role in acne treatment through modulation of the skin microbiome ...... It should be noted, however, that in the United States the Federal Drug ...... Felter, H. & Lloyd, J. 1898, "Phytolacca" in King's American Dispensatory Ohio Valley Co.,.
Selective inhibition of Proprionibacterium acnes by Calendula officinalis: a potential role in acne treatment through modulation of the skin microbiome Latifa Pelletier-‐Ahmed Abstract Recent investigations into the skin microbiome have revealed many potential beneficial effects for commensal skin bacteria like Staphylococcus epidermidis. These effects include anti-‐ inflammatory and antimicrobial effects. Current treatment of acne vulgaris frequently involves antimicrobial treatment that is non-‐selective against Proprionibacterium acnes resulting in eradication of other commensal bacterial like S. epidermidis. This study screened 6 proprietary hydroethanolic plant extracts: Calendula officinalis, Berberis vulgaris, Mahonia aquifolium, Phytolacca decandra, Lavandula angustifolia, and Echinacea purpurea/Echinacea angustifolia. The results of screening showed that Calendula was selectively able to inhibit P. acnes but not S. epidermidis. The minimum inhibitory concentration was 0.078 g/mL equivalent to weight of dried herb not dried extract. An aqueous extract of Calendula flowers was found to have the same selective antibacterial effects. The results indicate that Calendula has the potential to be a topical prebiotic for the treatment of acne.
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Table of Contents Abstract .................................................................................................................................. 1 Abbreviations ......................................................................................................................... 4 Introduction ........................................................................................................................... 5 Overview ......................................................................................................................................... 5 Acne vulgaris ................................................................................................................................... 7 Definition ............................................................................................................................................ 7 Pathogenesis ....................................................................................................................................... 9 Epidemiology and psychological consequences ................................................................................ 10 Conventional treatment .................................................................................................................... 10 Topical treatment .............................................................................................................................. 10 Oral Treatment .................................................................................................................................. 13 Antibiotic resistance .......................................................................................................................... 16 Complementary and alternative medicine ....................................................................................... 17 Propionibacterium acnes ............................................................................................................... 18 Staphylococcus epidermidis ........................................................................................................... 21 Current study – topical prebiotics .................................................................................................. 24 Phytolacca decandra L. ..................................................................................................................... 25 Calendula officinalis L. ....................................................................................................................... 27 Mahonia aquifolium (Pursh) Nutt. .................................................................................................... 29 Berberis vulgaris L. ............................................................................................................................ 30 Lavandula angustifolia L. .................................................................................................................. 31 Echinacea purpurea (L) Moench/ angustifolia DC. ............................................................................ 32 Methods ............................................................................................................................... 33 Preparation of ethanolic extracts ................................................................................................... 33 Bacterial Sensitivity Testing ........................................................................................................... 35 Minimum Inhibitory Concentration ................................................................................................ 35 Statistical analysis .......................................................................................................................... 37 Results and Discussion .......................................................................................................... 37 Preparation of extracts .................................................................................................................. 37 Bacterial Sensitivity ....................................................................................................................... 38 Minimum inhibitory concentration ................................................................................................ 41 Aqueous extraction of Calendula ................................................................................................... 42 Commensal bacteria ...................................................................................................................... 43 Bacterial resistance ........................................................................................................................ 44 Calendula ....................................................................................................................................... 45 Dosage ............................................................................................................................................... 46 Constituents ...................................................................................................................................... 46 Antimicrobial ..................................................................................................................................... 47 Anti-‐inflammatory ............................................................................................................................. 48 Antioxidant ........................................................................................................................................ 48 Wound healing .................................................................................................................................. 50 2
Angiogenic ......................................................................................................................................... 51 Immunostimulating ........................................................................................................................... 52 Skin parameters ................................................................................................................................ 52 Antitumour ........................................................................................................................................ 52 Case studies ....................................................................................................................................... 53 Clinical trials ...................................................................................................................................... 53 Safety ................................................................................................................................................ 55
Conclusion ............................................................................................................................ 56 Bibliography ......................................................................................................................... 57 Appendix I ............................................................................................................................ 73 Appendix II ........................................................................................................................... 75 Appendix III .......................................................................................................................... 78 Appendix IV .......................................................................................................................... 79 Appendix V ........................................................................................................................... 81 Appendix VI .......................................................................................................................... 90 Appendix VII ......................................................................................................................... 91 3
Abbreviations AMP Berberis Calendula CAM CFU COC COX DMSO Echinacea E. coli IL Lavandula LPS LTA Mahonia MIC MRSA OA P. acnes P. aeruginosa PDGF Phytolacca PI3-‐K PSM S. aureus S. epidermidis S. fecalis TLR TNF-‐α TTC UVB
antimicrobial peptides Berberis vulgaris Calendula officinalis chick chorioallantoic membrane colony forming unit combined oral contraceptive cyclooxygenase dimethyl sulfoxide Echinacea purpurea/Echinacea angustifolia Escherichia coli interleukin Lavandula angustifolia lipopolysaccharide lipoteichoic acid Mahonia aquifolium minimum inhibitory concentration methicillin-‐resistant Staphylococcus aureus oleanolic acid Proprionibacterium acnes Pseudomonas aeruginosa Platelet derived growth factor Phytolacca decandra Phosphoinositide 3-‐kinase phenol soluble modulin Staphylococcus aureus Staphylococcus epidermidis Streptococcus fecalis toll-‐like receptor tumour necrosis factor-‐alpha threshold for toxicological concern ultraviolet B
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Introduction
Overview The microbiome is the collection of microorganisms including bacteria, viruses and fungi that live on the exposed surfaces of the human body including the gastrointestinal tract, lungs, and skin. It is estimated that these organisms number as many as 100 trillion organisms, about 10 times the number of cells in the human body. Increasingly research is elucidating how these organisms have profound impacts on human physiology and health. Research has focused on the gut microflora, which plays a key role in digestion and immunity. Dysbiosis, or an increase in pathogenic microbes relative to beneficial ones, in the gut has been linked with conditions such as allergy, obesity, inflammatory bowel disease, diabetes mellitus, atherosclerosis and cancer (Mankowska-‐Wierzbicka et al., 2015). However, little is known about the skin microflora where it is estimated that there are one billion bacteria per square centimetre of human skin. Bacteria on the human skin can be transient, referring to contaminant bacteria that cannot colonise the skin; temporary residents, referring to bacteria that do not typically live on the skin but can colonise the skin; or resident referring to bacteria that continuously colonise the skin surface (Mankowska-‐Wierzbicka et al., 2015; Grice et al., 2008). The composition of these species depends on host conditions such as moisture, temperature, pH, ultraviolet radiation, growth substrate content and concentration, secretion of chemicals that inhibit growth, as well as the interrelationships between microorganisms (Christensen and Bruggeman, 2014; Grice et al., 2008; Bojar and Holland, 2004). Resident bacteria are mainly Gram-‐positive and have evolved to tolerate harsh environmental conditions such as low nutrients, acidity, desiccation, and continual shedding of epithelial cells. Resident species include Proprionibacteria, coagulase-‐negative Staphylococci, Micrococci, Corynebacteria and Acinebacteria (Krutmann, 2009). Knowledge of the skin microflora until recently has been limited to culture-‐dependent assays, however, it has been estimated that less than 1% of all bacteria species can be cultivated (Grice et al., 2008). Genetic sequencing of skin bacteria of the antecubital crease (inner elbow) has revealed 113 operational taxonomic units 1 in 7 divisions (in order of abundance): Proteobacteria mainly from the genera 1
Operational taxonomic unit: a species defined solely by its genetic material.
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Pesudomonas and Janthinobacterium; Actinobacteria mainly from the genera Kocuria and Proprionibacteria; Firmicutes; Bacteriodetes; and Cyanobacteria (Grice et al.; 2008). Further, metagenomic studies have shown that the relative abundance of phyla in the antecubital and popliteal (underside of knee) creases differs between individuals (Kong et al., 2012). Metagenomic studies challenge assumptions based on culture-‐based assays of bacterial abundance. However, metagenomic studies of the bioburden2 of Staphylococcus aureus found in the human nares supported the findings of culture-‐based assays (Kong et al., 2012). These findings suggest that our understanding of skin microbial ecology is in its infancy as new technologies begin to provide data on difficult-‐to-‐cultivate bacteria. Beneficial roles of resident bacteria include: inhibition of pathogenic species, further processing of skin proteins, free fatty acids, and sebum (Roth and James, 1988). Dysbiosis of the skin microbiota is associated with diseases such as atopic dermatitis or eczema, rosacea, psoriasis and acne vulgaris (Mankowska-‐Wierzbicka et al., 2015). Atopic dermatitis has been associated with a decreased production of antimicrobial peptides (AMPs) by keratinocytes and lesions frequently colonised and infected with S. aureus (Ong et al., 2002). The importance of understanding how our microbiota influence our health is increasingly important with growing urbanisation as theories emerge suggesting that decreased contact with natural environmental elements or decreased exposure to environmental microbes, the biodiversity and the hygiene hypotheses respectively, have negative impacts on microbiome diversity and health. Decreased exposure to environmental biodiversity was associated in a decreased biodiversity in Gram-‐negative gammaproteobacteria and an increased incidence of atopy or allergy (Hanski et al., 2012). Metagenomic studies have found decreased microbial diversity is associated with flare-‐ups of atopic dermatitis (Kong et al., 2012). Evidence now exists that begins to support the idea of a skin, gut and brain communication axis. In particular, the gut and skin have similar neuronal and inflammatory activity (Arck et al., 2010). This brings into question the generalised use of antibiotics and antimicrobial agents for the treatment of skin conditions when the full impact these products have on the skin and body, mediated via the microbiome, is not fully understood. Lai et al. (2009) suggest that with recent
2
Number of bacteria found on a non-‐sterilised surface.
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findings demonstrating the benefit of commensal bacteria in human health the indiscriminate use of topical and systemic antibiotics should be avoided.
Acne vulgaris Definition Acne vulgaris is a chronic inflammatory disease of the pilosebaceous unit or hair follicles associated with an oil gland. Acne lesions are characterised by excess production of sebum that is colonised by large numbers of Proprionibacterium acnes. Lesions can be non-‐inflammatory as open or closed comedones, or inflammatory as papules, pustules or nodules (Figures 1 and 2) (Walsh et al., 2016; Williams et al., 2012). Areas of the body rich in sebaceous glands include the head, chest and back (Bojar and Holland, 2004).
Figure 1. Changes to the sebaceous follicle in an acne lesion. A) a normal sebaceous follicle, B) a comedone, C) an inflammatory acne lesion with a ruptured follicular wall. Image taken from Williams et al. (2012).
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Figure 2. Different grades of acne: A) comedonal facial acne, B) moderate inflammatory facial acne, C) moderate inflammatory back acne, D) severe inflammatory facial acne, E) severe, inflammatory back acne. Image taken from Asai et al. (2016). With regards to clinical practice, however, there is no universally accepted grading scale for acne severity. Recently, 18 grading scales were ranked based on a range of criteria (Tan et al., 8
2013). One of the highest rated grading scales developed by Tan et al. (2007) is presented in Figure 3.
Figure 3. Grading scale for acne severity taken from Tan et al. (2007). Pathogenesis Four processes are involved in the pathogenesis of acne: inflammation and immune response leading to increased inflammatory mediators; altered keratinisation leading to abnormal follicular growth and differentiation; sebaceous gland hyperplasia and seborrhoea3 controlled by androgens; and colonisation of the follicle by P. acnes (Williams et al., 2012). However, a complete understanding of the molecular and cellular mechanisms that underlie acne have yet to be elucidated (Zaenglein et al., 2016). Abnormal keratinisation is the result of keratinocyte hyperproliferation and reduced desquamation leading to increased cohesion between keratinocytes. The accumulated keratinocytes combine with sebum, which causes a plug to form in the follicular duct. This leads to the formation of a comedone (Cunliffe et al., 2000). The formation of a biofilm by P. acnes may enhance the formation of microcomedones by forming a glue that holds corneocytes4 together to form a plug of the follicle (Burkhart and Burkhart, 2007). P. acnes flourish in the lipid-‐rich anaerobic sebaceous follicle comedone or non-‐inflammatory acne lesion. However, while the existence of distinct factors that contribute to acne formation is clear, the exact cause and effect relationships are not. Current topics of debate include whether comedone formation precedes or is the result of inflammation (Del Rosso and Kircik, 3 4
Increased sebum production. A keratinocyte in the terminal phase of differentiation.
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2013); whether the initial cellular infiltrate is neutrophilic or lymphocytic; or whether P. acnes initiates or exacerbates inflammation (Bojar and Holland, 2004). The oxidation of lipids found in sebum can also trigger the production of inflammatory mediators (Walsh et al., 2016). Other cutaneous microorganisms that are associated with the pilosebaceous follicle that may play a role in the pathogenesis of acne include Staphylococcus epidermidis and the yeast Malassezia spp. (Bojar and Holland, 2004). Epidemiology and psychological consequences Acne affects almost all people aged 15-‐17 years; it is moderate-‐severe in %15-‐20 of this age group. However, acne often persists into adulthood. Major side effects of acne are scarring and, if acne persists into adulthood, a negative impact on self-‐esteem (Williams et al., 2012). Psychological consequences of acne in adolescents include a decreased quality of life, lowered self-‐esteem and self-‐worth, increased feelings of uselessness, and lower body satisfaction. Acne is also linked to psychiatric disorders such as anxiety, depression and suicide ideation (Misery, 2011). Increased acne severity was associated with higher rates of suicide ideation5 among 4744 adolescents (Halvorsen et al., 2011). Conventional treatment Conventional treatment includes topical and oral treatments. Treatment regimens serve to prevent further acne formation rather than treat existing acne (Williams et al., 2012). Although there are numerous studies on acne treatment, there remain a large number of biases in published trials (Ingram et al., 2010). A lack of standardised assessment tools to characterise acne severity limits the comparability between studies. Population biases have left a research gap among the treatment of acne in people of colour (Zaenglein et al., 2016). Topical treatment Topical treatment is the first-‐line of therapy in conventional acne treatment (Zaenglein et al., 2016). Topical treatments include: benzoyl peroxide, topical retinoids, topical antibiotics, and salicyclic acid (Williams et al., 2012). 5
Self-‐reported thoughts related to suicide or suicide-‐related acts (Halvorsen et al., 2011).
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Benzoyl peroxide Benzoyl peroxide is a strong oxidising agent that produces free radicals that are toxic to bacterial cell membranes (Sagransky et al., 2009). It is antibacterial to several species of Proprionibacteria including P. acnes as well as Staphylococcus epidermidis, Staphylococcus hominus and the yeast Pityrosporum ovale (Cove and Holland, 1983). An indirect anti-‐ inflammatory action has been attributed to benzoyl peroxide through its cytotoxicity to neutrophils thus preventing the release of inflammatory cytokines (Hegemann et al., 1994). Further, it acts as a comedolytic and keratolytic agent (Sagransky et al., 2009). Benzoyl peroxide may cause hypersensitivity reactions, contact sensitization, burning, excessive erythema, dryness, scaling and peeling. As a strong oxidising agent it can bleach hair and clothes. It must be used for a minimum of 3 weeks to see results, with the maximum improvement occurring after 8 to 12 weeks. The incidence of adverse effects decreases with long-‐term use (Zaenglein et al., 2016, Supplement Table I; Sagransky et al., 2009). Benzoyl peroxide is frequently recommended as combination treatment with either topical retinoids or antibiotics. In particular, benzoyl peroxide has been shown to kill resistant strains of P. acnes (Leyden et al., 2008) and so can be used in combination with antibiotics to prevent the development of antibiotic resistance (Sagransky et al., 2009). However, in general few combinations have been adequately tested against relevant monotherapies (Williams et al., 2012). Salicyclic acid Salicyclic acid is an exfoliant used to control the symptomatic signs of acne. Shalita (1981) demonstrated efficacy for the treatment of salicyclic acid in mild to moderate acne; however, there are no studies that show it is superior to other products (Williams et al., 2012). Salicyclic acid is applied initially once per day and may be increased to 2 to 3 times per day. The main side effects are dryness, redness and peeling. Hypersensitivity reactions and salicylate toxicity may occur. Combination with other therapies may result in a cumulative irritant and drying effect (Zaenglein et al., 2016, Supplement Table II). Retinoids Topical retinoids are vitamin A analogues that bind to retinoic acid receptors, the main effect of 11
which is the prevention of the formation of the comedone (Zaenglein et al., 2016). They also have a strong effect against keratinisation and a moderate effect against inflammation (Williams et al., 2012). The use of retinoids can be limited by their side effects including dry skin, peeling, scaling, flaking, erythema, pruritis6, skin tenderness, and hyper-‐/hypopigmentation. There is increased risk for sunburn as retinoids interact with ultraviolet light resulting in photosensitivity. Exposure to wind and cold can also increase irritation (Zaenglein et al., 2016, Supplement Table VII). They also have a teratogenic 7 effect and are contraindicated in pregnancy. Women are encouraged to concomitantly take birth control (Williams et al., 2012). Antibiotics Topical antibiotics are believed to act directly through antibacterial action against P. acnes as well as through anti-‐inflammatory mechanisms. They have limited effect against non-‐inflamed acne lesions. The antibiotics primarily used in topical preparations are erythromycin and clindamycin, however, the effect of erythromycin may be declining due to antibiotic resistance (Williams et al., 2012). In response to growing concerns regarding antibiotic resistance topical antibiotics are not recommended as a monotherapy (Walsh et al., 2016). Clindamycin is currently the preferred topical antibiotic, although it is only supported by one randomised, double-‐blind, placebo-‐controlled 12-‐week clinical trial in 46 patients (Kuhlman and Callen, 1986). Side effects include severe colitis, dermatitis, folliculitis, photosensitivity, pruritis, erythema, dry skin and peeling. Clindamycin in combination with benzoyl peroxide was shown to be better than either alone in treating moderate to severe acne in a multicentre, randomised, double-‐blind trial with 2813 patients (Thiboutot et al., 2008). Side effects of the combined treatment include: erythema, peeling, drying, burning, and anaphylaxis (Zaenglein et al., 2016, Supplement Tables V & VI). Erythromycin is a topical treatment for mild to moderate inflammatory acne. Besides a risk for the development of antibiotic resistance there is also a risk for developing colitis caused by superinfection with Clostridium difficile. Topical erythromycin can also interact with cosmetic cleansing products especially those with abrasive, peeling and desquamating properties. To prevent antibiotic resistance erythromycin is recommended in combination with benzoyl 6 7
Skin itching. An agent that may cause birth defects.
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peroxide. Side effects of the combined treatments include: pseudomembranous colitis, urticaria, dryness, itching, burning sensation, erythema, skin discolouration, oiliness, and skin tenderness (Zaenglein et al., 2016, Supplement Tables III & IV). Oral Treatment Antibiotics Oral antibiotics are usually reserved for more severe acne (Williams et al., 2012). In addition to their antimicrobial properties they have substantial anti-‐inflammatory effects; however, their anti-‐inflammatory activity has only been established in vitro (Walsh et al., 2016). They are effective against inflammatory lesions but do not completely clear acne. They are used at low doses for long periods of time so there is an increased concern for antibiotic resistance. The evidence of the superiority of one type of antibiotic over another is limited; however, tetracyclines (tetracycline, minocycline, and doxycycline) are the preferred choice. The choice of which antibiotic to use should be evaluated based on the side effect profile and costs (Williams et al., 2012). For example, oral clindamycin is associated with serious gastrointestinal side effects and the liver and kidney function need to be monitored regularly with prolonged use (Walsh et al., 2016). Treatment with antibiotics for acne has been shown to increase the risk of developing upper respiratory tract infections (Margolis et al., 2005). Further, as increased understanding of the gut microbiome associates dysbiosis with diseases such as diabetes type II, obesity and allergic disease (Hernandez, 2016; Ipci et al., 2016; Turta and Rautava, 2016) established ideas regarding the risks and benefits of antibiotic use must re-‐ evaluated, particularly in vulnerable adolescent populations. The tetracycline class of antibiotics are considered the first line of treatment for moderate to severe inflammatory acne and are recommended as adjuncts to topical treatments. Tetracycline is the most recommended followed by minocycline and doxycycline. However, a Cochrane review did not find any evidence to support the use of one tetracycline over any other (Garner et al., 2012). Their mechanism of action is to inhibit bacterial protein synthesis by
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binding to the 30S ribosomal subunit. Demonstrated anti-‐inflammatory effects include inhibition of chemotaxis8 and metalloproteinase activity (Zaenglein et al., 2016). Tetracyclines are recommended for long-‐term use and are associated with a range of side effects that are covered in detail in Appendix 1. Common side effects include gastrointestinal disturbance and vaginal candidiasis. Darkening of tooth enamel has led to the contraindication of tetracyclines in children less than 8 years of age. Tetracycline is associated with adverse effects to the gastrointestinal system, teeth, skin, kidneys, liver, and blood as well as hypersensitivity reactions. Minocycline is most commonly associated with tinnitus, dizziness, and pigment deposition in the skin, mucous membrane and teeth; however, may also adversely affect the gastrointestinal system, genitourinary system, liver, respiratory system, kidneys, musculoskeletal system, and blood. Rarely it may cause drug-‐induced lupus and other hypersensitivity reactions. Doxycycline is associated with adverse effects to the gastrointestinal system, skin, kidneys, and blood as well as hypersensitivity reactions (Zaenglein et al., 2016, Supplement Tables XIV, XV & XVI). Combined oral contraceptives Combined oral contraceptives (COCs) that contain oestrogen (ethinyl estradiol) and progestogen are used because oestrogen suppresses sebaceous gland activity and reduces the formation of ovarian and adrenal androgens. Progestogen-‐only contraceptives should be avoided because they bind to both progesterone and androgen receptors. Third generation progestogens are more selective to progesterone receptors but carry an increased risk of thromboembolism (Williams et al., 2012). Five randomised, controlled trials have consistently demonstrated a significant improvement in acne vulgaris with COCs compared with placebo controls (Maloney et al., 2009; Plewig et al., 2009; Koltun et al., 2008; Lucky et al., 2008; Maloney et al., 2008). It should be noted, however, that in the United States the Federal Drug Administration (FDA) specifies that COCs should only be prescribed for acne in women who also desire contraception (Zaenglein et al., 2016). COCs containing ethinyl estadiol and a progestin are prescribed to treat acne in women after menarche. The contraindication and adverse effect profiles differ between drugs, which are covered in detail in Appendix II. COCs are primarily contraindicated in oestrogen or progestin 8
Attraction of cells, e.g. neutrophils, towards a chemical stimulus.
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sensitive cancers such as breast cancer, hepatic disease, and vascular disorders or diseases. Side effects associated with COCs can affect the cardiovascular system, gastrointestinal system, central nervous system, endocrine system, genitourinary system, blood, eyes, skin, liver and kidneys. COCs may also interact with a range of other drugs including drugs that may be used in acne treatment such as tetracyclines (Zaenglein et al., 2016, Supplement Tables XXIII, XIV, XXV, & XXVI). Isotretinoin Oral isotretinoin, made infamous under its trade name Accutane, is a vitamin A analogue that is very effective and results in a clinical cure 85% of the time. Relapse rates are 21% and are dose-‐ dependent. However, it is reserved for severe recalcitrant nodulocystic scarring acne because of the associated side effects (Williams et al., 2012). It is recommended for treatment for between 15 to 20 weeks. Side effects are covered in detail in Appendix III. Common side effects include cheilitis, dry skin, nose bleeds, secondary infection, temporary worsening of lesions, photosensitivity, increased serum lipids, and changes to the mucocutaneous, musculoskeletal and ophthalmic systems mimicking hypervitaminosis A. Less common side effects include inflammatory bowel disease, depression, anxiety, mood changes, cardiovascular risk factors, increased bone mineralization, increased scarring, and colonization with S. aureus. Isotretinoin may also interact with other acne drugs such as tetracyclines and COCs (Zaenglein et al., 2016; Williams et al., 2012). The link between isotretinoin and depression and suicide is disputed (Misery, 2011). Kontaxakis et al. (2009) reviewed the data linking isotretinoin with psychological effects. They concluded that there is a plausible link between isotretinoin and psychopathology not limited to depression, however, that more evidence is required to confirm a causal link. Isotretinoin is a known teratogen. In the United States hundreds of reports of congenital defects in exposed pregnancies were reported following isotretinoin’s initial release in 1982 (Dai et al., 1992). This led to the implementation of the risk management program iPLEDGE in the United States to prevent pregnancies exposed to isotretinoin. Despite this, however, there are still 150 isotretinoin-‐exposed pregnancies in the United States every year (Collins et al., 2014).
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Antibiotic resistance As the global use of antibiotics continues to rise, many countries have reported that more than 50% of P. acnes strains are resistant to topical macrolides9. Further, the incidence of P. acnes resistant bacteria found in patients has increased from 20% in 1978 to 62% in 1996. Both topical and oral antibiotics are of equal importance in the issue of antibiotic resistance. Part of the difficulty in detecting resistance is that it does not necessarily translate directly into treatment because acne is not exclusively an infectious disease. Therefore, resistance may manifest as no response or a reduced response to treatment (Walsh et al., 2016). An additional complication is that despite protocols to reduce antibiotic resistance, the adolescent population group remains notoriously difficult with regards to compliance (Williams et al., 2012). Resistance is not limited to P. acnes bacteria as topical antibiotics can affect all susceptible cutaneous bacteria. Resistant strains of S. epidermidis have been documented with an increase in the number of resistant strains following long-‐term antibiotic therapy (Nishijima et al., 2000). Mills et al. (2002) found that in 208 acne patients 87% had erythromycin-‐resistant coagulase-‐ negative Staphylococci, which increased to 98% after 12 weeks of therapy. No significant improvement was observed which the authors related to the additional presence of resistant P. acnes strains. One of the primary concerns regarding the development of resistant commensal bacteria such as P. acnes and S. epidermidis, is that they can become opportunistic pathogens in clinical settings where there are vulnerable populations such as the elderly or immune-‐compromised patients who are undergoing surgery. Moreover, what is of even greater concern is the increased incidence of resistant pathogenic bacteria as a result of antibiotic treatment for acne vulgaris. Topical and internal antibiotic use has been associated with an increase from 20%-‐85% in the number of tetracycline-‐resistant Streptococcus pyogenes found in the oropharynx (Levy et al., 2003) and increases in erythromycin-‐resistant S. aureus from 15%-‐40% have been found in the anterior nares following treatment with erythromycin (Mills et al., 2002).
9
A type of antibiotic that contains a lactone ring e.g. erythromycine
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Gentamicin and oxacillin resistant S. epidermidis isolates have been found among biofilm forming clinical strains (Kozitskaya et al., 2004). It has been shown in vivo that methicillin-‐ resistant genes can be transferred via horizontal gene transfer between S. aureus species (Wielders et al., 2001). Genome analysis indicates that horizontal gene transfer of methicillin-‐ resistant genes between S. epidermidis and S. aureus is possible (Gill et al., 2005; Hassan et al., 2004). Further, Staphylococci are noted for their adaptability and have been shown to have a high capacity for recombination indicating that S. epidermidis have the potential to evolve novel virulent and resistant traits (Zieburh et al., 2006). With the increasing number of methicillin-‐resistant Staphylococcus aureus (MRSA) related infections and deaths in a clinical setting the possibility of added collateral damage from methicillin-‐resistant S. epidermidis is a major concern. Moreover, commonly used antibiotics used to treat acne, clindamycin and doxycycline, are also used in the treatment of MRSA. Added to this the fact that resistant bacteria can easily spread through skin-‐to-‐skin contact between individuals who are receiving antibiotic acne treatment and vulnerable individuals who are not directly receiving treatment (Walsh et al., 2016). Complementary and alternative medicine Complementary and alternative medicine therapies are widely and popularly used; however, robust scientific evidence is lacking to support their usage in acne vulgaris (Williams et al., 2012). Over 100 plants have been implicated as having possible treatment effects in acne due to possessing antibacterial, anti-‐inflammatory, antioxidant and anti-‐androgen effects (Azimi et al., 2012). A single blind, randomised control trial compared tea tree oil with benzoyl peroxide in 124 patients and found that both significantly improved the number of inflamed and non-‐inflamed acne lesions. Tea tree oil was slower acting but presented with fewer side effects (Basset et al., 1990). A randomised, double-‐blind controlled trial found a 5% tea tree oil topical preparation to be more effective than control in 60 patients with regards to acne severity and total numbers of acne lesions (Enshaieh et al., 2007).
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Internally, Berberis vulgaris aqueous extract was shown to significantly reduce numbers of inflamed and on non-‐inflamed lesions and acne severity over placebo in 49 adolescents (Fouladi, 2012). In a novel approach to acne treatment taking into account the complex interrelationships of the microbiota, Bockmuhl et al. (2006) have looked into the role of herbs as prebiotics. The concept of prebiotics is well known in the gut where they are described as non-‐digestible food components that selectively support the growth of certain bacteria that can play a role in supporting human health (Gibson and Roberfroid, 1995). Bockmuhl and his team screened 100 plant extracts, 5 of which (Pinus sylvestris, Ribes nigrum, Lamium album, Thea sinensis, and Panax ginseng) showed the ability to inhibit P. acnes and promote the growth of S. epidermidis. A combination of P. sylvestris and R. nigrum extracts showed the best results. Expanding on this work they tested an extract combination of P. sylvestris, R. nigrum, and P. ginseng on the forehead of 11 female volunteers for 21 days. They used fluorescence in situ hybridization to observe bacterial populations on the skin to avoid the biases of bacterial culture assays. Although the small sample size was not statistically significant the results showed a trend towards decreasing the P. acnes population while the total bacterial population remained unchanged. Unpublished work by this team showed that the herbal formulation was better tolerated on the skin compared with conventional topical antibiotics. The authors hypothesized that prebiotic treatment has the theoretical potential to rebalance the skin microflora for long-‐ term improvement. However, further research is required to confirm this hypothesis. Several preliminary studies have found positive results looking at the role of supplements in acne. An observational study found a reduction in acne lesions with zinc combined with antioxidants (Sardana and Garg, 2010). A prospective, randomized, open-‐label trial found a reduction in acne lesions with probiotic treatment (Jung et al., 2013).
Propionibacterium acnes P. acnes is a gram-‐positive bacterium. It is non-‐motile having a typical coryneforme10 shape with irregular, short branching. They are not strict anaerobes as described in much of the literature because they can tolerate the presence of oxygen. They have been previously 10
Having a rod-‐like or club-‐like shape.
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classified as Corynebacterium spp., Bacillus spp., and anaerobic diphtheroids. Their structurally resilient Gram-‐positive cell wall makes them resistant to desiccation, osmotic shock and mechanical stress (Bojar and Holland, 2004). P. acnes is a commensal skin bacteria that is found in the sebaceous follicles of those with and without acne (Walsh et al., 2016; Dessinioti et al., 2010). P. acnes is involved in the early and late pathophysiological processes of acne (Walsh et al., 2016) including the induction and maintenance of inflammation. However, the exact mechanism of its involvement in acne pathogenesis is controversial (Dessinioti et al., 2010). Moreover there are distinct strains of P. acnes with some strains having been associated with more severe acne (Beylot et al., 2013). Key mechanisms through which P. acnes is involved in the pathogenesis of acne are production of lipases, proteases, and hyaluronidases; production of chemotactic agents; activation of classical and alternate complement pathways; induction of inflammatory cytokines TNF11-‐α, IL12-‐1α, IL-‐1β, IL-‐8; increases in the expression of TLR213, TLR4 and MMP14-‐9; and modulation of keratinocyte differentiation (Dessinioti et al., 2010; Ingram et al., 1980; Puhvel and Sakamoto, 1980; Webster and Leyden, 1980; Scott et al., 1979; Webster et al., 1979; Hoeffler, 1977; Puhvel and Reisner, 1972). Further, the formation of a biofilm may enhance the formation of microcomedones by binding corneocytes together to form a plug of the follicle (Burkhart and Burkhart, 2007). P. acnes has been shown to be involved in increased proliferation of keratinocytes with effects on keratinocyte differentiation. It activates the innate immunity through TLR2 and protease-‐ activated receptors, which induce the production of inflammatory cytokines and MMPs (Beylot et al., 2013; Del Rosso and Kircik, 2013; Jugeau et al., 2005; Shibata et al. 2009; Kim et al., 2002). The drug retinoic acid downregulates TLR2 thereby decreasing the release of inflammatory cytokines induced by P. acnes ligands (Liu et al., 2005 found in Dessinioti et al., 2010). 11
Tumour necrosis factor. Interleukin. 13 Toll-‐like receptors are mammalian analogues to toll proteins first identified in Drosophila. They are transmembrane proteins and form part of the innate immune response against pathogens such as bacteria, fungi and parasites. Pathogenic ligands that bind to the extracellular domains of TLRs result in nuclear translocation of the transcription factor nuclear factor kappa-‐B (NFκB) which regulates gene expression of many genes involved in the immune response including inflammatory cytokines (Dessinioti et al., 2010). 14 Matrix metalloproteinase: involved in the breakdown of the extracellular matrix and include collagenases, gelatinases, stomelysins, and matrilysins (Dessinioti et al., 2010). 12
19
The production of inflammatory cytokines by P. acnes followed by the initiation of the inflammatory cascade has been demonstrated in human sebocytes (Huang et al., 2015) and keratinocytes (Schaller et al., 2005; Graham et al., 2004; Heymann, 2006). In monocytes P. acnes has been shown to stimulate cytokines such as IL-‐1β, IL-‐8, IL-‐12 and TNF-‐α (Sugisaki et al., 2009; Kim et al., 2002; Vowels et al., 1995). Although, proinflammatory cytokines such as IL-‐1α and β and TNF-‐α have been found in healthy sebaceous glands as well (Antilla et al., 1992; Boehm et al., 1995). The relationship between acne pathogenesis and different inflammatory cytokines induced by P. acnes has also been explored. IL-‐1α has been shown to cause hypercornification (Guy et al., 1996; Guy et al. 1998). Il-‐8 is chemotactic towards neutrophils, which release lysosomal enzymes disrupting the epithelium and leading to increased inflammation (Hoeffer et al., 1976; Webster et al., 1980b). IL-‐12 leads to a shift towards a TH15-‐1 mediated immune response suggesting a possible autoimmune mechanism (Kim et al., 2002). TNF-‐α is involved in the maturation and migration of Langerhans cells (a type of dendritic cell) from the epidermis into the lymphatic circulation for presentation of antigens to T cells in local lymph nodes. This plays a vital part in the induction of the cutaneous immune response (Kimber et al., 2002; Caux et al., 1992; Kaplan et al., 1992). P. acnes modulates integrin and filaggrin protein expression. Integrins are involved in the proliferation and differentiation of keratinocytes and filaggrin plays a key role in the keratinisation process. Dysregulation of these processes: hyperproliferation; abnormal differentiation; and decreased desquamation possibly the result of premature termination of the keratinsation process, lead the formation of comedones (Jarrouse et al., 2007). Certain strains of P. acnes have been shown in keratinocytes to induce human β-‐defensin-‐2 mRNA, a type of antimicrobial peptide (Nagy et al., 2005). Modulation of antimicrobial peptide may play a role in acne pathogenesis, however, is also suggestive of the complex dynamics of the skin microbiome.
15
T helper cell.
20
Staphylococcus epidermidis Coagulase-‐negative S. epidermidis is a commensal skin bacteria that has been shown to co-‐ colonise with P. acnes in acne lesions (Fitz-‐Gibbon et al., 2013; Nishijima et al., 2000). S. epidermidis is involved in the protection of skin from infections and other environmental insults. Theoretically, S. epidermidis could play at role in skin barrier function and the development of innate immune responses (Krutmann, 2009). S. epidermidis has recently gained notoriety in a clinical setting where it can become an opportunistic pathogen in clinical environments (Christensen and Bruggeman, 2014). Nosocomial infections are usually associated with medical devices such as an intravascular or intrathecal16 catheter, urinary catheters and pace maker electrodes. Patients most likely to be affected are long-‐term care patients such as elderly patients, critically ill patients and immunocompromised patients. The ability to form biofilms has been identified as a key feature of pathogenic S. epidermidis strains. Biofilms are communities of microorganisms that secrete polysaccharides and proteins to form an extracellular matrix that can stick to surfaces such as metal or plastic. Biofilms serve to protect microorganisms from environmental stressors including antibiotics (Figure 4) (Ziebuhr et al., 2006).
Figure 4. Factors involved in the initial adherence and biofilm formation in Staphylococci. Taken from Ziebuhr et al. (2016). 16
Introduction into the spinal canal or subarachnoid space.
21
S. epidermidis produces compounds that inhibit the growth of non-‐native potentially pathogenic micro-‐organisms (Christensen and Bruggeman, 2014). Phenol soluble modulins (PSMs) are a family of peptides that are produced by almost all Staphylococci species. Δ-‐toxin a PSM in S. epidermidis was shown (in vitro and in vivo in mice) to interact with host antimicrobial peptides to enhance antimicrobial activity against Group A Streptococcus (Cogen et al., 2010). Bacteriocins are another group of peptides or proteins that have antibacterial activity. Lantibiotics are a class of bacteriocin peptides containing the amino acids lanthionine or methyllanthionine and are frequently produced by S. epidermidis (Christensen and Bruggeman, 2014). Pep5, epicidin 280, epilancin K7 and epidermin are all well characterised lantibiotics. Pep5 and epicidin are both produced by clinical strains of S. epidermidis. Pep5 and epidermin are both of interest in their potential to inhibit biofilm-‐producing strains of S. epidermidis in clinical environments (Bastos et al., 2009). S. epidermidis was also shown to modulate keratinocyte expression of antimicrobial peptides human β-‐defensin-‐2 and human β-‐defensin-‐3 as well as enhance inhibition of group A Streptococcus and S. aureus by cell lysate (Li et al., 2013; Lai et al., 2010). A recent study by Xia et al. (2016) has looked at the ability of Staphylococcal lipoteichoic acid (LTA) to reduce the P. acnes induced production of inflammatory cytokines interleukin (IL)-‐6 and tumour necrosis factor (TNF)-‐α in mouse ears. Their research demonstrated that this response was specific to keratinocytes rather than monocytes. The authors postulated this to be due to the need of keratinocytes to recognise commensal skin bacteria while monocytes are normally in a sterile environment. LTA was shown to induce the production of micro RNAs (miRNA), which are small noncoding single-‐stranded RNAs that regulate gene expression on a post-‐transcriptional level by preventing gene translation (Ambros, 2004). Only the inhibition of miR-‐143 abrogated the inhibitory effect of LTA on P. acnes induced production of IL-‐6 and TNF-‐α in keratinocytes. The mechanism of action was deduced through further experimentation showing firstly that LTA binding to the toll-‐like receptor 2 (TLR2) induced the production of miR-‐143 in human and murine keratinocytes as well as mouse ears. Further, TLR2-‐deficient keratinocytes were not 22
induced by LTA to produce miR-‐143. TLRs are well known for their role in producing proinflammatory signals in response to microbial stimuli (Lai et al., 2009). P. acnes was shown to be unable to induce an inflammatory response in TLR2-‐deficient mice. This confirms previous work that has indicated the role of TLR2 in inflammation induced by P. acnes (Kim et al., 2002). It was then demonstrated that miR-‐143 targets the 3’-‐UTR17 region of the TLR2 itself, decreasing the expression of TLR2. Overexpression of TLR2 by human keratinocytes abrogated the anti-‐inflammatory effect of LTA on P. acnes induced production of IL-‐6 and TNF-‐α. Finally, injection of mouse ears with miR-‐143 antagomir18 before being injected with P. acnes and LTA restored the inflammatory response that was inhibited by injection of P. acnes and LTA only. Figure 5 shows clearly the relationship of S. epidermidis mediated posttranscriptional regulation of TLR2 expression preventing the binding of ligands from P. acnes that lead to the production of inflammatory cytokines. The main limitation of this study, as admitted by the authors, was that the LTA used was sourced from S. aureus and not S. epidermidis, which is currently not commercially available. However, LTA sourced from S. aureus and S. epidermidis have been shown to have similar structure and function (Weidenmaier et al., 2004).
Figure 5. Staphylococcus epidermidis inhibits inflammation induced by Proprionibacterium acnes. Staphylococcal lipoteichoic acid binds to TLR2, which induces the production of miR-‐143 by keratinocytes. MiR-‐143 binds to the 3’UTR region of TLR2 mRNA preventing its translation into protein. The decreased production of TLR2 by keratinocytes results in a decreased 17
Untranslated region. Oligonucleotides that are used to prevent microRNA binding to mRNA.
18
23
production of inflammatory mediators TNF-‐α and IL-‐6 induced by the binding of P. acnes ligands to TLR2. Image taken from Skabytska and Biedermann (2016). Lai et al. (2009) also showed previously that Staphylococcal LTA suppressed skin inflammation during wound repair through TLR2-‐dependent inhibition of TLR3 mediated production of IL6 and TNF-‐α. Their findings are suggestive of the delicate balance required for maintaining skin homeostasis subsequent to injury. The presence of anti-‐inflammatory bacterial products is of value in preventing undesirable levels of inflammation that could impede wound healing. S. epidermidis has also been shown to inhibit the growth of P. acnes through the fermentation of glycerol in vitro. Further, inhibition of P. acnes did not occur in the absence of fermentation. Glycerol is a naturally produced metabolite on the human skin that can be metabolised to short chain fatty acids. This suggests a possible role for topical applications that provide substrates for fermentation in the treatment of acne (Wang et al., 2014). Different strains of S. epidermidis have been shown to have different levels of antimicrobial activity towards P. acnes. However, no difference in activity was detected between S. epidermidis collected from healthy skin versus acne-‐affected skin. Therefore the role of S. epidermidis in inhibiting P. acnes in an in vivo environment remains to be elucidated. In particular, it was shown that differences in environment, agar medium versus broth, resulted in different antimicrobial activity (Christensen et al., 2016). Overall, more work needs to be done to confirm the role of S. epidermidis in skin health in humans. However, these studies are suggestive of the complexity of interactions between resident microbes and hosts, and how these interactions can support and maintain health.
Current study – topical prebiotics There are two key reasons as to why alternatives to the current acne protocol should be considered: the growth of antibiotic resistance, and adverse effects caused by current drug treatments. The popularity of natural products for acne treatment makes research into this area important and vital for public interest. As our understanding of the complex interactions that occur between our body and the microbiome increase, holistic and novel ways of understanding and treating pathology become validated.
24
The current study draws strongly on the idea of a topical prebiotic proposed in the work of Bockmuhl et al. (2006) that identified several herbs that act selectively to inhibit P. acnes while leaving S. epidermidis populations intact. A prebiotic strategy would involve an antimicrobial strategy to reduce P. acnes overgrowth while leaving commensal bacterial such a S. epidermidis unharmed (Krutmann, 2009). Therefore, the current study proposes to identify herbs that selectively reduce P. acnes without inhibiting the growth S. epidermidis. Six herbs were selected for initial screening. Herbs were chosen based on meeting one or all of three criteria: traditional use in acne vulgaris, evidence for antibacterial properties and clinical evidence for use in acne. Other additional relevant factors in acne treatment such as antioxidant properties were considered where data on the original selection criteria was lacking. Phytolacca decandra L.
Figure 6. Phytolacca decandra. Photo credit Peter Jarrett.
25
Phytolacca decandra L. synonymous with P. americana, commonly known as pokeweed, belongs to the Phytolaccaceae family native to the Americas, but now distributed in Africa and Asia (Figure 6) (Patra et al., 2014). The eclectics19 traditionally used Phytolacca topically and internally for pustular skin conditions (Felter and Lloyd, 1898). One can infer that this could include pustular, inflammatory acne. P. americana crude methanolic extract strongly inhibited Porphyromonas gingivalis and Streptococcus mutans, bacteria implicated in periodontal inflammation and disease, however, the effect on Escherichia coli was negligible (Patra et al., 2014). A methanol extract of a related species P. dodecandra from the Ethopian tradition of plant-‐ based medicine has been shown to inhibit the growth of Pseudomonas aeruginosa but not S. aureus and E. coli (Tadeg et al., 2005). Hydromethanolic extract of the same species had a minimum inhibitory concentration (MIC) of 250 mg/mL for Streptococcus pyogens and P. aeruginosa but no effect on S. aureus, E. coli and Proteus vulgaris (Taye et al., 2011).
19
th
Eclectic medicine was a branch of American medicine that emerged in the 19 century that drew on a range of herbal medicines.
26
Calendula officinalis L.
Figure 7. Calendula officinalis. Photo credit Peter Jarrett. Calendula officinalis L. is commonly known as pot marigold and belongs to the Asteraceae family (Figure 7). It is native to Asia and southern Europe (Basch et al., 2006). Calendula has been shown to be antibacterial against S. aureus, which is a causative agent in skin infections and aggravates conditions such as psoriasis and atopic dermatitis (Roopashree et al., 2008). A calendula extract tested on oral biofilms did not show any antibacterial activity against Gram-‐ positive Streptococci bacteria, Actinomyces viscosus or Lactobacillus casei (Modesto, Lima and de Uzeda, 2000). Dumenil et al. (1980) found amongst dry flower extracts of water, 45% and 80% ethanol, only the 80% ethanol extract showed antibacterial activity against E. coli, P. aeruginosa, S. aureus and Streptococcus fecalis at concentrations of 50 mg/mL and 25 mg/mL for S. aureus and S. 27
fecalis. A fresh plant extract using water and acetone showed antibacterial activity against S. aureus at concentrations of 10 mg/mL and against S. fecalis at 50 mg/mL. Methanolic and ethanolic extracts were shown to have some antibacterial effects against Gram-‐positive and Gram-‐negative bacteria including S. aureus, but not as much as the antibiotic ciprofloxacin at the same concentration (Efstratiou et al., 2012). Ethanolic and aqueous extracts of Calendula flowers were shown to inhibit coagulase-‐positive Staphylococci, coagulase-‐negative Staphylococci, and P. aeruginosa. No inhibitory effect was demonstrated in Enterococci spp. (Mathur and Goyal, 2011). Aqueous and ethanolic flower extracts were shown to inhibit the growth of E. coli, Salmonella typhi, Klebsiella pneumoniae, Enterobacter aerigenes but not Agrobacterium tumefaciens (Bissa and Bohra, 2011). Antibacterial activity against Gram-‐positive bacteria was demonstrated from the hexanic fraction of an ethanolic extract of Calendula flowers but not the dichloromethane fraction. Different strains of S. aureus showed MICs ranging from 0.19 mg/mL to 4.37 mg/mL (Parente et al., 2011). Oleanolic acid (OA) and its glycoside and glucuronide derivatives from Calendula were tested for antibacterial activity. OA was shown to have the most antibacterial activity and demonstrated antibacterial activity against S. epidermidis. Data for any antibacterial activity of the OA glycosides or glucuronides was not reported. Free OA is found in the roots (not the flowers) of Calendula derived from the deglycosylation of accumulated OA glycosides (Szakiel et al., 2008).
28
Mahonia aquifolium (Pursh) Nutt.
Figure 8. Mahonia aquifolium. Photo credit Peter Jarrett. Mahonia aquifolium (Pursh) Nutt. synonymous with Berberis aquifolium is commonly known as Oregon grape and belongs to the Berberidaceae family (Figure 8). Mahonia is native to western North America and was traditionally used in the treatment of dermatological disease (Wiesenauer and Ludtke, 1996). Mahonia contains the alkaloid berberine with known antimicrobial activity (Cernakova and Kostalova, 2002). One study tested 20 strains of coagulase-‐negative staphylococci including 14 strains of S. epidermidis and 20 strains of P. acnes against a crude extract from the stem and bark of Mahonia as well as against the main protoberberine alkaloids, berberine and jatrorrhizine. The MICs indicating the lowest concentration for inhibiting microbial growth were determined. P. acnes was inhibited the most by the crude extract and alkaloids relative to the Staphylococci species. One strain of S. epidermidis was not inhibited by any agent at the highest tested concentration of 500 µg/mL. This was in contrast to the antibiotic ciprofloxacin which inhibited all strains of staphylococci. The study concluded that the crude extract and protoberberine alkaloids of Mahonia were inferior to antibiotic drugs currently used in clinical practice (Slobodnikova et al., 2004).
29
The hydroxylated alkaloids jatrorrhizine and magnoflorine extracted from the stem bark of Mahonia were shown to have antioxidant effects in ethanolic and liposomal bilayer environments. The antioxidant properties were more potent in the liposomal bilayer owing to the lipophilic nature of the compounds. Berberine was only shown to have modest antioxidant properties, which was related to its chemical structure lacking a hydroxyl group available for hydrogen donation in redox20 reactions (Rackova et al., 2004). Berberis vulgaris L.
Figure 9. Aerial parts of Berberis vulgaris, taken from Zarei et al. (2015). Berberis vulgaris L., commonly known as barberry, is native to Europe, North Africa and Asia (Figure 7) (Zarei et al., 2015). The one of the key constituents of Berberis is the isoquinoline alkaloid berberine. Berberine is well known for its antibacterial properties against a range of bacteria including methicillin-‐resistant S. aureus (Kang et al., 2015; Peng, et al., 2015; Azimi et al., 2012, Yu et al., 2005). Berberine has been shown to be bacteriostatic against S. epidermidis and inhibit biofilm formation at doses smaller than the MIC (Wang et al., 2009). Further, in vivo berberine has been shown to restore intestinal gut flora following treatment of Clostridium difficile infection with the antibiotic vancomycin, suggesting bacterial modulating capabilities (Lv et al., 2015).
20
Reduction-‐oxidation.
30
A 4-‐week placebo-‐controlled trial in 49 adolescents found significant improvements in total acne lesions and acne severity in participants given 600 mg capsules per day of an aqueous extract of Berberis (Fouladi, 2012). Lavandula angustifolia L.
Figure 10. Lavandula angustifolia. Photo credit Peter Jarrett. Lavandula angustifolia L., commonly known as lavender, is in the Lamiaceae family and is native to the Mediterranean (Figure 10). Lavandula is traditionally indicated in acne and has plausible theoretical indications for acne due to anti-‐inflammatory and antimicrobial properties (Basch et al. 2004). Most studies on Lavandula have been done using the essential oil only, which limits the conclusions that can be made regarding the whole plant extract. Several studies have demonstrated the antibacterial properties of Lavandula essential oil against MRSA (Ester et al., 2014; Roller et al., 2009). One study demonstrated antibacterial activity of Lavandula essential oil varieties from Poland against non-‐MRSA S. aureus. Different varieties of Lavandula were shown to have different chemical profiles relative the volatile oils present. The different varieties demonstrated different antibacterial strengths (Adaszynska et
31
al., 2013). Other studies have also demonstrated the antibacterial activity of Lavandula against Staphylococcus ssp. including S. epidermidis (Sienkiewicz et al.; Sokovic et al. 2010). An ethnobotanical study from Turkey looked at the whole plant ethanolic exract from a related species; Lavandula stoechas. The extract was shown to have no antibacterial activity against S. epidermidis. A relevant traditional use for this species is topically on inflamed wounds (Uzun et al., 2004). Echinacea purpurea (L) Moench/ angustifolia DC.
Figure 11. Echinacea purpurea. Photo credit Peter Jarrett. Echinacea purpurea (L) Moench and Echinacea angustifolia DC. are both commonly referred to as Echinacea (Figure 11). They are in the Asteraceae family and are native to central North America. Traditional indications and theoretical indications with limited evidence include treatment for acne. Traditional usage with precedence and recommendations by modern practitioners include topical application to promote wound healing (Basch et al., 2005). A proprietary 65% ethanol extract of Echinacea purpurea aerial parts and root, Echinaforce® by A. Vogel Bioforce AG, was found to significantly inhibit the growth of P. acnes bacteria. Further, the same inhibitory effect was not shown for S. aureus, although the data was not shown. P. 32
acnes was also shown to stimulate the production of the inflammatory cytokines 1L-‐6 and 1L-‐8 on trachea-‐bronchial and lung derived human epithelial cell lines. Incubation of the epithelial cells and P. acnes with a 1:100 dilution of Echinacea extract resulted in an inhibition of cytokine production. This suggests the potential for Echinacea in treating two key aspects in the known pathogenesis of acne, inflammation and P. acnes proliferation (Sharma et al. 2011). Methods
Preparation of ethanolic extracts Six proprietary alcoholic herbal extracts were selected for the initial bacterial sensitivity test. These were Phytolacca decandra, Calendula officinalis, Berberis aquifolium, Berberis vulgaris, Lavandula angustifolia and a blend of Echinacea purpurea (60%) and Echinaea angustifolia (40%). Table 1 shows the tincture strengths (drug extract ratio), percentage alcohol, manufacturer, batch number and expiration date of the selected herbs. The concentrations of the equivalent amount of dried or fresh herb (g) per mL were 1.0 g/mL for Phytolacca, 0.625 g/mL for Calendula, and 2.5 g/mL for the other four plants. However, the amount of plant extract obtained from a fresh plant does not account for the water weight, which is 80% on average (Bone, 2003). Therefore it should be noted that the amount of plant material used in fresh plant extraction of Lavender would be significantly less than that used in a dry plant extraction. Mediherb has high standards of quality assurance, which includes following guidelines published by the British Pharmacopoeia (British Pharmacopoeia Commission, 2016), to ensure the identity of the plant used, and the quality of the product. The guidelines published by the British Pharmacopeia for Calendula can be found in Appendix IV. The quality control specifications completed by Mediherb for Calendula tincture are presented in Appendix V. The certificate of analysis for Calendula 1:2 tincture batch number 157038 (Table 1) is presented in Appendix VI.
33
Plant Phytolacca decandra Calendula officinalis Mahonia aquifolium Berberis vulgaris Lavandula angustifolia Echincea purpurea (60%)/ Echinacea angustifolia (40%)
Drug extract ratio 1:5 (dry)
Ethanol (%) Manufacturer
Batch number
45
6978/1
Expiration date (m/y) 07/18
1:2 (dry)
90
157038
04/16
1:2 (dry)
25
157202
06/16
1:2 (dry)
45
1:2 (fresh)
45
1:2 (dry)
60
Phyto Pharmaceuticals, UK Mediherb, Australia Mediherb, Australia Rutland Biodynamics, UK Rutland Biodynamics, UK Mediherb, Australia
130656-‐ 07/18 12/3 150716-‐7/2 06/20 160305
06/16
Table 1. The drug extract ratio (amount of herb (g) relative to extraction solvent (mL), whether the extraction used fresh or dry plant material, the percentage (%) ethanol used as an extraction solvent, the product manufacturer and country of origin, batch number and expiration date (month/year). A total of 5 mL of each sample was placed in a nitrogen evaporator TurboVap® LV produced by Caliper Life Sciences at 60°C for 2 hours. Nitrogen is an inert gas so no oxidative changes should have occurred in the herbal extracts. The samples were then freeze dried. Five days later the dried herbal residues were dissolved in either water or dimethyl sulfoxide (DMSO). Phytolacca, Mahonia, Berberis, Lavandula, and Echinacea were dissolved in 1 mL deionised water. Calendula was dissolved in 2mL water and 2 mL DMSO. The new concentrations of the equivalent amount of dried or fresh herb (g) per mL were 1 g/mL for Phytolacca, 0.625 g/mL for Calendula, and 2.5 g/mL for the other four plant extracts.
34
Bacterial Sensitivity Testing Staphylococcus epidermidis NCIMB 12721 and Proprionibacterium acnes NCIC 737 were tested for bacterial sensitivity. S. epidermidis and P. acnes were prepared in a broth with a turbidity of 0.5 McFarlands equivalent to 1.5x10^8 CFU (colony forming units) per mL. Three replicates were performed to test the bacterial sensitivity of P. acnes and S. epidermidis to Phytolacca, Calendula, Mahonia, Berberis, Lavandula, and Echinacea, a negative control of 50% water/ 50% DMSO, and a positive control of ampicillin with a disk potency of 25 µg. Large plates were prepared using a Muller-‐Hinton standardized agar medium at a depth of 4 mm. To perform the well-‐diffusion assay seven wells were made in each plate and 50 µL of each sample was placed in a single well in the agar medium. The ampicillin disk was placed in the centre of the plate and sensitivity was tested using the Kirby-‐Bauer disk diffusion method. P. acnes was incubated in anaerobic conditions using a gas pack (Thermo Scientific brand AnaeroGen™ 2.5 L) and air tight container. Both P. acnes and S. epidermidis were incubated at 37°C. S. epidermidis was incubated for 24 hours and P. acnes was incubated for 92 hours because it is a slower growing bacteria. The diameters of the inhibition zones were manually measured with a ruler against black paper to improve colony visibility. After 96 hours the zones of inhibitions in the plates containing P. acnes overlapped making measurement impossible. The experiment was repeated with small plates containing a single well or disk. In the second experiment only two replicates were completed.
Minimum Inhibitory Concentration Herbal extract Calendula 90% ethanolic extract (drug extract ratio 1:2) produced by Mediherb, Australia, batch number 157038, expiration date 04/16 was selected for further testing. A sample of 5 mL of Calendula was evaporated using the nitrogen evaporator TurboVap® at 25⁰C. Following nitrogen evaporation the sample was freeze dried.
35
After 6 days the evaporated herbal residue was dissolved in 1mL of DMSO. A total of 400 µL of herbal extract dissolved in DMSO was added to 400µL deionised water. The new concentration of the equivalent amount of dried herb (g) per mL was 1.25 g/mL. Five two-‐fold serial dilutions were made from the original concentration of 1.25g/mL. The dilutions were made in 50% DMSO and 50% sterile deionised water. The dilutions were 0.625 g/mL, 0.31 g/mL, 0.16 g/mL, 0.078 g/mL and 0.039 g/mL. The original concentration, the 5 dilutions and a 50% DMSO/ 50% water control were each added to a well in a single large plate. Three replicates each were made against P. acnes and S. epidermidis. The plates were incubated at 37⁰C, S. epidermidis for 24 hours and P. acnes for 4 days. After the incubation period the diameter of the inhibition zones were manually measured with a ruler.
Preparation of aqueous extraction Calendula flower heads were purchased from Neal’s Yard, (Covent Garden, London, UK), batch number 9350. A total of 5.09 g of raw material were soaked for 15 minutes at ambient temperature in 150 mL deionised water. The flower heads were then heated to boiling (~100°C) and were maintained at boiling temperature for 10 minutes. The water and flower heads were agitated frequently to ensure adequate mixing. The aqueous extraction was filtered through No.3 Whatman filter papers (thick, medium speed paper with high retention) via vacuum filtration. The aqueous extract was then placed in a rotary evaporator for 5 hours to concentrate the solution. The final concentration of the equivalent weight of dried herb (g) per mL of water was 0.625 g/mL.
36
Statistical analysis Standard deviation of the mean was used to assess statistical significance of values. Means and standard deviation were calculated using the STANDEV function in Microsoft Excel version 14.0.7165.5000.
Results and Discussion
Preparation of extracts A major disadvantage to dissolving the tinctures in water is the loss of constituents that are not soluble in water. In particular the dried residue of Berberis had a significant amount of black resinous material that did not dissolve. The water extract of Phytolacca also had a visible precipitate. The advantage of this method was to not inhibit the growth of the bacteria due to potential antibacterial properties of the solvent (e.g. ethanol).
37
B
A
C
D
E
F
G
H
I
Figure 12. Bacterial sensitivity of S. epidermidis (A) and P. acnes (B-‐I) to Phytolacca (1), Calendula (2), Mahonia (3), Berberis (4), Lavandula (5), Echinacea (6), DMSO (7) and ampicillin (Amp.). 38
Bacterial Sensitivity
InhibiUon Diameter (mm)
60 50 40 30
P. acnes
20
S. epidermidis
10 0
Figure 13. Zones of bacterial inhibition measured by total diameter (mm) minus the well diameter (6 mm). Means plus standard deviations of the mean are based on 2 replicates for P. acnes and 3 replicates for S. epidermidis. Calendula, Mahonia, Berberis, Lavandula and Echinacea all inhibited P. acnes. Berberis showed the greatest inhibition of 30±5.7 mm. Mahonia, Berberis, Lavandula, and Echinacea all inhibited S. epidermidis (Figure 12). Again, Berberis showed the greatest inhibition with a value of 13±1 mm (Figure 13). The inhibition diameters presented exclude the well diameter (6 mm), which would have falsely affected the mean and standard deviation where one of three replicates was measured as zero. Only Calendula inhibited P. acnes showing an inhibitory diameter of 10±0 mm, but not S. epidermidis suggesting selective inhibition of P. acnes over S. epidermidis (Figure 13). However, it should be noted that Calendula had the lowest concentration equivalent to dried herb, 0.625 g/mL compared with 2.5 g/mL for Berberis. This suggests that higher concentrations of Calendula could show larger inhibitory diameters. The negative control DMSO showed no antibacterial effect on either P. acnes or S. epidermidis (Figure 1) indicating that the DMSO used as a solvent for the dried Calendula extract did not affect the bacterial inhibition shown by Calendula. 39
Ampicillin showed significantly higher inhibitory diameters than all the plant extracts, 55.5±0.7 mm and 14.3±1.2 mm for P. acnes and S. epidermidis respectively (Figure 1). The inhibitory diameter for S. epidermidis including the well diameter was 20±1.2 mm, less than interpretive standard of 28 mm for Staphylococcus spp., indicating possible resistance (CLSI, 2012, p74). However, it should be noted that 25 µg of the single compound ampicillin is not directly comparable to plant extracts at concentrations equivalent to dried plant material. For Calendula 50 µg at a concentration of 0.625 g/mL is equivalent to 31.3 mg of dried herb. Further, the amount of crude dried extract, which contains a range of phytochemicals extracted from a dried herb, is variable and depends on the extraction method (Nizynski et al., 2015). Efstratiou et al. (2012) found that the amount of dried extract obtained from ethanolic extraction was 17.4 g/100 g dried petals and Fonesca et al. (2010) found that for a 50% hydroethanolic extraction of the dried flowers the dry weight yield was 14.9±3.2%. From this data, theoretically the amount of dried extract in 31.3 mg dried plant material could range from 5.45 mg to 3.66mg or 5400 µg to 3700 µg. Again this theoretical yield would contain hundreds of phytochemicals rather than a single concentrated compound. Therefore, it is only possible to conclude that an unknown concentration of the crude extract derived from 31.3 mg of dried Calendula flowers is less antibacterial when compared to 25 µg of Ampicillin. Whether the antibacterial constituents of Calendula are less antibacterial than ampicillin remains unknown.
40
Minimum inhibitory concentration
A
B
Figure 14. Minimum inhibitory concentration of Calendula ethanolic extract on P. acnes (A). Diluted concentrations are 1.25 g/mL (1), 0.625 g/mL (2), 0.31 g/mL (3), 0.16 g/mL (4), 0.078 g/mL (5), and 0.039 g/mL (6). Well 7 is the negative control DMSO. The same extracts showed no inhibitory effects towards S. epidermidis (B).
InhibiUon Diameter (mm)
18 16 14 12 10 8 6 4 2 0 1.25
0.625
0.31
0.16
0.078
0.039
Calendula Extract DiluUons (g/mL)
Figure 15. Inhibition diameters for serial dilutions of Calendula extract applied to P. acnes. Concentration of extract is the equivalent dried weight of Calendula per mL of distilled water. For clarity the inhibition diameter is the total diameter (mm) minus the well diameter (6 mm). Means and standard deviations are based on 3 replicates. 41
Concentrations of Calendula showing two-‐fold serial dilutions demonstrated a linear antimicrobial effect on P. acnes (Figure 15). The Calendula extract did not inhibit S. epidermidis growth. DMSO at a concentration of 50% also did not inhibit P. acnes indicating that it did not influence the antimicrobial effect of the Calendula extract (Figure 14). The MIC was 0.078 g/mL equivalent to dried herb (Figure 15). Mathur and Goyal (2011) showed the MIC for coagulase-‐negative Staphylococci for an ethanolic extract of Calendula flowers was 15.0 mg/mL. This suggests that considerably higher concentrations equivalent to higher quantities of crude extract might inhibit S. epidermidis.
Aqueous extraction of Calendula A
B
Figure 16. Calendula aqueous extract, at 0.034 g/mL (1) and 0.625 g/mL (2) equivalent to dried herb weight, applied to agar medium seeded with either P. acnes (A) or S. epidermidis (B). Zones of inhibition can be seen on the plate with P. acnes, but not on the plate with S. epidermidis. A concentration of 0.625 g/mL equivalent to dried herb was obtained from aqueous extraction. Three replicates applied to cultures of P. acnes and S. epidermidis showed an inhibitory diameter of 10.3±0.58 mm for P. acnes and 0 mm for S. epidermidis (Figure 16). These results show the same degree of bacterial inhibition for an aqueous extract as compared with a
42
hydroethanolic extract (10±0 mm). These results indicate that the antibacterial compounds in the Calendula extract are soluble in both water and ethanol. The original infusion 0.034 g/mL equivalent to dried herb also showed some bacterial inhibition with an inhibitory diameter of 3.3±0.58 (minus the 6 mm well). The ethanol extract at a concentration of 0.039 g/mL equivalent to dried herb did not show any inhibition. This could suggest that there are some differences in the constituents in the aqueous extraction that are responsible for the bacterial inhibition. Another possibility is that the antibacterial activity at that concentration was enhanced due to the presence of volatile oils. A major limitation with rotary evaporation is the potential loss of active constituents such as volatile oils through the process of heating and removing water vapour, as volatile oils evaporate at lower temperatures than water. The essential oil of Calendula has been shown to be antifungal (Gazim et al., 2008). However, antibacterial assays on the essential oil have not been done. One of the major essential oil constituents of Calendula flowers α-‐thujene (Okoh et al., 2008) has been shown to be antibacterial as one of the main constituents in an essential oil mix in Juniperis excelsa (Sela et al., 2015), although no data exists as to the antibacterial properties of α-‐thujene alone. Mathur and Goyal (2011) found the MIC for coagulase-‐negative Staphylococci for an aqueous extract of Calendula flowers to be 17.5 mg/mL. Again, this suggests that considerably higher concentrations equivalent to higher quantities of crude extract might inhibit S. epidermidis.
Commensal bacteria The main topical antibiotics used in acne treatment erythromycin and clindamycin have been shown to be antimicrobial against both P. acnes and S. epidermidis found in acne lesions. Further, both strains but especially S. epidermidis can become resistant following long-‐term antibiotic therapy (Setsuko et al., 2000). The current study has demonstrated that in vitro Calendula ethanolic and aqueous extracts have the potential to selectively inhibit P. acnes over S. epidermidis. Theoretically this would allow the skin to retain the anti-‐inflammatory and antimicrobial effects of S. epidermidis colonisation (Christensen and Bruggeman, 2014; Lai et al., 2009).
43
Further, theoretical effects of commensal bacteria on programming of innate immunity are relevant and warrant further investigation. The lysate from the non-‐pathogenic Gram-‐negative bacterium Vitreoscilla filiformis was found to alleviate symptoms of atopic dermatitis possibly through a reduction in S. aureus and a direct immunomodulatory effect on the skin (Gueniche et al., 2008). An in vivo study by Volz et al. (2014) looked at the potential mechanism for the reduction of inflammation by V. filiformis which induced a tolerogenic dendritic cell phenotype characterised by anti-‐inflammatory IL-‐10 production and the induction of type 1 regulatory T cells. The effect of V. filiformis lysate on dendritic cells is evocative of the role bacteria play, developed through evolutionary processes, in programming healthy immune responses.
Bacterial resistance Bacterial resistance continues to be a growing problem worldwide. As bacterial resistance of P. acnes and S. epidermidis as a result of antibiotic treatment of acne is on the increase (Walsh et al., 2016) novel approaches to acne treatment must be considered. Alternative therapies such as herbal extracts provide promising avenues of research. Although this study did not identify a specific antimicrobial compound in Calendula, it is important to look at the broader picture of phytochemical extracts. Proponents of phytotherapy argue that phytochemicals work synergistically to enhance efficacy of documented effects. Recent research into the berberine-‐containing plant Hydrastis canadensis found that the antibacterial properties of berberine were enhanced by a whole plant extract of the aerial plants. Constituents found in the aerial parts induced efflux pump inhibition in S. aureus, which was not induced by berberine or other known alkaloids canadine and hydrastine (Ettefagh et al., 2011). This research takes into account the complexity of plant extracts and their mechanisms with the promise of complex synergistic strategies that may serve as effective models to combat and reduce the incidence of antibiotic resistance. 44
Calendula Written historical accounts of Calendula used as a medicinal plant can be found in the writings of Pliny and Virgil (Macht, 1955). Historically used in the 12th century for impetigo (bacterial skin infection) by Hildegarde von Bingen (Dumenil et al., 1980). Written references to Calendula can also be found in the 13th and 14th centuries and interpretation of these writings suggests wound-‐healing capabilities (Patrick et al., 1996). In Ayurvedic medicine Calendula has traditionally been used for the treatment of blepharitis, eczema, gastritis, minor burns, warts, sprains and wounds (Mathur and Goyal, 2011). Modern actions of Calendula include vulnerary
21
, anti-‐inflammatory, styptic
22
, and
antimicrobial (Bone, 2003). The German Commission E lists actions for Calendula as wound healing and anti-‐inflammatory (American Botanical Council, 2013). Research into Calendula plant extracts have demonstrated antimicrobial, anti-‐inflammatory, would-‐healing, angiogenic, antioxidant, immunostimulant and antitumor effects, as well as improvement in skin parameters (Agatonovic-‐Kustrin et al., 2015; Efstratios et al., 2012; Parente et al., 2012; Akhtar et al., 2011; Fronza et al., 2009; Preethi et al., 2008; Ukiya et al., 2006; Varlijen et al.; 1989). All of these properties have a potential role in the treatment of pathological processes of acne such as inflammation, keratinocyte hyperproliferation, overgrowth of P. acnes, and healing following the rupture of an acne lesion. Clinical trials supporting wound-‐healing and anti-‐ inflammatory properties of Calendula topical preparations have looked at reduced severity in radiotherapy-‐induced dermatitis and oropharyngeal mucositis, and diaper dermatitis as well perineal healing after episiotomy23 (Kodian and Amber 2015; Babee et al., 2013; Eghdampour et al. 2013; Panahi et al., 2011). Only one clinical trial has looked at antimicrobial effects with regards microbial adherence to sutures following the extraction of unerupted maxillary third molars (Faria et al., 2010).
21
Promotes healing of wounds when applied locally (Bone, 2003). Helps to stop bleeding when applied locally (Bone, 2003). 23 Surgical cutting of the perineal mucles during the second stage of labour to prevent tearing during the passage of the foetus through the vagina. 22
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Dosage The German Commission E recommends 2-‐4 mL tincture24 per day and 1-‐2g of flower heads in 150 mL of boiling water up to 3 times a day. For topical use the equivalent of 2-‐5 g of flower heads in 100g of ointment can be used (American Botanical Council, 2013). Bone (2011) recommends a dosage of 1.5-‐4.5 mL per day of a 1:225 liquid extract from flower heads based on the recommendations from the British Herbal Pharmacopoeia. Constituents More than 150 chemicals have been isolated from different fractions and subfractions of Calendula using lipophilic, water-‐alcoholic, and supercritical CO2 extractions. The chemical groups included: minerals, carbohydrates, lipids, tocopherols, amino acids, phenolic acids, tannins, coumarins, flavonoids, quinones, steroids and sterols, monoterpenes, sesquiterpenes, triterpenes, and resins. Extracts made using maceration of plant material and percolation with ethanol and water as a solvent were found to contain compounds that were polar and medium polar such as flavonoids, coumarins, carotenoids, terpenoid glycosides, phenolic acids, and tannins (Martins et al., 2014; Andersen et al., 2010; Fonseca et al., 2010; Roopashree et al., 2008). More detailed information regarding Calendula chemical composition can be found in Appendix VII. Carotenoids have been extracted from dried petals (Kishimoto et al., 2005; Bako et al., 2002). Carotenoids are precursors to vitamin A and retinoids in animals and humans. They are known to be antioxidant, immune-‐enhancing, and antimutagenic. The carotenoid content changes when fresh Calendula flowers are dried. The main carotenoid in dried petals has been found to be lutein at 15-‐25%. In alcoholic tinctures the carotenoid content has been shown to decompose (Bako et al., 2002). Major components of Calendula are the triterpenoid esters including the faradiol esters: lauryl, myristoyl, and palmitoyl (Hamburger et al., 2003). Diethyl ether extracts found the highest 24
Hydroethanolic extract with a ratio of dried herb (g) to ethanol and water (mL) being equal or greater than 1 to 3 (1:3). This means that 3 mL of extract is equivalent to 1 g of herb. 25 The ratio of dried herb (g) to ethanol and water (mL), 2 mL of extract is equivalent to 1 g of dried herb.
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levels of triterpenoids in the ligulate flowers (182 mg/g), which was nearly twofold higher than those found in the flower heads (107.4 mg/g) (Nizynski et al., 2015). Anti-‐inflammatory properties have been attributed to the triterpenoids in Calendula (Ukiya et al., 2006; Neukirch et al., 2005; Della Loggia, 1994). Other constituents extracted from Calendula with associated properties include flavonoids as antioxidants, amino acids accelerating wound healing, essential oil as antifungal and antibacterial, saponins as antitumour, calendin and calendulin as anticancer and lymphocyte-‐ stimulating, and polysaccharides as immunostimulant (Muley et al., 2009). Calendulasaponins A, B, C and D and ionone glucosides (officinosides A and B) have been found to have hypoglycaemic, gastric emptying inhibiting and gastroprotective effects (Marukami et al., 2001). Antimicrobial The ethanolic and methanolic extracts of Calendula dried petals showed antifungal activity against Candida spp., Aspergillus spp. and Exophala dermatitidis comparable to the antifungal agent fluconazole at a concentration 10x greater (Efstratios et al., 2012). These findings are in contrast to the findings of Chakraborthy (2008) who used a dried leaf extract and did not find antifungal activity. Discrepancies in the results may be a result of the different phytochemical profiles of the flowers and the leaves; for example, the essential oil from the flowers of Calendula has been shown to have antifungal activity (Gazim et al., 2008). The antifungal properties may play a role in managing Malassezia spp. overgrowth in acne lesions. However, the exact role of Malassezia spp. remains to be elucidated (Bojar and Holland, 2004). Antibacterial activity of Calendula extracts has been demonstrated for a range of pathogenic bacteria such as S. aureus and Streptococci bacteria (Efstratiou et al., 2012; Bissa and Bohra, 2011; Mathur and Goyal, 2011; Parente et al., 2011; Roopashree et al., 2008; Modesto et al. 2000; Dumenil et al. 1980). However, to the best knowledge of the researcher no previous studies have investigated the antibacterial properties of Calendula extract towards P. acnes and S. epidermidis. One study investigated the effects of the triterpenoid oleanolic acid against S. epidermidis and found antibacterial effects. However, it should be noted that oleanolic acid is found naturally occurring in the roots of Calendula (Szakiel et al., 2008). 47
Anti-‐inflammatory From early pathogenesis to exacerbation of existing acne lesions inflammation plays a major role in acne vulgaris. Inflammatory events have been found in the earliest stages of acne formation (Jeremy et al., 2003). The anti-‐inflammatory properties of Calendula could be of significant benefit to the treatment of acne. However, further studies using in vitro and in vivo acne models are required to establish this link. An in vivo study looked at acute and chronic inflammation models in mice. The Calendula ethanolic extract produced by maceration (a common extraction process used by herbalists) was administered as a dried residue orally at ranges of 100, 250 and 500 mg/kg. Rat paw oedema in carrageenan-‐induced (acute) and formalin-‐induced (chronic) inflammation was significantly reduced. In vitro, murine lung fibroblast cytotoxicity induced by the macrophage supernatant of lipopolysaccharide (LPS) treated mice was significantly reduced when mice were concomitantly treated with Calendula extract. The serum of LPS and Calendula treated mice had reduced levels of the inflammatory cytokines IL-‐1β, IL-‐6 and TNF-‐α. Similarly, spleen from LPS and Calendula treated mice showed reduced COX-‐226 expression as determined by gel electrophoresis (Preethi et al., 2008). In vivo studies cannot be extrapolated to human studies; however, it should be noted that doses of 250 mg/kg and 500 mg/kg are considerably higher than what are recommended internally for humans (American Botanical Council, 2013). An in vivo study in rats looked at oral and intracolonic delivery of Calendula hydroethanolic extract in the treatment of an acetic acid-‐induced ulcerative colitis model. Complete resolution of inflammatory changes was seen in rats treated with a 3000 mg/kg oral extract and a 20% intracolonic gel. Levels of malondialdehyde were also decreased which serves as a marker for tissue damage, inflammation and lipid peroxidation (Tanideh et al., 2016). Antioxidant Chronic inflammation increases oxidative stress (Preethi et al., 2008). Further, the oxidation of lipids found in sebum can trigger the production of inflammatory mediators (Walsh et al., 2016). In this way the antioxidant properties of Calendula could play a role in acne treatment and inflammation reduction. Again, research using acne models is required to confirm this link. 26
Cyclooxygenase-‐2, enzyme that converts arachidonic acid to prostaglandin E2. A proinflammatory marker.
48
Calendula extracts produced by supercritical CO2 extraction and Soxhlet ethanolic extraction demonstrated anti-‐oxidant activity. Some of this activity may have been due to the presence of the antioxidant chamazulene found in both extracts (Agatonovic-‐Kustrin et al., 2015). Fonesca et al. (2010) looked at antioxidant properties of a 50% hydroethanolic extract in vitro and in vivo. The Calendula extract showed antioxidant activity comparable to quercetin. Using glutathione as an indication of UVB 27 -‐induced epidermal oxidative stress, irradiated mice treated with 150 – 300 mg/kg of Calendula extract showed glutathione levels similar to those of untreated mice. Further, Calendula treated irradiated mice showed a change in matrix metalloproteinase activity. As MMPs are involved in both pro-‐ and anti-‐inflammatory processes the authors suggest that this change may be reflective of the potential protective effect of Calendula extract has on UV-‐induced skin damage, although further research needs to be done to confirm this idea. In a similar study, rats subjected to UVB radiation were treated with a cream containing 4% and 5% Calendula essential oil. Results suggested antioxidant effects with decreased levels of lipid peroxidation marker enzymes and increased levels of endogenous antioxidants such as glutathione and superoxide dismutase found in the skin (Mishra et al.,2012). However, the results are not directly comparable to water and ethanol extractions as the levels of volatile oils in herbal teas and tinctures would be considerably lower. Preethi et al. (2006) showed that an ethanol extraction of Calendula flower heads had free radical scavenging activity and inhibited lipid peroxidation in vitro. In vivo in mice doses of 100 and 250 mg/kg inhibited the generation of superoxide by macrophages and resulted in significant increases in levels of catalase, glutathione reductase and glutathione. Catalase is a heme protein that catalyses the decomposition of hydrogen peroxide to water and oxygen, thereby protecting the cell from oxidative damage. Glutathione reductase is involved in the regeneration of glutathione, a non-‐protein antioxidant involved in the process of detoxification. In vitro Calendula propylene glycol extract demonstrated antioxidant activity in polymorphonuclear leukocytes at concentrations as low as 0.20 µg/mL (Braga et al., 2009).
27
Ultraviolet B.
49
Wound healing Numerous in vitro and in vivo studies have looked at the benefit of Calendula in wound healing. Demonstration of wound healing effects has included increased epithelisation time via phagocytosis, increased granulation, and increased metabolism in processes related to tissue regeneration (Basch et al., 2006). Considering that the formation and rupture of inflammatory acne lesions as well severe cystic and nodulocystic acne can lead to significant scarring, any treatment that supports wound healing and reduces the potential for scar formation would be of benefit. However, further research in acne models is required to support this link. One study used a scratch wound assay, an in vitro model using mouse fibroblasts. Proliferation and migration of fibroblasts are measured, both of which are necessary for wound granulation and re-‐epithelisation and therefore wound healing. Platelet derived growth factor (PDGF) was used as a positive control as it has been shown to increase the formation of granulation tissue and improve the rate of wound healing. Both ethanolic and hexane extracts of Calendula flowers demonstrated increased proliferation and migration of fibroblasts similar to that of PDGF. The triterpenoid faradiol palmitate was shown to contribute to this process (Fronza et al., 2009). An in vivo study in mice fed an ethanolic Calendula extract showed improved granuloma formation in thermal burns. Hydroxy proline, which acts as a marker for extracellular matrix, levels were significantly higher in extract treated granuloma tissue than controls. Acute phase proteins haptoglobin and oromucoid were decreased in extract-‐treated mice. Histopathological analysis showed decreased infiltration of lymphocytes and no plasma cells or polymorphs compared with controls. Mechanisms put forward for the improved granuloma formations were increased synthesis of collagen and decreased catabolism of collagen due to the presence of flavonoids that can create artificial cross-‐linkages between collagen molecules (Chandran and Kuttan 2008). An 80% ethanolic extract of Calendula was mixed into a gel at 5% and 10%. Applied to an oral mucositis chemotherapy-‐induced model in hamsters; the Calendula preparations significantly reduced oral mucositis over vehicle control and untreated control (Tanideh et al., 2013). A limitation of this study was a failure to precisely quantify the Calendula extract.
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Ethanolic extracts of Calendula flowers were applied to a cutaneous wound rat model and macroscopic observation showed faster wound healing than the control treated with distilled water. However, changes were not quantitatively measured and significance was not discussed. Microscopic evaluation of wounds showed significant decreases in fibrin and hyperaemia indicating anti-‐inflammatory circulatory alterations in the Calendula group. Further an increase in collagen indicating fibroplasia was found in the Calendula group. Immunohistochemical evaluation showed increased numbers of blood vessels in the dermis of rats treated with Calendula indicating neovascularization (Parente et al., 2011). In vitro treatment of a wound-‐healing model in rats using a lamellar gel emulsion containing Calendula oil suggested this method of application was superior to Calendula oil alone. Results demonstrated superior re-‐epithelialization, neovascularisation, increased collagen indicative of fibroblast proliferation, and modulation of the inflammatory response compared with controls (Okuma et al., 2015). An Achilles tendon injury model in rats showed that after 7 days, rats treated with a topical Calendula preparation had increases in collagen and non-‐collagen protein, and increased changes in collagen organisation at the wound site (Aro et al., 2015). Calendula tincture stimulated fibroblast proliferation and migration mediated by a PI3K 28-‐ dependent pathway, suggesting a mechanism for enhanced wound healing (Dinda et al., 2015). Angiogenic An aqueous extraction of Calendula flowers was shown to be highly angiogenic in a chick chorioallantoic membrane (CAM) assay, which serves as a model for angiogenesis. Increased levels of hyaluronan were found in areas of increased neovascularisation suggesting induction by Calendula extract. Hyaluronan plays a role in the wound healing process through influencing such processes as cellular locomotion and proliferation. The authors conclude that neovascularisation via induction of hyaluronan is a possible mechanism for the wound healing properties of Calendula (Patrick et al., 1996). Similar findings using the CAM assay were found using an ethanolic extract of Calendula flowers (Parente et al., 2012). 28
Phosphoinositide 3-‐kinase
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Immunostimulating Polysaccharides were shown to have an immunostimulating effect on granulocytes, which demonstrated increased phagocytosis in vitro (Varlijen et al., 1989). Local application of Calendula, which enhances lymphocyte function, could play a role in wound-‐healing effects observed in Calendula. Further studies are required to establish a link as well as determine the benefit in acne. Skin parameters A one-‐sided blind study of 21 participants demonstrated changes in skin parameters following treatment with a topical water-‐in-‐oil emulsion containing Calendula extract. Significant changes were found in increased skin tightness required for preventing damage and increased skin hydration required for normal metabolism (Akhtar et al., 2011). Improvements in skin parameters that supports normal metabolism could help to prevent abnormal keratinisation and seborrhoeic processes. However, further studies are required to establish this link. Antitumour Early work looking at cytotoxic and antiproliferative effects in cancer cell lines could suggest a possible role in arresting the keratinocyte hyperproliferation characteristic of acne pathogenesis. However, much work is needed to confirm this hypothesis. In particular research with acne models in vitro and in vivo. Triterpene glycosides isolated from Calendula flowers demonstrated cytotoxicity in vitro against cancer cell lines. They were most effective against colon cancer, leukaemia, and melanoma cells (Ukiya et al., 2006). Aqueous laser-‐activated calendula flower extract was shown in vitro to inhibit the proliferation of a range of human and murine tumour cells. The inhibition ranged from 70 -‐ 100%. The mechanism was identified as cell cycle arrest between G0 and G1 phase and caspase-‐3 induced apoptosis. The same extract induced proliferation of human peripheral blood lymphocytes and natural killer cells (Medina et al., 2006). 52
Case studies An 18-‐year-‐old male with recurrent cheilitis29 that did not respond to standard treatment was successfully treated with a 10% Calendula ointment without recurrence following treatment (Roveroni-‐Favaretto et al., 2009). This case study supports the use of Calendula in decreasing inflammation and supporting skin healing. Interesting cheilitis is a side effect of oral isotretinoin (Zaenglein et al., 2016, Supplement Table XXIX) suggesting a possible role in the treatment of adverse effects. However, case studies remain anecdotal and further clinical studies are required to determine the applicability of these results to a broader population. Clinical trials Several clinical trials have looked at the effect of topical Calendula by virtue of its anti-‐ inflammatory and antioxidant properties in the treatment of radiotherapy-‐induced dermatitis. A review by Kodian and Amber (2015) discuss flaws in these studies such as a lack of vehicle control, a lack of measurement of the level of ionising radiation via dosimeter, and a lack of taking account significant factors that affect radiotoxicity to the skin such as skin type, presence of skin folds and area irradiated. They found it difficult to compare studies that have looked at acute radiation-‐induced dermatitis because the reporting outcomes were inconsistent, in particular those assessing the level of severity of toxic skin reactions. A well-‐reported single-‐blind study by Pommier et al. (2004) looked at 254 breast cancer patients. Taking into account factors such as skin type and breast size, they demonstrated a significant decrease in the incidence of acute dermatitis with topical Calendula application. However, a randomised blinded Swedish study that looked at 420 breast cancer patients receiving radiotherapy also did not find a significant difference between the group receiving a proprietary Calendula cream and that receiving standard cream. The study took into account BMI30 and dose of radiotherapy. However, did not take into account skin type (Sharp et al., 2013). 29
A chronic condition producing dry, inflamed, crusted and sometimes fissured lips. BMI: body mass index, measures height to weight ratio to indicate underweight, normal weight and overweight patients. 30
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A recent Brazilian double-‐blind controlled trial looked at acute radiodermatitis in 51 head and neck cancer patients. Compared with an essential fatty acid ointment, Calendula ointment significantly reduced the severity of acute radiodermatitis (Schneider et al., 2015). The study failed to describe the details of the Calendula preparation, there is also a question of how genuinely similar the two preparations were with regards to adequate blinding. Other factors like skin type and area irradiated were not taken into account. A randomized controlled trial of 20 patients with head and neck cancer treated with radiotherapy or a combination of chemotherapy and radiotherapy were treated with a Calendula mouth gel to prevent and treat radiotherapy-‐induced oropharyngeal mucositis31. Patients were treated with a 2% Calendula extract (obtained from ethanol extraction) oral gel mouthwash or a vehicle control. Over 8 weeks the Calendula mouth gel scored significantly better on week 2, 3 and 6 on the Oral Mucositis Assessment Scale and 3 members of the Calendula group did not get any mucositis. Finally, 19.41 ± 4.34 mg/L of the flavonoid quercetin were measured and the antioxidant activity of the Calendula extract was found to be comparable to the known antioxidant gallic acid. The authors postulated that the mechanism by which Calendula may improve mucositis is as an antioxidant that prevents free radical damage subsequent to radiotherapy-‐induced cell death and injury (Babee et al., 2013). This was a well-‐reported study, however, the small sample size (20) and the large number of drop-‐ outs/ excluded participants (9 in total) throughout the study, strongly limits the significance of the results. One study compared the use of proprietary Calendula ointment (1.5% extract) and Aloe vera cream in the treatment of diaper dermatitis on 66 children less than 3 years of age. The study found a significant reduction in the presence of diaper dermatitis in the Calendula group (Panahi et al., 2011). This study was relatively small and there was a lack of vehicle control, which is a considerable setback when different vehicles are used. Ointment and creams are absorbed differently by the skin and likely affect skin barrier function differently. Of lesser note the type of Calendula extract was not specified which limits the reproducibility of the study.
31
Inflammation of the oral mucosa that results in atrophy, redness, swelling and ulceration (Babee et al., 2013).
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Eghdampour et al. (2013) compared Aloe vera ointment and Calendula ointment with a treatment-‐as-‐usual control in perineal healing after episiotomy in 111 primiparous32 women in Iran. Significant improvements in redness, oedema, ecchymosis33, and discharge were seen in the Aloe vera and Calendula groups after the 5th day of treatment. This was a partially blinded study, however, significant limitations include a lack of vehicle control and a failure to describe the Aloe vera and Calendula ointments. Further, the publication contains significant grammatical errors that limit effective communication. A Brazilian trail on 18 patients following surgical extraction of unerupted maxillary third molars looked at the antimicrobial properties of mouthwashes containing Calendula against microbial adherence to sutures. The Calendula extract trended towards microbial inhibition but was not more significant than the positive control chlorhexidine digluconate (Faria et al., 2010). This was an unblinded study with limitations in sample size that limit the significance of the results. Further, antimicrobial reductions looked at global CFU/mL without distinguishing between beneficial and pathogenic bacteria. These studies indicate general effectiveness for Calendula as an anti-‐inflammatory and wound-‐ healing agent. Higher quality trials are needed to provide further evidence to this effect. With regards to acne, specific trials in acne patients are required to determine the potential role for Calendula in acne treatment. Safety The Cosmetic Ingredient Review Expert Panel regards Calendula as safe for use topically when used at recommended concentrations of 0.0001% to 0.8% for flower extract and 0.02% to 0.1% for flower oil. It is used in 295 cosmetic products in the United States (Andersen et al., 2010). This is well within the dosage recommended by the German Commission E (American Botanical Council, 2013) based on the theoretical yield of Calendula flowers discussed previously in the Bacterial Sensitivity section. However, it is of note that no adverse reactions were found in 34 children treated for diaper dermatitis with an ointment containing 1.5% Calendula extract for up to 10 days (Panahi et al., 2011). 32
Giving birth for the first time. Bruising.
33
55
The threshold for toxicological concern (TTC) is a risk assessment approach used for regulatory purposes in the food industry that has recently been adapted for the cosmetic industry. TTC recommendations depend on published data regarding known chemical constituents and are conservative recommendations that are one to three orders of magnitude below the daily-‐ recommended exposure limit. The TTC for topical application of Calendula is 18 g per day of a preparation containing 0.1% of the extract assuming an 80% absorption factor, which has a safety factor of greater than 20-‐fold (Re et al.,2009). It is commonly stated that Calendula should be used with caution in people who have allergies or hypersensitivity to the Asteraceae family (Basch et al., 2006). However, there is little evidence to support this recommendation (Leach, 2008). Calendula extract was found to be non-‐cytotoxic in vitro on lung fibroblast and liver cells at concentrations less than 15mg/mL (Okuma et al., 2015).
Conclusion The results indicate that Calendula 90% hydroethanolic and aqueous extracts both selectively inhibit P. acnes over S. epidermidis. To the best knowledge of the author this is the first time antibacterial properties of Calendula extracts have been tested against P. acnes or S. epidermidis. These results suggest that Calendula may be a potential topical skin prebiotic for the treatment of acne. Further, anti-‐inflammatory, antioxidant, and wound healing properties combined with a long tradition of topical use make it an ideal candidate for low risk acne treatment. Further research is needed to determine the effectiveness of a topical prebiotic strategy in the treatment of acne, as well as specific research in vitro, in vivo and in clinical trials as to the effectiveness of Calendula in treating acne. Additional factors including different strains of P. acnes and S. epidermidis should be considered.
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Appendix I Side effects associated with tetracycline antibiotics prescribed as adjunctive treatment in moderate to severe acne, modified from Zaenglein et al., 2016, Supplement Tables XIV, XV & XVI. Tetracycline antibiotic Tetracycline
Minocycline
Adverse effects and toxicities Gastrointestinal: anorexia, nausea, epigastric distress, vomiting, diarrhoea, glossitis, black hairy tongue, dysphagia, enterocolitis, inflammatory lesions with candida overgrowth in the anogenital region, oesophagitis, or oesophagitis, oesophageal ulceration Teeth: permanent discoloration during tooth development, enamel hypoplasia Skin: maculopapular and erythematous rashes, exfoliative dermatitis, oncholysis, nail discoloration, photosensitivity Renal: dose-‐related rise in blood urea nitrogen Liver: hepatotoxicity and liver failure Hypersensitivity reactions: urticarial, angioneurotic oedema, anaphylaxis, anaphylactoid sickness-‐like reactions such as fever, rash or arthralgia Blood: haemolytic anaemia, thrombocytopaenia, thrombocytopenic purpurea, neutropaenia, eosinophilia Other: bulging fontanels in infants, raised intracranial pressure General: fever, discoloration of secretions Gastrointestinal: anorexia, nausea, vomiting, diarrhoea, dyspepsia, stomatitis, glossitis, dysphagia, enertocolitis, pseudomembranous colitis, pancreatitis, inflammatory lesions with moilial overgrowth in the oral and anogenital regions, oesophagitis, and oesophageal ulcerations Genitourinary: vulvovaginitis Hepatic toxicityL hyperbilirubinaemia, hepatic chelstatis, increases in liver enzymes, fatal hepatic failure, jaundice, hepatitis including autoimmune, liver failure Skin: alopecia, erythema nodosum, hyperpigmentation of nails, pruritis, toxic epidermal necrolysis, vasculitis, maculopapular and erythematous rashes, exfoliative dermatitis, fixed drug eruptions, lesions occurring in the glans penis have causes balanitis, erythema multiforme, Stevens-‐Johnson syndrome, photosensitivity, pigmentation of the skin and mucous membranes Respiratory: cough, dyspnoea, bronchospasm, exacerbation of asthma or pneumonitis Renal: interstitial nephritis, dose-‐related rise in blood urea nitrogen, reversible acute renal failure Musculoskeletal: arthralgia, arthritis, bone discoloration, myalgia, joint stiffness and joint swelling 73
Doxycycline
Hypersensitivity reactions: urticarial, angioneurotic oedema, polyarthralgia, anaphylaxis/anphylactoid reactions including shock and death, anaphylactoid purpurea, myocarditis, pericarditis, exacerbation of systemic lupus erythmatosus and pulmonary infiltrates of eosinophilia, transient lupus-‐like syndrome, and serum sickness-‐like reactions Blood: agranulocytosis, haemolytic anaemia, thrombocytopaenia, leukopaenia, neutropaenia, pantcytopaenia, eosinophilia Central nervous system: convulsions, dizziness, hypoaesthesia, paraesthesia, sedation, vertigo, bulging fontanels in infants and benign intracranial hypertension (pseudotumor cerebri) in adults, headache Oral/ Teeth: enamel hypoplasia, tooth discoloration, oral cavity (including tongue lip and gum) discoloration Other: thyroid cancer, abnormal thyroid function, tinnitus, decreased hearing Gastrointestinal: anorexia, nausea, vomiting, diarrhoea, glossitis, dysphagia, enterocolitis, inflammatory lesions with monilial overgrowth in the anogenital region, hepatotoxicity, oesophagitis, oesophageal ulcerations Skin: toxic epidermal necrolysis, Stevens-‐Johnson syndrome, erythema multiforme, maculopapular and erythematous rashes, exfoliative dermatitis, photosensitivity Renal: dose-‐related rise in blood urea Hypersensitivity reactions: urticaria, angioneurotic oedema, anaphylaxis, anaphylactoid purpura, serum sickness, pericarditis, exacerbation of systemic lupus erythematosus Blood: haemolytic anaemia, thrombocytopaenia, neutropaenia, eosinophilia Other: bulging fontanels in infants, raised intracranial pressure
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Appendix II Contraindication and side effect profiles for combined oral contraceptives (COCs) containing oestrogen (ethinyl estradiol) and a progestin, adapted from Zaenglein et al., 2016, Supplement Tables XXIII, XIV, XXV, & XXVI. COC Ethinyl estradiol/ norgestimate
Contraindications Blood pressure: systolic > 160 mmHg, diastolic > 100 mmHg Carcinoma of the breast or endometrium Circulatory: cerebral vascular disease, coronary artery disease, deep vein thrombosis, thromboembolic disorders, valvular heart disease with complications, diabetes with vascular involvement Surgery with prolonged immobilization Hepatic: cholestatic jaundice or pregnancy, iatrogenic jaundice, hepatic adenoma, hepatic carcinoma, hepatocellular disease with abnormal liver function Hypersensitivity Genital bleeding (undiagnosed) Ethinyl Anaphylactic reaction or estradiol/ angioedema norethindrone Circulatory: arterial acetate/ thromboembolic disease ferrous (stroke or myocardial fumarate infarction), cerebral vascular disease, coronary artery disease, deep vein thrombosis, thromboembolic disease, pulmonary embolism Carcinoma of the breast or endometrium Hepatic: cholestatic jaundice of pregnancy, jaundice with previous pill use, hepatic
Adverse effects and toxicities Cardiovascular: oedema, varicose vein aggravation Central nervous system: depression, migraine, mood changes Skin: cholasma, melasma, erythema nodosum Endocrine: amenorrhoea, breakthrough bleeding, breast pain or tenderness, fluid retention, infertility Gastrointestinal: abdominal bleeding, abdominal cramps, appetite changes, nausea, weight changes, vomiting Genitourinary: cervical ectropion, cervical secretion, vaginal candidiasis, vaginitis Blood: decreased folate, porphyria exacerbation Hepatic: cholestatic jaundice Other: anaphylaxis, lupus exacerbation
Central nervous system: headache, depression, nervousness, mood disorder Endocrine: breast pain, irregular menstruation, menorrhagia, weight changes Genitourinary: urinary tract infections, vaginitis, abnormal uterine bleeding Gastrointestinal: abdominal pain, nausea, vomiting, diarrhoea, dyspepsia Infection: viral infection Respiratory: sinusitis
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adenoma, hepatic carcinoma, hepatic disease Undiagnosed genital bleeding Pregnancy Ethinyl Renal dysfunction estradiol/ Adrenal insufficiency drospirenone Hypertension, uncontrolled Circulatory: cerebrovascular disease, coronary artery disease, deep vein thrombosis, pulmonary embolism, hypercoagulopathies, thrombogenic valvular disease, thrombogenic rhythm disease Breast cancer or other oestrogen-‐ or progestin-‐ sensitive cancer Headaches with focal neurologic symptoms, migraines if ≥ 35 years Pregnancy Smoking if ≥ 35 years Undiagnosed uterine bleeding Hepatic dysfunction, hepatic tumours benign or malignant Ethinyl Adrenal insufficiency estradiol/ Breast cancer or other drospirenone/ oestrogen-‐ or progestin-‐ levomefolate sensitive cancer Circulatory: cerebrovascular disease, coronary artery disease, deep vein thrombosis, pulmonary embolism, hypercoagulopathies, thrombogenic valvular disease, thrombogenic rhythm disease of the heart Diabetes with vascular disease Headaches with focal neurologic symptoms, migraines if ≥ 35 years Hepatic: tumours benign or malignant, hepatic disease
Cardiovascular: oedema, varicose vein aggravation, increased risk of arterial thromboembolism, cerebral thrombosis, hypertension, myocardial infarction Gastrointestinal: abdominal bloating, abdominal cramps, nausea, weight changes, vomiting Central nervous system: depression, migraine Skin: melasma, allergic rash Endocrine: amenorrhoea, breakthrough bleeding, breast changes, infertility, carbohydrate tolerance decreased, spotting Genitourinary: cervical ectropion, cervical secretion, vaginal candidiasis Blood: decreased folate, porphyria exacerbation Hepatic: cholestatic jaundice Ocular: contact lens intolerance, corneal curvature changes Other: anaphylaxis, systemic lupus erythematosus exacerbation Endocrine: weight increase, hyperkalaemia, impaired glucose tolerance Cardiovascular: arterial thromboembolism, deep vein thrombosis, hypertension, myocardial infarction Gastrointestinal: abdominal pain, nausea, vomiting, gallbladder disorder, pancreatitis Hepatic: cholasma, cholestasis, neoplasm of liver Neurologic: headache, haemorrhagic cerebral infarction, migraine, thrombotic stroke Blood: thromboembolic disorder, porphyria exacerbation Psychiatric: depression, irritability, labile affect (episodes on uncontrollable 76
Uncontrolled hypertension crying) Pregnancy Reproductive: break through bleeding, Renal impairment breast tenderness, disorder of Smoking if ≥ 35 years menstruation, reduced libido, cervical Undiagnosed uterine bleeding dysplasia Immunologic: anaphylaxis Eyes: thrombosis of retinal vein Respiratory: pulmonary embolism
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Appendix III Side effects of isotretinoin, adapted from Zaenglein et al., 2016, Supplement Table XXIX. Adverse effects and Toxicities Cardiovascular: chest pain, oedema, flushing, palpitation, stroke, syncope, thrombosis Central nervous system: aggressive/violent behaviour, depression, emotional instability, fatigue, headache, psychosis, suicidal ideation/attempts, stroke, pseudotumour cerebri, seizure Skin: alopecia, cheilitis, cutaneous allergic reaction, dry nose, dry skin, eruptive xanthomas, nail dystrophy, photosensitivity Endocrine: abnormal menses, elevated glucose, increased cholesterol, hyperuricaemia, elevated triglycerides Gastrointestinal: bleeding and inflammation of gums, colitis, oesophagitis, inflammatory bowel disease, nausea, pancreatitis Blood: agranulocytosis, anaemia, neutropaenia, pyogenic granuloma, thrombocytopaenia Hepatic: raised liver enzymes (AST and ALT), hepatitis, raised lactate dehydrogenase Musculoskeletal: arthralgia, arthritis, back pain, hypertrophy of bone, increased creatinine kinase, rhabdomyolysis Ocular: dry eyes, optic neuritis Otic: hearing loss Respiratory: bronchospasms, epitaxis
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Appendix IV
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Appendix V The Mediherb quality control specifications for Calendula tincture.
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Appendix VI The certificate of analysis for Calendula tincture used in this study.
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Appendix VII Table showing a summary of the chemical composition of Calendula from the literature adapted from Re et al. (2009). Component class
Main fraction and % concentration
Sub-fraction Potassium (≈6.0%) Sodium (≈1.7%
Mineral matter
Major elements as salts
Magnesium (≈0.9%) Calcium (≈0.5%) Iron (0.15%) Arabinogalactan PSII 25 kDa (arabinose, galactose)
Carbohydrates
Arabinogalactan PSIII 35 kDa (arabinose, galactose)
12–25%, dry matter
Mucilage (1.5%): rhamnoarabingalactan PSI 15 kDa (arabinose, galactose, rhamnose) 9-Hydroxy-trans-10,cis-12octadecadienic-acid Capric acid Caprylic acid Dimorphecolic acid Lauric acid Fatty acids mainly as esters (5%, dry matter)
Linoleic acid Linolenic acid Myristic acid Palmitic acid Palmitoleic acid Pentadecanoic acid
Lipids
Stearic acid Calendic acid C32H62 Dotriacontane Hentriacontane Hydrocarbon/paraffin/waxes (0.015%, fresh petals)
Heptacosane Hexacosane Nonacosane Octacosane Tetratriacontane Tritriacontane Lignin
Phenolic compounds
Phenolic acids in free and esterified forms (0.1%, dry matter)
Caffeic acid Chlorogenic acid Coumaric acid
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Component class
Main fraction and % concentration
Sub-fraction Ferulic acid Gentisic acid trans-O-Coumaric acid O-Hydroxyphenylacetic acid 4-Coumaric acid 4-Hydroxy benzoic acid Protocatechuic acid Quinic acid Salicylic acid (traces) Sinapinic acid Syringic acid Vanillic acid Veratric acid Astragalin Hyperoside Calendoflaside Calendo flavo side Calendo flavobio side Isoquercitrin Isorhamnetin Isorhamnetin-3-neohesperidoside Isorhamnetin-3-O-(2″,6″-dirhamnosyl)glucoside Isorhamnetin-3-O-(2″-rhamnosyl)glucoside Isorhamnetin-3-O-glucoside Isorhamnetin-3-rhamnosyl-(1,2)rhamnoside
Flavanoids (
