625
0.0625
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Isotretinoin
Kocuria sp.
0.156
EP
A. nilotica M. chamomilla
S. aureus
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P. acnes
ND means not detected
3.3. Antioxidant assay by DPPH-Free radical scavenging activity DPPH is a rapid and sensitive method to evaluate the antioxidant capacity of plant extracts. It is based on the decolourization of DPPH in the presence of antioxidants in the tested sample. The color change from purple to yellow shows the strong antioxidant capacity of the sample. The percentage values of antioxidant activity of selected plant extracts at different concentration 50-200 µg/ml were shown in figure 3. In this assay, ascorbic acid gave 10
ACCEPTED MANUSCRIPT antioxidant activity with IC50 value of 35.45µg/ml. The results of the present investigation indicated that the extracts of R. officinalis, A. nilotica and A. indica exhibited noteworthy
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antioxidant activity with the IC50 value of 109.3, 136.9 and 108 µg/ml, respectively.
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Figure 3: Percentage of antioxidant activity of plant extracts These results are representative of the antioxidant experiment in triplicates with mean ± SEM. Statistical significance against Ascorbic acid as a control *P<0.05, **P<0.01; ***P<0.001. (*** denotes significant value)
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4. Conclusion
The results of this study indicate that extracts of R. officinalis, M. Chamomilla and A. nilotica
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show antibacterial activity with less MIC against acne causing bacteria. Additionally R. officinalis and M. Chamomilla also showed good antioxidant activity along with A. indica extract. As these are the natural extracts, so they have no or less side effects. These can also be an alternative and better option for resistant acne causing bacteria. The topical anti-acne formulations can be developed using these plant extracts individually or in combination. 5. Acknowledgement All authors are thankful to the Head of the Department, Life Science Department, Gujarat University, Ahmedabad, Gujarat, for providing all the facilities for this research work.
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