Table of contents
Table of figures
Table of tables
List of abbreviations
Anti-acne activity of medicinal and herbal sources: an overview
2. Review of literature
4. Materials and Methods
4.1 Study area
4.2 Collection of samples
4.3 Pure culture preparation
4.4 Morphological and biochemical tests
4.5 Extract preparation
4.6 Antimicrobial activity
4.8 Statistical analysis
5. Results and discussion
Table of figures
Figure 1. Map of Kerala showing the sample collection point. Authors own work.
Figure 2. Details of the isolated Staphylococcus sp. a), b), c) and d) growth of the organism on various culture media, e) and f) microscopic examinations. Authors own images.
Figure 3. Details of antimicrobial activity of various extracts against Staphylococcus sp. And zone of inhibition a) concentration 50 µl, b) concentration 100 µl, c) concentration 150 µl, d) concentration 200 µl. Authors own images.
Table of tables
Table 1. Various biochemical tests of the isolated bacterial strains (isolate 1; Staphylococcus sp. and 2; Pseudomonas sp.).
Table 2. Antimicrobial activity of various extracts against Staphylococcus sp. and zone of inhibition (mm).
List of abbreviations
Abbildung in dieser Leseprobe nicht enthalten
Firstly, we thank God Almighty whose blessing were always with us and helped us to complete this project work successfully.
We wish to thank our beloved Manager Rev. Fr. Dr. George Njarakunnel, Respected Principal Dr. Joseph V.J, Vice Principal Fr. Joseph Allencheril, Bursar Shaji Augustine and the Management for providing all the necessary facilities in carrying out the study. We express our sincere thanks to Mr. Binoy A Mulanthra (lab in charge, Department of Biotechnology) for the support. This research work will not be possible with the co-operation of many farmers.
Lastly, we extend indebt thanks to patents, friends, and well-wishers for their love and support.
Prem Jose Vazhacharickal, John Joseph, Jiby John Mathew, Sajeshkumar N. K and E. S Sumayya
Anti-acne activity of medicinal and herbal sources: an overview
Prem Jose Vazhacharickal1, John Joseph, Jiby John Mathew, Sajeshkumar N. K and E. S Sumayya
Acne is the most common skin disorder with a very high prevalence. Acne may be treated with a combination of remedies including over – the counter skin care, and medications, and chemical or laser procedures. All these treatments are comparatively costlier. Hence in the current study, attempts are being made to search for new and cheaper remedy for acne. Here anti acne activity of various samples [Paste, cucumber, tomato, multani mitty (fullers earth) ,Guava leaf, turmeric and ointment(control)] was studied against Staphylococcus sp. The most effective samples used is guava leaf.
Keywords: Acne; Staphylococcus; Biochemical identification.
Acne vulgaris is one of the commonest skin disorders, for which dermatologists are still struggling since years to treat successfully. It mainly affects adolescents, through it may present at any age. It is almost a universal disease occurring in all races and affecting 95% of 16-year-old boys and 83% of 16-year-old girls to some degree. The incidence of severity of acne, peak at 40% in 14-17-year-old girls and 35% in boys aged 16-19 year.
Acne by definition is a multi-factorial chronic inflammatory disease of pilosebaceous units. It affects the skin of the face, neck and upper trunk. These particular sebaceous follicles have capacious follicular channels and voluminous, multi-acninar sebaceous glands. Acne develops when these specialized follicles undergo pathogenic alteration that results in the formation of non-inflammatory lesions [comedons] and inflammatory lesions [papules, pustules and nodules].
Staphylococcus epidermidis and Propionibacterium acne are considered as the major skin bacteria that cause the formation of acne. Propionibacterium acne, a gram-positive an anaerobic pathogen, plays an imp role in the pathogenesis of acne.
It is implicated in the development of inflammatory acne by its capability to activate complements and by its ability to metabolize sebaceous triglycerides into fatty acids, which chemotactically attract neutrophils. Staphylococcus epidermidis, an aerobic organism, is usually involved in super facial infections within the sebaceous unit.
Modern acne therapy has been designed to interrupt the pathogenic pathway at one or more points. Tropical or Systemic therapy is available for the treatment of acne, which includes comedolytic agents and antibiotics and various anti-inflammatory drugs and Systemic therapy includes antibiotics, zinc and hormones. The excessive use of antibiotics for long period has led to increased resistance in acne causing bacteria; Propionibacterium acne and Staphylococcus epidermidis against a number of antibiotics used to treat acne.
The area’s most vulnerable to acne are the largest oil secreting glands present in the face, back and trunk. It is a chronic inflammatory disease of multifactorial etiology affecting more than 85% of teenagers and frequently continues into adulthood.
The influencing factors of acne include excess sebum secretion, hyper keratinization of the hair follicle, oxidative stress and the release of inflammatory mediators. Acne is related to abnormalities in sebaceous gland function particularly in teenage. The hyper secretion of hormone androgen stimulates higher sebum secretion in sebaceous gland. The secreted sebum normally contains a mixture of lipids; squalene wax and cholesterol both in free and in ester forms and triglycerides that naturally provide a skin barrier function. However, the resulting abnormalities in sebaceous glands due to hormonal effects alter sebum composition and decrease linoleic acid content. Thus, the normal skin barrier function is impaired. In addition, the deficiency of linoleic acid in the follicle promotes the growth of normal flora like P. acnes. Proliferation of Propionibacterium acne leads to inflammatory lesions and severe acne.
In addition, Staphylococcus epidermidis and Pitryosporum ovale are present in acne lesions. Although the other microorganisms are present in acne lesions, the prevalent bacterium implicated in the clinical course of acne is Propionibacterium acnes. It is a gram positive, anaerobic bacterium that mostly resides in the pilosebaceous follicles of the skin.
Although P. acnes area member of the normal skin commensal, it plays a critical role in the development of acne [both inflammatory and non-inflammatory] when it becomes overgrown and colonizes the pilosebaceous unit.
Staphylococcus epidermidis, an aerobic organism, usually involves in superficial infections within the sebaceous unit. These factors provide a potential target for the treatment.
Propionibacterium acnes and Staphylococcus epidermidis are the target sites of antiacne drugs. Long term use of antibiotics against acne is outdated because of exacerbated antibiotic resistance. The development of antibiotic resistance is multifactorial, including the specific nature of the relationship of bacteria to antibiotics, how the antibacterial is used, host characteristics, and environmental factors. To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as alternative treatments for diseases.
The aim of this study is to find anti- acne activity of Staphylococcus sp against medicinal and herbal sources.
2. Review of literature
Propionibacterium acnes and Staphylococcus epidermidis have been recognized as pus-forming bacteria triggering an inflammation in acne (Chomnawang, 2005; Lertsatitthanakorn, 2008). Propionibacterium acnes is an anaerobic pathogen, which plays an important role in the pathogenesis of acne by inducing certain inflammatory mediators and comedogenesis (Jain and Sangal, 2009).
Propionibacterium acnes are found on the skin of neonates, but true colonization begins 1-3 years prior to sexual maturity. During this time, number of Propionibacterium acnes rise from fewer than 10/cm2 to about 106/cm2, chiefly on the face and upper thorax. In the lipid-rich microenvironment of the hair follicle, Propionibacterium acnes produce inflammatory mediators that result in papules, pustules and late rnodulocystic lesions that are typical of inflammatory acne Propionibacterium acnes increase sebum production and inflammation at the site of pilosebaceous follicles. It was reported that lipid mediators are able to interfere with sebocytes differentiation and sebogenesis of acne through the activation of pathways related to peroxisome proliferators-activated receptors (Halliwell and Gutteridge, 1998). Propionibacterium acnes is a relatively slow growing aero tolerant anaerobic gram-positive bacterium. This bacterium primarily lives on the fatty acid present in the sebaceous glands or sebum secreted by follicles. It has the ability to produce propionic acid and catalase in presence of indole, nitrate or both in dole and nitrate. When a pore is blocked by the secretion, the anaerobic bacterium starts over growing and secretes chemical which break down the wall of the pore and leads to acne lesion. Propionibacterium resembles Corynebacterium in morphology and arrangement.
Since Propionibacterium acnes is a common resident of the pilosebaceous glands of the human skin and acne is caused in part from an infection, it was suppressed with topical and oral antibiotics. Antibiotics were primarily used in treatment against acne for more than 40 years (Tzellos, 2011).
Normally used topical and oral antibiotics are Clindamycin, Erythromycin, Triclosan, Tetracycline, Minocycline and Metronidazole. The most frequently used topical antibiotic for acne was Clindamycin which is available as a solution, lotion or gel at 1% strength. Salicylic acid is also used in treatment of acne as it was found to be one of the good cleansers that has both anti ¬inflammatory and mild comedolytic effects. It is used in treatment of mild acne or as an adjunctive agent (David, 2003). Physical treatment in the form of lesion removal, photo-therapy is also helpful in few of them (Leyden, 1997). Now a day's new therapeutic modalities and various permutation and combinations have been designed in topical agents; that include benzoyl peroxide, antibiotics, Retinoids etc. These combinations when tried on patients increase the frequency and severity of dryness, scaling, erythema, burning, stinging and itching (Draelos, 2010). Minocycline has an increased risk of severe adverse effects compared to other tetracyclines. It may induce hypersensitivity reactions affecting the liver, lungs, kidneys or multiple organs (Drug Reaction with Eosinophilia and Systemic Symptoms [DRESS] syndrome) in the first weeks of treatment and with long-term treatment, may cause autoimmune reactions (Systemic Lupus Erythematosus, autoimmune Hepatitis). In addition, Central Nervous System (CNS) symptoms such as dizziness are more frequent compared to other tetracyclines. Long-term treatment may also induce hyperpigmentation of the skin.
Therapy with these medications generally lasts from several weeks to months, but may sometimes even continue for years. In particular, this long-term exposure to oral antibiotics creates tremendous selective pressures for the emergence of resistant strains of Propionibacterium acnes (Ross, 2001).
In fact, resistance to Propionibacterium acnes develops in 50% of individuals following treatment with both topical and oral antibiotics (Esperson, 1998). The rate of Propionibacterium acnes resistance corresponds to the length of treatment (Tan, 2005). In a prospective study of 151 patients, the rate of resistance in patients who had never been on antibiotics was 0, compared to 6.25% in patients on short-term (6-18 weeks) antibiotics and 21.6% in patients on longer periods of antibiotics.
Since the use of antibiotics has resulted in resistant strains of Propionibacterium acnes and also the many side effects associated with its prolonged use resulted in people opting for holistic treatment. Medicinal plants are known to have enormous therapeutic capabilities that modern medicine is searching for. Thus, it became an alternative therapy for consumers, which is cost effective when compared to modern treatment involving antibiotics.
Herbs are safe, efficacious and multifunctional. The ingredients in topical acne treatments, particularly herbs and naturally derived compounds, have received considerable interest as they have fewer adverse effects than synthetic agents (Keren, 2003)
The anti-bacterial and anti-fungal activity of four seaweeds namely Sargassum binderi, Amphiroasp., Turbinaria conoides and Halimeda macroloba Decaisne from the east coast of Gulf of Thailand was studied. The test organisms used in the study were Staphylococcus aureus, S. epidermidis, Propionibacterium acnes, Proteus mirabilis and Candida albicans. it was found that aqueous and ethanolic extracts of all the four species were found to have antimicrobial activity. However aqueous extract of T. Conoides demonstrated the maximum activity. Therefore, aqueous extract of T. Conoides was further evaluated for its anti-inflammatory effect using EPP-induced ear edema and carrageenin-induced hind paw edema tests. Results revealed anti-inflammatory activity of aqueous extract of T. Conoides comparable to that of phenylebutazol and acetylsalicylic acid (Walailuck, 2011). Studies on anti-oxidant and anti-biotic activity of Selagineila involvens extract (SIE) revealed to have an antioxidant effect in a dose-dependent manner in the hydroxyl radical-mediated oxidation test. Thus, SIE could be used as a safe non-antibiotic that can be used to treat acne. It was also known to reduce the non-specific initiation and augmentation phase of the inflammatory response of Propionibacterium acnes (Joo, 2008).
Herbal plants and its parts are effective alternatives used to treat acne. Herbal medicines are recommended since these are powerful cleansers that could clear your body of unwanted harmful toxins and enrich the body with useful nutrient minerals.
Ayurveda, which is one of the oldest system of traditional medicine of India is now regaining its pace, the herbs in Ayurveda used to treat acne are Neem, Tea tree oil, Sandal wood, Burdock root, Red clover, Aloe vera, Calendula and Lavender. All these plants are well known for their anti-microbial activity. Calendula officinalis, Cassia tora and Momordica charantia are well known herbs used in ayurvedic traditional medicine for their effectiveness against wide range of diseases including skin infections as they have diverse secondary metabolites that is responsible for their antibacterial activity (Roopashree and Raman, 2008).
In a study, the antimicrobial activity of Indian medicinal plants against Propionibacterium acnes that caused acne vulgaris was evaluated. Extracts of Rauwolfia serpentine (roots), Piper nigrum (seeds), Azadirachta indica (leaves), Ficus religiosa (leaves), Euphorbia hirta (roots), Ocimum sanctum (leaves), Phyllanthus niruri (whole plants), Cardiospermum halicacabum (leaves), Mordica charantia (fruits), Casaurina equisetifolia (fruits), Cynodon dactylon (leaves) and Jasmimum sambac (flowers) were tested for antimicrobial activity by agar well diffusion and broth dilution method. The results from the disc diffusion method showed that 07 medicinal plants could inhibit the growth of Propionibacterium acnes. Among these Azadirachta indica, Momordica charantia, Casuarina equisetifolia, Rauwolfia serpentina, Cardiospermum halicacabum, Phyllanthus niruri and Piper nigrum had strong inhibitory effects. Based on broth dilution method, Rauwolfia serpentina extract had the greatest antimicrobial effect. Piper nigrum and Momordica charantia showed outstanding antimicrobial properties against Propionibacterium acnes based on the agar well diffusion assay. In bioautography assay, the Rauwolfia serpentine extract produced strong inhibition zones against Propionibacterium acnes. Phytochemical screening of Rauwolfia serpentine revealed the presence of alkaloids (berberine and harmane) which could possibly be responsible (Harisaranraj, 2010) for inhibition.
Medicinal plants belonging to families Liliaceae, Rutaceae, Zingiberaceae, Myrtaceae, Lamiaceae etc. contains alkaloids, tannins, flavonoids, terpenoids, volatile oil and essential oil which are reported to have significant effect against acne causing bacterial (Deepak, 2011).
A stable anti-acne preparation was developed in a study by determining the main chemical constituents of volatile oil from leaves of Eucalyptus globules and Psidium guajava. Volatile oils from these plants leaves were extracted and analysed for their main chemical components. Antimicrobial activity of the oils was determined by agar diffusion and micro dilution method. These volatile oils showed good anti-microbial activity against Propionibacterium acnes. The main constituents present in the volatile oils of Eucalyptus and Guava leaves were gamma-terpentine and alpha-pinene. These results suggested that the preparations incorporating the volatile oils of Eucalyptus and Guava could be used in anti-acne formulation (Sirivan, 2008).
Bioactivity studies on Java citronella oil (Cymbopogon winterianus) revealed good anti-acne activity with potential for use in the development of topical anti-acne preparations. Jasmimum auriculatuma small herb of the family Oleaceae found growing wild in South India and the Western peninsula was reported to have antioxidant and antibacterial activities. The alcohol free defatted extract of Jasmimum auriculatum leaves has been reported to contain the essential oils lupeol and Jasminol that has a potent anti-acne activity.
Lemon grass oil contained citral as the major constituent and it showed a good anti-acne activity. Thus, suggesting that lemon grass can be used to treat acne vulgaris (Faiyazuddin, 2010). The dried herbs assessed were Chrysanthemum morifolium Juhua), Lonicera japonica (honeysuckle), Jasmimum sambac (jasmine), Lavandulaan gustifolia (lavender), Rosa damascene (rose), Osmanthus fragrans (osmanthus), Eucommia ulmoides (duzhong), Gynostemma pentaphyllum (jiaogulan), Cymbopogon citrates (lemon grass) and Ilex paraguariensis (yerba mate). The herbs were ground and extracted with methanol in a series of steps. They were tested against Propionibacterium acnes by determining Minimum inhibitory concentration (MIC).
Many herbal plants were screened for acne treatment; the plants included Ocimum, Tabernaemontana dinaricata, Melaleuca alternifolia (Tea tree) and Aloe vera. These were being made in the form of topical formulations. In-vitro antibacterial studies were performed against Propionibacterium acnes. This was carried out by well diffusion method and Tetracycline was used as standard. Ocimum was developed in hydroalcoholic extract, Tabernaemontana dinaricata, Tea tree and Aloe vera was developed in ethanolic extract. Ocimum sanctum and Tea tree showed the greater zone of inhibition (Sawarkar, 2010).
The antibacterial activity of oriental herbal extracts of Angelica dahurica and Glycyrrhiza glabra was studied against Propionibacterium acnes. Glycyrrhiza glabra gave more promising results and therefore could be helpful in prevention and treatment of acne lesions. Some plant extracts exhibited both antimicrobial and anti-inflammatory effects against Propionibacterium acnes (Nam, (2003).
Three herbal extracts namely Rosa damascene, Eucommia ulmoides and Ilex paraguariensis were used in a study with 3 concentrations (0.5, 1 and 2 mg/mL). Results of the studies showed that both Eucommia ulmoides and Ilex paraguariensis possessed antimicrobial and anti-inflammatory effects against Propionibacterium acnes.
Pitikamardhini an herbal formulation, which consists of plants such as Rubia cordifolia, Symplocos racemosa, Acorus calamus, Coriandrum sativum and Citrus limonis was effective in curing Acne vulgaris. A clinical trial was made which gave good results and also improved facial complexion. Present studies revealed that Pitikamardhini could be used in clinical treatment of acne (Burade, 2009).
The antibacterial activity of plant extract against Propionibacterium acnes and S. Epidermidis was studied by cup plate method. In this method, the macerated fruit and fruit decoction of Terminalia chebula and Terminalia bellarica (belonging to Combretaceae family) was used individually and in combination. The combination of 20% gave the best zone of inhibition (around 26 mm) (Sawarkar, 2011). Terminalia bellarica was studied for its anti-microbial activity against Staphylococcus aureus, Streptococcus pneumoniae, Salmonella typhi and many others by crude and methanolic extracts of the seed by disc diffusion method. The zone of inhibition was around 15.5 to 28.0 mm for crude extract and 14.0 to 30.0 mm for methanolic extract. The study suggested that methanolic extract was better than the crude one. The results also indicate that T. Bellarica dry fruit possesses potential broad spectrum antimicrobial activity (Elizabeth, 2005).
The current research work is based on the following hypothesis
1) Herbal extracts show a promising effect on acne treatment compared to chemical ointments.
2) These herbal extracts differ in their anti-acne activities.
4. Materials and Methods
4.1 Study area
Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts. The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al ., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November.
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