Anti-acne activity of Achillea ‘ Moonshine ’ petroleum ether extract

Achillea millefolium (yarrow) is a traditionally used plant to treat wounds. The present study was conducted to evaluate the anti-acne activity of Achillea ‘Moonshine’, a hybrid variety of Achillea. The plant was extracted in four solvents petroleum ether, ethyl acetate, ethanol and water. These extracts were screened for anti-microbial, free radical scavenging, anti-tyrosinase, anti-inflammatory activity and cytotoxicity assays necessary to characterize its anti-acne activity. The most promising activity was determined in the petroleum ether extract. The minimum inhibitory concentration (MIC) value for the petroleum ether extract was 0.83 mg/ml against Propioni bacterium acnes and 0.37 mg/ml against Staphylococcus. The minimum bactericidal concentration (MBC) value for petroleum ether was 0.83 and 0.75 mg/ml for P. acnes and Staphylococcus epidermidis, respectively. Though the ethyl acetate had a high flavonoid and phenolic content it was observed that the IC50 values for the petroleum ether extract for free radical scavenging activity was 64.81 μg/ml, which was higher than ethyl acetate. Petroleum ether also showed tyrosinase inhibition at 0.033 mg/ml. The extract was also able to decrease the inflammatory cytokines like TNF-α and IL-8, and showed no cytotoxicity against dermal fibroblasts. These results suggest presence of active anti-acne phytochemicals in the petroleum ether extract, making it a novel plant candidate for the treatment of acne.


INTRODUCTION
Achillea millefolium Linn.commonly known as yarrow is a flowering plant from the Asteraceae family, and is represented by about 85 different species.It is widely found in Asia, North America and Europe (Li et al., 2011;Moradi et al., 2013).Traditionally, yarrow is purported to be a diaphoretic, astringent, tonic, stimulant and mild aromatic (Benedek et al., 2007).The plant has a long history as a powerful 'healing herb', used topically for wounds, cuts and abrasions (Benedek et al., 2007).Previous studies have shown yarrow to have potential cosmetic uses (Chandler et al., 1982) including skin surface rejuvenation effects.However, the majority of the studies have been performed on the flowers of yarrow.Various species of A. millefolium used in garden traditions have been superseded with cultivars and hybrids with improved qualities.One such hybrid is Achillea 'Moonshine' which is a cross between Achillea 'Taygetea' and Achillea clypeolata.Achillea 'Taygetea' is an ornamental plant native to Europe while Achillea clypeolata is native to Balkan Peninsula.Both the plants have yellow colored flowers and are drought resistant.However, there has not been much research on individual species.Over and above, hybridization and intraspecific variability have complicated the taxonomy.As a result of this, most of the plants of genus 'Achillea' are referred to as Yarrow and there is no clear identification of individual species.For the following study, the most popular Achillea that is used in the North American gardens Achillea 'Moonshine' was used.
Achillea 'Moonshine' is an herbaceous perennial plant, 1 to 2 ft high and blooms between June to September, and is resistant to drought and dry soil.It is known for its fern-like, aromatic, silvery to gray-green foliage and its tiny, long-lasting, bright lemon-yellow flowers which appear in dense, flattened, compound corymbs (to 2 to 3" across).The present research is aimed towards the study of anti-acne activity in the leaves and stem extracts of Achillea 'Moonshine'.
Acne vulgaris (cystic acne or simply acne) is a common human skin disease, occurring mostly during the onset of puberty.It is generally characterized by areas of skin with seborrhea, comedones, papules (pinheads), pustules (pimples), nodules and possibly scarring.The known causes for acne are infection by bacteria such as Propionibacterium acnes and Staphylococcus epidermidis, hormonal changes, genetic predisposition to acne, adverse effects of cosmetics, medications, or other factors.Hence, all scientific attempts in developing antiacne drugs focus on addressing the above mentioned causes of acne (Toyoda and Morohashi, 2001).
Acne occurs due to blockage in the follicles resulting in the formation of comedones.The sebaceous glands become clogged with sebum and dead skin cells.Under these conditions, bacterium Propionibacterium acne can cause inflammatory lesions in the dermis around the comedones or microcomedo resulting in redness and scarring.The inhibition of P. acnes decreases the rupturing of comedones into the surroundings and helps to inhibit the progression of acne.Another organism which is implicated in the etiology of acne is a common skin micro flora, Staphylococcus epidermidis which promotes pustules and nodule formation.Thus, for screening anti-acne compounds the inhibition of both these organisms is of prime consideration in treating acne (Knor, 2005;Tanghetti, 2013).
Another factor that causes increased chances of acne is oxidative stress.The skin is exposed to oxidative stress induced by reactive oxygen species (ROS) both endogenously and externally.ROS play an important role in production of inflammatory mediators such as interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) by monocytes/macrophages during the progression of acne.Also, excessive and repeated stimulation by invading organisms such as P. acnes and S. epidermidis causes over production of ROS (Akamatsu and Horio, 1998).These ROS causes considerable damage to the tissues leading to increase in inflammatory responses.Thus, reduction of ROS by antioxidants and free radical scavengers as well as inhibition of these inflammatory mediators is necessary to prevent damage at the cellular and the tissue levels.Post inflammatory hyper pigmentation (PIH) characterized by pigmentation in dermis or epidermis is commonly seen in acne sufferers.It causes discoloration of skin following the inflammation caused during acne.Increase in pigmentation is due to alternations in the melanocytes, and thus, post-acne treatment requires clearing of affected skin area (Davis and Callender, 2010).A potential anti-acne candidate should thus also have an ability to clear the melanin depositions.
Many oral and topical antibiotics are used for the management and treatment of acne vulgaris.However, the prolonged use of these antibiotics has created a problem of antibiotic resistance among the acne inducing bacteria (Humphrey, 2012).Also, the long term use of topical agents causes skin irritation, leading to decrease in patient compliance and treatment failure.To overcome these drawbacks, natural products have been studied extensively as alternatives to complement the existing therapies for the treatment of acne because they provide a largely unexplored source of drug discovery and development.
In this study, the antibacterial activity of Achillea 'Moonshine' extracts against the two acne causing organisms (P.acnes and Staphylococcus epidermidis), free radical scavenging activity, anti-tyrosinase, anti-inflammatory potential as well as cytotoxicity against human skin cells to identify the most potent extract possessing anti-acne activity were determined.

METHODOLOGY Collection of plant material
Eight full bloomed Achillea 'Moonshine' plants were collected from Holly Days Nursery Inc. near Philadelphia (United States) in June, 2013.

Preparation of extracts
The leaves and stems were washed three times with distilled water and grounded into fine powder using a simple grinder after they were air dried for 3 days.The plant material was then extracted with four solvents of increasing polarity (petroleum ether, ethyl acetate, ethanol and water) by maceration.The solvent was evaporated and the extracts were stored at 4°C till further use.

Determination of anti-microbial activity against P. acnes and S. epidermidis Bacterial strains and media
The bacterial strains incorporated in this study were P. acnes (ATCC) and S. epidermidis (ATCC).P. acnes was incubated in brain-heart infusion media for 48 h at 37°C under anaerobic conditions in an anaerobic jar, while S. epidermidis was incubated in Muller Hinton broth for 24 h at 37°C, and their densities were adjusted to approximately 0.5 McFarland Standard.

Anti-microbial activity by disc diffusion method
Sterile 5 mm-Whatman No. 1 filter paper discs were used in the disc diffusion assay.The discs were soaked separately with 30 µl of each of the extract at a concentration of 6 mg/ml in dimethyl sulfoxide (DMSO) for organic solvents and sterile water for the water extract.Negative control discs were soaked in DMSO and distilled water for organic solvent and water extracts respectively.Erythromycin (60µg/disc) was used as positive control.These discs were placed on Mueller-Hinton agar plates and brain heart infusion agar plates, previously swabbed with S.epidermidis and P. acnes respectively at a concentration of 10 6 Colony Forming Units (CFU)/ml.The plates were incubated at 37°C for 24 h in case of S. epidermidis and 48 h anaerobically for P. acnes.Anti-bacterial activity was defined as the diameter (mm) of the clear inhibitory zone formed around the discs.

Minimum inhibitory concentration by tube dilution method
Tube dilution method was used to determine the minimum inhibitory concentration (MIC) against P. acnes and S. epidermidis.The extracts were dissolved in DMSO and were serially diluted from concentration 5 to 0.1 mg/ml in sterile culture tubes containing 0.2 ml sterile nutrient broth.The tubes were inoculated with 100 μl of bacterial suspension (approximately 10 6 CFU/ml) in their respective broths and incubated at 37°C for 24 h in the case of S. epidermidis and 48 h anaerobically for P. acnes.After incubation, the tubes were examined for growth by visually observing the turbidity.MIC was defined as the minimum concentration that resulted in no growth of the bacteria.The test was carried out in triplicates (Chomnawang et al., 2005;Kim et al., 2008).

Minimum bactericidal concentration (MBC)
The MBC was determined by pipetting out 0.1 ml bacterial culture from MIC tubes which had no bacterial growth and plating it on to Muller Hinton agar for S. epidermidis and on Brain heart infusion agar plate in case of P. acnes for 24 h and 48 h respectively.After incubation, the concentration at which there was no single colony of bacteria was taken as MBC.The tests were carried out in triplicates.

Determination of total phenolic contents in the plant extracts
The concentration of phenolics in plant extracts was determined using Folin-Ciocalteu method (Pourmoradet al., 2006).The reaction mixture was prepared by mixing 0.5 ml of methanolic solution of extract in concentration of 1mg/ml, 5 ml of 10% Folin-Ciocalteu's reagent dissolved in water and 4 ml 1M NaHCO 3 .The absorbance was measured at 765 nm.The total phenolic content was calculated from a linear calibration curve with commercial gallic acid.The content of phenolics in extracts was expressed in terms of gallic acid equivalent (µg of gallic acid/ml of extract).The experiments were carried out in triplicates.

Determination of total flavonoid content in the plant extracts
The content of flavonoids in the examined plant extracts was determined using spectrophotometric method (Pourmorad et al., 2006).Briefly, 0.5 ml of methanolic solution of the extract in the concentration of 1 mg/ml was mixed with 1.5 ml methanol, 0.1 ml of 10% AlCl 3 solution, 0.1 ml of 1M potassium acetate and 2.8 ml distilled water.The samples were incubated for 30 min at room temperature, and the absorbance was measured at 415 nm.The amount of flavonoid was calculated from a liner calibration curve with commercial quercetin.The flavonoid content was expressed in terms of quercetin equivalent (µg of quercetin/ml of extract).The experiments were carried out in triplicates.

Determination of free radical scavenging activity
The free radical scavenging activity of the extracts was determined on the ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals (Wu et al., 2009).The stock solution of extracts was prepared in methanol to achieve the concentration of 1 mg/ml.Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.25, 15.62 µg/ml respectively.0.1 ml from each dilution was mixed with 0.9 ml of methanolic solution of DPPH (0.4% w/v).After 30 min incubation in dark at room temperature, the absorbance was recorded at 517 nm.Control sample contained all the reagents except the extract.Percentage scavenging was calculated using equation: Where, Ac is absorbance of control (DPPH in methanol); As is the absorbance of the sample/extract.Extract concentration providing 50% inhibition (IC 50 ) was calculated from the graph of percentage scavenging against extract concentration.Tests were carried out in triplicate.

Determination of anti-tyrosinase activity
Tyrosinase inhibition activity in order to evaluate the ability of the extracts to reduce the post inflammatory hyper pigmentation was determined by spectrophotometric assay in a 96 well plate (Baurin et al., 2002).70 µl of extract in DMSO was mixed with 30 µl mushroom tyrosinase (313 Units/ml).The mixture was incubated for 10 min at 37°C.L-tyrosinase (2 mM) in potassium phosphate buffer (50 mM, pH 6.5) was added and the resultant solution was again incubated for 20 min at 37°C.The final absorbance was read at 490 nm.Percentage inhibition was calculated by Where, Ac is absorbance of control; As is the absorbance of the sample.Extract concentration providing 50% inhibition (IC 50 ) was calculated from the graph of percentage inhibition against extract concentration.Tests were carried out in triplicate.

Cell line and culture media
Human monocytic THP-1 cells (ATCC TIB-202) were cultured in RPMI supplemented with heat inactivated fetal bovine serum while normal human dermal fibroblasts (Lonza CC2155) were cultured in FGM™-2 BulletKit™ (Lonza) and were incubated in a humidified atmosphere with 5% CO 2 at 37°C.

Measurement of cytokine production
To determine the effect of the petroleum ether extract on production of pro inflammatory cytokines (TNF-α and IL-8), human monocytic THP-1 cells (10 6 cells/ml) in a serum free media was stimulated with 100 µg/ml P. acnes (wet weight), alone or in combination of different concentrations (50 and 100 µg/ml) of the extract and were incubated for 18 h at 37°C.The culture supernatants were harvested.The concentrations of IL-8 and TNF-α in the supernatant were measured by ELISA (Kim et al., 2008).

Cytotoxicity assay
Normal human dermal fibroblasts (6000cells/well) were seeded in a 96 well plate and the extract treatment began 10 h after seeding.The general viability of the cultured cells was determined by XTT assay in which XTT is reduced to its formazan derivative.The cells were incubated with 50 and 100 µg/ml petroleum ether extract in DMSO for 18 h in a humidified atmosphere with 5% CO 2 at 37°C.20µl of activating reagent was added to 1ml XTT and each well was treated with 50 µl of this activated XTT and was incubated for 5 h.The absorbance of the wells was measured at 475 nm and 660 nm.
The tests were carried out in triplicates (Kim et al., 2008).

Data analysis
Data are expressed as mean ± standard deviation or as percentages for anti-microbial, free radical scavenging, total phenolic and flavonoid content determination and anti-tyrosinase activity.Statistical significance was determined by one way ANOVA using GraphPad Prism 6 software for free radical scavenging, antityrosinase, anti-inflammatory activity and cell viability assay (Graphpad Software, Inc, La Jolla, California).Differences were considered significant at P value of less than 0.05.

Disc diffusion assay
Disc diffusion assay was performed to determine the antibacterial activity of the extracts against P. acnes and S. epidermidis (Table 1).The assay was performed as a pilot study to identify the active extracts.The water extract did not show any anti-microbial activity against both the organisms, and so was eliminated from further studies.The ethanolic extract did not show any activity against S. epidermidis but was active against P. acnes.

MIC determination by tube dilution
The anti-microbial activity of the extracts against the two acne causing organisms, P. acnes and S. epidermidis is shown in Table 2.All the extracts showed anti-microbial activity against the two acne causing organisms.The lowest MIC against P. acnes was observed in the petroleum ether extract (0.83 mg/ml) whereas that for S. epidermidis was also observed in the petroleum ether extract (0.37 mg/ml).MIC is the lowest concentration of the extract that will inhibit the visible growth of the bacteria while MBC is the lowest concentration of the plant extract that is required to kill the bacteria.As shown in the results, all the extracts show MBC value no more than four times the MIC values, and so all the extracts were considered to have antimicrobial properties.The results reported are performed in triplicates (French, 2006).

Total phenolic and flavonoid content
Total phenolic content was estimated by gallic acid (Table 3), and expressed as micrograms of gallic acid equivalent (GAE)/ml of extract.The highest phenolic content was determined in the ethyl acetate extract (360.3±0.082µgGAE/ml extract).The petroleum ether extract had the lowest amount of phenolic content.Total flavonoid content was estimated by quercetin (Table 3) and expressed as micrograms of quercetin equivalent (QE)/ml of extract.The highest flavonoid content was determined in the ethyl acetate extract (308±0.015µg QE/ml extract).The petroleum ether extract had the lowest amount of flavonoid content.

Determination of free radical scavenging activity
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging assay was performed to evaluate the free radical scavenging activity.The % radical scavenging for all the three varieties is shown in Figure 1 and Table 4. DPPH is a stable free radical.On accepting hydrogen from a corresponding donor, its solutions lose the characteristic deep purple (λ max 515-517 nm) color.The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.The petroleum ether extract had the highest free radical scavenging activity (IC 50 : 64.81 µg/ml).BHT (butylated hydroxyl toluene; IC 50 :31.05µg/ml) was used as a reference compound.The extract concentrations have a significant level (P < 0.05) of free radical scavenging activity compared with the control.

Determination of anti-tyrosinase activity
The % inhibition of tyrosinase by the extracts is shown in Figure 2 and Table 5.The IC 50 value for benzoic acid, the reference compound, was 0.021 mg/ml.The petroleum

Measurement of cytokine production
The petroleum ether extract of Achillea 'Moonshine' was examined for its activity against inflammation induced by P. acnes in terms of inhibitory effects of cytokine production.As shown in the Figure 3, cells treated with bacteria showed an increase in TNF-α and IL-8 production.However, co-cultures of cells with bacteria and extracts (50 µg/ml) suppress the production of these cytokines.There was an elevated level of TNF-α cytokine production in 100 µg/ml of extracts which is was observed in some other studies as well (Kim et al., 2008).

Cytotoxicity assay
A potential candidate to be used in an anti-acne formulation should not be cytotoxic to human skin when applied topically.Figure 4 shows that the cell viability for human dermal fibroblasts is not significantly different for the cells grown with and without the plant extracts, suggesting no cytotoxicity.

DISCUSSION
The anti-acne screening presented in this study provides a plant candidate that is commonly grown in major parts of the world.To be qualified as plant with anti-acne properties, several different properties such as MIC and MBC against P. acnes as well S. epidermidis was performed.Other tests such as free radical scavenging, cytotoxicity test and anti-inflammatory tests were used to determine the extract which had the most potential of reducing and curing acne.Some of the published antiacne studies that are cited here have used plant material that is not easily available; hence our research provides data that gives insights to a unique property of yarrow that is known historically to treat wounds (Chandler et al., 1982).Anti-microbial activity assay, both by disc diffusion and tube dilution method for different extracts from Achillea 'Moonshine' showed that the petroleum ether extract had the highest potency against the main acne causing bacteria.Hence, the study is interested to know about the properties of petroleum ether extract in alleviating other causes and effects of acne.
The alcoholic fractions, usually rich in flavonoids have been used previously for anti-microbial treatments.For example, methanolic fraction from Terminalia chebula and Terminalia bellarica used in comibination showed more anti-microbial activity against several microbes including P. acnes and S. epidermidies (Greeshma et al., 2006).Batubara et al. (2009) studies on anti-acne potency in Indonesian medicinal plants also showed higher anti-microbial activity in the alcohol fractions.However, this study that the petroleum ether extracts from Achillea showed a high potency against acne causing organisms.Petroleum ether extracts from medicinal plants have been shown to contain majorly alkaloids and tannins (Ghumare et al., 2014).Tannins have natural astringent properties, and have been used topically to treat acne (Bedi et al., 2002).Alkaloids such as achilleine isolated from A. millefolium L. have been used as homeostatic and could be possibly one of the compounds in the petroleum ether extract contributing to the anti-acne effects (Miller et al., 1954).
This study also reported free radical scavenging activity more in the petroleum ether extract (Table 4).Generally, the free radical scavenging activity is attributed to flavonoids and phenolic compounds which are high in ethyl acetate extracts (Table 3).But, in this study the petroleum ether fraction had the highest radical scavenging activity, which indicates presence of certain potent non polar compounds such as fatty acids, certain flavones and steroids with high radical scavenging potential.Free radical scavenging activity was high in ethyl acetate extracts but this extract did not show high potency in killing P. acnes and S. epidermidis compared to petroleum ether extracts which is one of the most important factors in anti-acne treatment.
The tyrosinase inhibition activity was significant in the petroleum ether extract compared to the control.Compounds such as fatty acids and steroids easily solubilize in petroleum ether.These non-polar phytochemicals act as tyrosinase inhibitors by either competitively inhibiting the enzyme or by chelating the copper ion at the catalytic site of the enzyme.For some lipids, the inhibitory action is proposed due to binding of the compound to some site of the tyrosinase, except the catalytic site.Polyphenols are also considered to be potent tyrosinase inhibitors, but in this case, these compounds are not as effective as the non-polar phytochemicals because the petroleum ether extract which generally contains non polar compounds was more effective than the other extracts in which the polyphenols are soluble.
Petroleum ether extracts also showed antiinflammatory effects that contribute in the properties of A. 'Moonshine' to qualify as a treatment for acne.The cytotoxicity tests on human dermal fibroblasts also showed Achillea to be safe for topical use.Based on the different anti-acne screens the petroleum ether extracts demonstrated to have the best potential as an anti-acne agent.

CONCLUSION AND RECOMMENDATION
Based on the current findings, the petroleum ether extract of Achillea 'Moonshine' showed the most promising antiacne activity.It had potent anti-microbial, free radical scavenging, anti-tyrosinase and anti-inflammatory activity.The extracts were also nontoxic to the dermal fibroblasts making it a good candidate for topical application.The plant is easily available in North America, Asia and Europe.These results and the increased availability of Achillea 'Moonshine' makes it an ideal novel plant candidate for the treatment of acne.Further studies need to be focused on isolation and characterization of the phytoconstituents responsible for anti-acne activity.

Table 1 .
Antimicrobial activity of various Achillea 'Moonshine' extracts by disc diffusion assay.

Table 2 .
Antimicrobial activity against P. acnes and S. epidermidis.

Table 3 .
Total phenolic and flavonoid content in various extracts.

Table 4 .
% Free radical scavenging activity of various extracts.