In Vitro Antimicrobial Activity of Gel Containing the Herbal Ball Extract Against Propionibacterium Acnes

Scientia Pharmaceutica

Article In Vitro Antimicrobial Activity of Gel Containing the Herbal Ball Extract against Propionibacterium acnes

Chutima Jantarat 1,2,*, Pornpak Sirathanarun 2, Tatsanee Chuchue 2, Atthaphon Konpian 2, Gorawit Sukkua 2 and Prutthicha Wongprasert 2 1 Drug and Cosmetics Excellence Center, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand 2 School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand; [email protected] (P.S.); [email protected] (T.C.); [email protected] (A.K.); [email protected] (G.S.); [email protected] (P.W.) * Correspondence: [email protected]; Tel.: +66-756-728-30

Received: 5 January 2018; Accepted: 22 February 2018; Published: 28 February 2018

Abstract: The herbal ball has been used as a Thai traditional medicine for relieving many diseases including acne. However, the application process of the herbal ball in practice is complicated and time consuming. The objective of this work was to utilize an herbal ball extract to formulate a gel to reach a more favorable use of the herbal ball for acne treatment. An herbal ball consisting of Andrographis paniculata, Centella asiatica, the Benchalokawichian remedy and the stem bark powder of Hesperethusa crenulata was prepared. The obtained herbal ball was steamed and squeezed to obtain the extract. Gel formulations containing the herbal ball extract at concentrations of 0.1, 1 and 5% w/w were prepared based on a carbomer gel. The herbal ball extract had antioxidant (EC50 = 219.27 ± 36.98 µg/mL) and anti Propionibacterium acnes activities (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) = 31.25 µg/mL). The 5% w/w gel formulation had antimicrobial activity against P. acnes, showing an inhibition zone value of 10.00 ± 1.00 mm. This indicates that the developed gel formulation has potential for acne treatment. In comparison to the traditional method of herbal ball usage, the application of herbal ball extract in the form of gel should be more convenient to use.

Keywords: herbal ball; gel; Propionibacterium acnes; antimicrobial activity; Andrographis paniculata; Centella asiatica; Benchalokawichian remedy; Hesperethusa crenulata

1. Introduction Acne vulgaris (acne) is one of the most common skin disorders in the general population around the world. In Thailand, it has been found to be the highest incidence of out-patients seeing dermatologists from 2013–2015 according to a study by the Institute of Dermatology, Thailand [1]. Acne affects more than 80% of adolescents and young adults and can also be seen in individuals in both adults and children [2]. Although it is not life threatening, acne often causes physical and psychological problems such as poor self-image, depression and anxiety. Acne is caused by many factors related to the hyperactivity of the sebaceous gland, follicular epidermal hyperproliferation and inﬂammation caused by pathogens. The well-known pathogen linked to the skin condition of acne is Propionibacterium acnes (P. acnes), an anaerobic, gram-positive bacterium. There are a variety of effective acne treatments. Topical therapy is considered as the standard treatment for mild and moderate acne. Some common medications include benzoyl peroxide, antibiotics, retinoids and salicylic acid. Oral antibiotics are suitable for the treatment of moderate and severe acne. Some antibiotic therapies for acne include tetracycline, doxycycline, minocycline, erythromycin, trimethoprim-sulfamethoxazole, trimethoprim and azithromycin [1]. However, the use of antibiotics for acne treatment causes bacterial resistance. The prevalence of P. acnes resistant to clindamycin, tetracycline,

Sci. Pharm. 2018, 86, 8; doi:10.3390/scipharm86010008 www.mdpi.com/journal/scipharm Sci. Pharm. 2018, x, x FOR PEER REVIEW 2 of 9 erythromycin,Sci. Pharm. 2018, 86 trimethoprim, 8 ‐sulfamethoxazole, trimethoprim and azithromycin [1]. However, the2 use of 9 of antibiotics for acne treatment causes bacterial resistance. The prevalence of P. acnes resistant to clindamycin, tetracycline, doxycycline and erythromycin has occurred in many countries worldwide [3]. Isotretinoin,doxycycline anda group erythromycin of retinoids, has is occurred an efficacious in many treatment countries for worldwide severe acne; [3 however,]. Isotretinoin, the application a group of ofretinoids, this treatment is an efficacious when pregnant treatment must for severe be avoided acne; however, as it can the cause application abortion of this and treatment birth defects. when Phytotherapypregnant must is be an avoided alternative as it cantreatment cause abortionfor acne andand birthhas gained defects. more Phytotherapy attention isrecently an alternative due to severaltreatment advantages for acne and such has as gained low side more effects, attention its availability recently due in to local several areas advantages and is normally such as low low cost. side Manyeffects, herbs its availability in Thailand in local have areas been and proven is normally to be low able cost. to Manytreat acne—for herbs in Thailand example, have Centella been proven asiatica to, Zingiberbe able to montanum treat acne—for, Curcuma example, longaCentella, Garcinia asiatica mangostana, Zingiber and montanum Andrographis, Curcuma paniculata longa, Garcinia [4,5]. These mangostana have activityand Andrographis in different paniculata mechanisms[4,5]. These including have activity antibacterial, in different antioxidant, mechanisms and/or including anti‐inflammation antibacterial, effectsantioxidant, [6]. Benchalokawichian, and/or anti-inflammation a Thai herbal effects remedy, [6]. Benchalokawichian, is one of the medicines a Thai listed herbal in remedy, the Thailand is one Nationalof the medicines List of Essential listed in Medicines the Thailand that National is typically List used of Essential for relief Medicines of fever [7]. that It is is composed typically used of roots for fromrelief offive fever plants [7]. Itincluding is composed Ficus of racemosa roots from, Capparis five plants micracantha including, FicusClerodendrum racemosa, Capparispetasites, micracantha Harrisonia, perforateClerodendrum and Tiliacora petasites, triandraHarrisonia in perforate equal amountsand Tiliacora of each. triandra Recently,in equal it has amounts also been of each. used Recently, to treat itacne has andalso several been used skin to disorders treat acne [8]. and Several several herbs skin disorders may be used [8]. Several in combination herbs may to be obtain used inbetter combination result and to hasobtain nowadays better result been and developed has nowadays for use been in the developed form of an for herbal use in ball. the form of an herbal ball. The herbal ball or herbal herbal compress compress ball (Figure 1 1)) isis aa Thai traditionaltraditional treatmenttreatment thatthat usesuses aa roundround herbal ball of selected herbs. The selected herbs can be used either in fresh or dried forms. The herbal ball has been used for relieving many diseases including acne. Its pharmacological activity depends on the the herb herb composition. composition. To To prepare prepare an an herbal herbal ball, ball, selected selected herbs herbs are arechopped chopped into into small small pieces pieces and wrappedand wrapped in a inmuslin a muslin cloth cloth and and bundled bundled together together as asa sphere. a sphere. To To use use the the herbal herbal ball, ball, it it must must be steamed for about 15–20 min, then applied onto the the skin with a gentle pressing action to allow the active ingredients ingredients to to be be absorbed absorbed [9]. [9]. The The herbal herbal ball ball treatment treatment is isvery very popular popular in inspas, spas, especially especially in thein the case case of ofinducing inducing deep deep relaxation, relaxation, relieving relieving stress stress and and fatigue. fatigue. However, However, for for treating treating chronic diseases including acne, the herbal ball treatment must be applied every every day day for for a long time, so it is not suitable as it takes takes many many processes processes and time for treatment. treatment. One One herbal herbal ball ball can can be be used used repeatedly repeatedly only 3–4 times before being discarded. After After the the first ﬁrst use, it must be stored in a refrigerator until the next time of use. Therefore, the adaptation of of using an herbal ball from the traditional method to a formulation containing the herbal ball extract would be more convenient.

Figure 1. Herbal ball. Figure 1. Herbal ball.

The objective of this work was to prepare a gel formulation containing the herbal ball extract to be used Theinstead objective of using of the this herbal work ball was in to the prepare traditional a gel method formulation for acne containing treatment. theThe herbalherbal ball containedextract to four be used types instead of herbs of including using the Andrographis herbal ball paniculata in the traditional, Centella asiatica method, the forBenchalokawichian acne treatment. remedyThe herbal and ball the contained stem bark four powder types ofof herbsHesperethusa including crenulataAndrographis (Thanaka), paniculata which, Centellahave different asiatica, mechanismsthe Benchalokawichian to treat acne. remedyAndrographis and paniculata the stem and bark Centella powder asiatica of Hesperethusa have antibacterial crenulata and antioxidant(Thanaka), effectswhich have[6], the different Benchalokawichian mechanisms toremedy treat acne.has anAndrographis antibacterial paniculata effect [10]and andCentella Thanaka asiatica has haveanti‐ inflammatoryantibacterial and and antioxidant antioxidant effects effects [6], [11]. the BenchalokawichianThe herbal ball was remedy extracted has anin antibacterialthe same way effect as [the10] traditionaland Thanaka use. has The anti-inﬂammatory obtained extract and was antioxidant further used effects to prepare [11]. The the herbal gel formulation ball was extracted and the in thegel formulationsame way as was the tested traditional for physicochemical use. The obtained properties extract was and furtheranti P. acnes used activity. to prepare the gel formulation and the gel formulation was tested for physicochemical properties and anti P. acnes activity.

Sci. Pharm. 2018, 86, 8 3 of 9

2. Materials and Methods

2.1. Materials 2,2-Diphenyl-1-picrylhydrazyl (DPPH), gallic acid and tetracycline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carbopol® 940 was supplied by BF Goodrich (Cleveland, OH, USA). Propylene glycol and triethanolamine were supplied from Fisher Scientific (Leicestershire, UK). Propionibacterium acnes (DMST 14916) was obtained from the Department of Medical Science, Ministry of Public Health, Thailand. Brain heart infusion (BHI) media were purchased from Himedia laboratories (Mumbai, India). Clindalin® gel (clindamycin 1% gel, Union Drug Laboratories Ltd., Bangkok, Thailand) was purchased from a drugstore. Distilled and sterile water was used across the study. All other chemicals and solvents were of analytical grade and used as received. Andrographis paniculata and Centella asiatica leaves were collected as fresh herbs from the Krasaesin district, Songkhla, Thailand. The stem bark powder of Hesperethusa crenulata or Thanaka was obtained from the Thongphaphum district, Kanchanaburi, Thailand. The Benchalokawichian remedy was purchased as the dried powder from the Thaprachan herb shop, Bangkok, Thailand.

2.2. Preparation of Herbal Ball and Herbal Ball Extract The herbal ball was prepared by using four types of dried herbs including Andrographis paniculata leaves, Centella asiatica leaves, the Benchalokawichian remedy in powder form and Thanaka. Fresh leaves of Andrographis paniculata and Centella asiatica were cleaned and dried in a hot air oven (UF 110, Memmert GmbH + Co. KG, Schwabach, Germany) at 45 ◦C, then roughly ground with a mortar and pestle. Dried herbs (10 g each) were mixed together and wrapped in a muslin cloth and tied up together in a spherical shape. The herbal ball was then moistened by dipping into water for 2–3 s and steamed by placing over hot water in a water bath (WNB 29, Memmert GmbH + Co. KG) at 80 ◦C for 15 min. Subsequently, the herbal ball was squeezed by hand to obtain the crude extract solution. The herbal ball was repeatedly extracted another three times. The obtained extract solution was poured together and centrifuged at 9000 rpm at 25 ◦C for 5 min. The supernatant was ﬁltered through Whatman® grade No. 1 ﬁlter paper and dried by freeze-dryer (GAMMA 2-16 LSCplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). After freeze-drying, a dried herbal ball extract of 6.74% of the total weight of the dried herbs was obtained. The dried herbal ball extract was kept in the desiccator until used.

2.3. Evaluation of Herbal Ball Extract

2.3.1. Antioxidant Activity The antioxidant activity of the herbal ball extract was evaluated by the DPPH assay. The procedures were conducted in the same manner as described by Sharma et al. [12]. The DPPH was dissolved in ethanol and further diluted to make the final concentration of 0.4 mM. The sample solution was prepared by dissolving the herbal ball extract in water and diluted to obtain five concentrations from 10 to 300 µg/mL. Gallic acid was used as the positive control. It was dissolved in ethanol and diluted to obtain five concentrations from 1 to 8 µg/mL. A 100 µL aliquot of each sample solution was added to 100 µL of DPPH solution in a 96-well plate. The solution was mixed by being gently shaken and kept in the dark for 30 min. The absorbance at 517 nm was measured using a microplate reader (BioTek, BioTek Instrument, Inc., Winooski, VT, USA). The antioxidant activity percentage (AA%) was evaluated using the following equation:

AA% = (1 − As/A0) × 100 (1) where As and A0 are the absorbance at 517 nm of sample solution and blank DPPH solution, respectively. Sci. Pharm. 2018, 86, 8 4 of 9

The half maximal effective concentration (EC50), which is the concentration of the sample exhibiting 50% antioxidant activity, was calculated from the curve plotting between the sample concentration (x axis) and the AA% (y axis) [13].

2.3.2. Anti Propionibacterium acnes Activity The anti P. acnes activity of the herbal ball extract was tested by determining the minimum inhibitory concentration (MIC) by the broth dilution method and the minimum bactericidal concentration (MBC) following the MIC assay. P. acnes were grown in a BHI broth under anaerobic conditions using the Anaerocult A Mini system (Merck, Darmstadt, Germany) at 37 ◦C for 72 h. The turbidity of bacterial suspension was adjusted by using McFarland No. 0.5 to obtain about 1 × 108 colony-forming unit (CFU)/mL. The herbal ball extract (0.05 g) was dissolved in sterile water and the volume adjusted to 100 mL to obtain a concentration of 500 µg/mL as a stock solution. The stock solution was ﬁltered through a 0.45 µm sterile ﬁlter before being diluted with BHI broth in a 2-fold dilution series in glass test tubes at six concentrations from 250 to 7.81 µg/mL. The positive control was tetracycline, which was diluted in a 2-fold dilution series to achieve six concentrations from 0.24 to 0.0075 µg/mL. The P. acnes suspension (50 µL) was added in each sample solution (1 mL). The samples were then incubated in an anaerobic environment using the Anaerocult A Mini system (Merck, Darmstadt, Germany) at 37 ◦C for 48 h. The lowest concentration with a clear culture observed was detected as the MIC. Negative controls were performed with the same procedure as the sample but without adding the P. acnes. For determination of MBC, the dilution representing the MIC and three of the more concentrated sample dilutions were plated on a BHI agar. A sample dilution of one of the less concentrated MIC sample dilutions was used as the positive control. The agar plates were incubated in an anaerobic environment using the Anaerocult A Mini system (Merck, Darmstadt, Germany) at 37 ◦C for 48 h. The lowest concentration of the sample that showed no visible growth was detected as the MBC.

2.4. Preparation of Gel Formulation Containing the Herbal Ball Extract Gel formulations containing the herbal ball extract were prepared in three concentrations including 0.1, 1 and 5% w/w using carbomer as the gel base. The herbal ball extract (0.1, 1, or 5 g) was dissolved in about one-third of the sterile water used in the formula. The herbal ball extract solution was filtered through a 0.45 µm sterile filter and mixed with another part of sterile water to obtain a solution with a total volume of 95 mL. Carbopol® 940 (1 g) and propylene glycol (3 mL) were mixed in a mortar. The herbal ball extract solution was then added and stirred continuously to ensure complete hydration. Triethanolamine (about 1–2 mL) was then added dropwise under gentle stirring until a gel was formed.

2.5. Evaluation of Gel Formulation Containing the Herbal Ball Extract

2.5.1. Physicochemical Properties Gel formulations containing the herbal ball extract were subjected to tests for appearance, color, odor, homogeneity and pH. The appearance, color and homogeneity were tested by visual inspection. The homogeneity was graded as +++ good, ++ fair and + poor. The pH was determined using a digital pH meter (Cole-Parmer, London, UK).

2.5.2. Anti Propionibacterium acnes Activity The anti P. acnes activity of the gel formulations containing the herbal ball extract was evaluated by using the agar well diffusion method. The BHI agar was inoculated by spreading P. acnes over the entire agar surface and left to dry. A hole with a diameter of 6 mm was punched aseptically with a sterile tip. The gel formulations were introduced into the well. Then, agar plates were incubated in an anaerobic environment using the Anaerocult A Mini system (Merck, Darmstadt, Germany) at 37 ◦C for Sci. Pharm. 2018, 86, 8 5 of 9

48 h. Gel base and Clindalin® gel (clindamycin 1% gel) were used as the placebo and positive control, respectively.Sci. Pharm. 2018 The, x anti, x FORP. PEER acnes REVIEWactivity was investigated as the diameters of the clear zone. 5 of 9

2.5.3.48 Stability h. Gel base Study and Clindalin® gel (clindamycin 1% gel) were used as the placebo and positive control, respectively.The gel formulations The anti P. wereacnes activity subjected was to investigated accelerated as stabilitythe diameters tests of by the using clear azone. freeze-thaw cycle for six cycles. For each cycle, the samples that were packed in the plastic tube were stored at 4 ◦C for 24 h, 2.5.3. Stability Study then stored at 30 ◦C for 24 h. After the completion of six cycles, the gel formulations were evaluated for physicochemicalThe gel formulations properties were when subjected compared to accelerated to the freshly stability prepared tests by formulations.using a freeze‐thaw cycle for six cycles. For each cycle, the samples that were packed in the plastic tube were stored at 4 °C for 3. Results24 h, then and stored Discussion at 30 °C for 24 h. After the completion of six cycles, the gel formulations were evaluated for physicochemical properties when compared to the freshly prepared formulations. 3.1. Antioxidant Activity of the Herbal Ball Extract 3. Results and Discussion The DPPH assay is a reliable method to determine the antioxidant activity of the samples. The antioxidants3.1. Antioxidant can Activity stabilize of the the Herbal free Ball DPPH Extract radicals due to the proton donating ability. The reduction of free DPPHThe DPPH radicals assay can is be a reliable detected method by spectroscopy to determine at the 517 antioxidant nm based activity on the of purple the samples. color changingThe to yellow.antioxidants In this can study, stabilize the herbal the free ball DPPH extract radicals was due sterilized to the proton by being donating ﬁltered ability. through The reduction a 0.45 µm of sterile ﬁlterfree before DPPH testing. radicals By can ignoring be detected sterilization, by spectroscopy microbial at 517 contaminants nm based on of the the purple sample color might changing be obtained. to The antioxidantyellow. In this activity study, the of herbal the herbal ball extract ball extract was sterilized and the by positive being filtered control through gallic a 0.45 acid μ arem sterile shown in Figurefilter2. The before AA% testing. of the By herbal ignoring ball sterilization, extract and gallicmicrobial acid contaminants increased with of increasingthe sample concentration.might be The valuesobtained. within The antioxidant the examined activity concentrations of the herbal rangedball extract from and 25–60% the positive for the control herbal gallic ball acid extract are and shown in Figure 2. The AA% of the herbal ball extract and gallic acid increased with increasing 2–85% for the gallic acid. In addition, it was observed that the antioxidant activities of both the herbal concentration. The values within the examined concentrations ranged from 25–60% for the herbal ball extract and gallic acid within the examined concentrations were related to concentration as a linear ball extract and 2–85% for the gallic acid. In addition, it was observed that the antioxidant activities relationship.of both the The herbal calculated ball extract EC50 andof the gallic herbal acid ball within extract the examined and gallic concentrations acid were 219.27 were± related36.98 µtog/mL and 4.97concentration± 0.16 µ asg/mL, a linear respectively. relationship. TheThe calculated herbal ball EC extract50 of the had herbal lower ball extract antioxidant and gallic activity acid than the gallicwere acid219.27 by ± about36.98 μ 40-fold.g/mL and This 4.97 would ± 0.16 μ beg/mL, generally respectively. observed The forherbal the ball crude extract plant had extract lower when comparedantioxidant to the activity pure antioxidant than the gallic compound. acid by about However, 40‐fold. This when would compared be generally to the observed antioxidant for the activity of thecrude herbal plant ball extract extract when obtained compared from to the several pure herbsantioxidant composition compound. with However, that of when the individual compared herb to the antioxidant activity of the herbal ball extract obtained from several herbs composition with extracts reported in the literature, the herbal ball extract had better activity. The EC50 value of the DPPHthat radical of the individual scavenging herb activity extracts of reported the water in the extract literature, of Andrographis the herbal ball paniculata extract hadand betterCentella activity. asiatica The EC50 value of the DPPH radical scavenging activity of the water extract of Andrographis paniculata were 418.51 µg/mL [14] and 300 µg/mL [15], respectively. This implied that the extract obtained and Centella asiatica were 418.51 μg/mL [14] and 300 μg/mL [15], respectively. This implied that the fromextract the herb obtained combination from the could herb combination induce more could effective induce activity more effective than the activity extract than obtained the extract from the individuals.obtained Moreover,from the individuals. Thanaka Moreover, might also Thanaka increase might the antioxidant also increase activity the antioxidant of the herbalactivity ball of the extract. Antioxidantherbal ball activity extract. is one Antioxidant of the mechanisms activity is underlyingone of the phytotherapy’smechanisms underlying treatment phytotherapy of acne. Althoughʹs the antioxidanttreatment of activityacne. Although of the herbalthe antioxidant ball extract activity was of not the high,herbal itball might extract help was to not improve high, it themight overall resultshelp of to acne improve treatment the overall by synergizing results of acne it with treatment anti P. by acnes synergizingactivity. it with anti P. acnes activity.

FigureFigure 2. Antioxidant 2. Antioxidant activity activity percentage percentage (AA%) of of (a ()a )the the herbal herbal ball ball extract extract and and (b) the (b) positive the positive control gallic acid at various concentrations. control gallic acid at various concentrations.

Sci. Pharm. 2018, x, x FORSci. PEERPharm. REVIEW 2018, x, x FOR PEER REVIEW 6 of 9 6 of 9

3.2. Propionibacterium3.2. acnesPropionibacterium Activity of Herbal acnes Ball Activity Extract of Herbal Ball Extract P. acnes is responsibleP. acnes for isthe responsible inflammation for theprocess inflammation in acne formation. process in The acne herbal formation. ball extract The herbal ball extract showed anti P. acnesshowed activity. anti The P. acnes MIC activity.against P.The acnes MIC was against equal P. to acnes the MBC.was equal The valueto the wasMBC. 31.25 The value was 31.25 μg/mL. In comparisonμg/mL. to Inthe comparison positive control to the tetracycline positive control (MIC tetracycline= 0.06 μg/mL), (MIC the = 0.activity06 μg/mL), of the the activity of the herbal ball extractherbal was about ball extract500‐fold was lower. about It has500 ‐beenfold lower.recommended It has been that recommended the threshold thatMIC the values threshold MIC values for plant extract andfor plantpure compoundsextract and pureshould compounds be 100 μg/mL should and be 10 100 μg/mL, μg/mL respectively and 10 μg/mL, [16]. respectivelyThe [16]. The herbal ball extract,herbal therefore, ball extract,could be therefore, considered could to have be considered high antimicrobial to have high activity antimicrobial against P. activityacnes. against P. acnes. When the activityWhen of the the herbal activitySci. ballPharm. of extract the2018 ,herbal x , xfound FOR ballPEER in extractREVIEWthis study found was in comparedthis study with was itscompared herb with its herb 6 of 9 Sci. Pharm. 2018, 86, 8 6 of 9 components (Andrographiscomponents paniculata (Andrographis and Centella paniculata asiatica and) reported Centella inasiatica the literature,) reported it in was the found literature, that it was found that the herbal ball extractthe herbal had better ball 3.2.extractactivity. Propionibacterium had The better MIC activity.of the acnes water TheActivity extractMIC of of Herbal of the An waterdrographis Ball Extractextract paniculata of Andrographis and paniculata and 3.2.Centella Propionibacterium asiatica againstCentella acnes P. acnesasiatica Activity were against of Herbal0.625P. acnes P. mg/mL Ballacnes is Extractresponsible were and 0.6255 mg/mL, mg/mLfor the respectively inflammationand 5 mg/mL, [4]. The processrespectively combination in acne [4]. offormation.The combination The herbal of ball extract natural substancesnatural with similar substances biologicalshowed with anti similaractivity P. acnes biological that activity. could activity provide The MIC that additive against could activityprovide P. acnes against additivewas equal acne activity to‐ the against MBC. Theacne value‐ was 31.25 P. acnes is responsible for the inﬂammation process in acne formation. The herbal ball extract causing bacteria wascausing also bacteriafoundμ ing/mL. was the alsostudy In comparisonfound by Julianti in the to etstudy theal. [17].positive by Julianti The controlBenchalokawichian et al. tetracycline[17]. The Benchalokawichian (MICremedy = 0.06 μg/mL), remedy the activity of the showed anti P. acnes activity. The MIC against P. acnes was equal to the MBC. The value was was reported to bewas effective reported against toherbal be a e varietyffective ball extract ofagainst bacteria was a about variety including 500 of‐fold bacteria both lower. gram including It positivehas been both and recommended gram negative positive that and the negative threshold MIC values 31.25 µg/mL. In comparison to the positive control tetracycline (MIC = 0.06 µg/mL), the activity of bacteria [10], however,bacteria for [10], antimicrobial however,for plant extractforactivity antimicrobial and against pure compoundsP. activity acnes, itagainst hasshould not P. bebeenacnes 100 , reported. μit g/mLhas not and The been 10 μ reported.g/mL, respectively The [16]. The the herbal ball extract was about 500-fold lower. It has been recommended that the threshold MIC BenchalokawichianBenchalokawichian remedy mightherbal also remedy ballbe beneficial extract, might therefore, foralso acne be beneficial treatment could be forconsidered caused acne bytreatment bacteria.to have caused high The antmain byimicrobial bacteria. Theactivity main against P. acnes. values for plant extract and pure compounds should be 100 µg/mL and 10 µg/mL, respectively [16]. anti P. acnes activityanti ofP. theacnes herbal activityWhen ba lltheof extract theactivity herbal should of ba the llcome extractherbal from shouldball Andrographis extract come foundfrom paniculata Andrographisin this, which study paniculata was compared, which with its herb The herbal ball extract, therefore, could be considered to have high antimicrobial activity against P. acnes. contains andrographolide,contains andrographolide,an activecomponents principle an(Andrographis of active Andrographis principle paniculata paniculata of Andrographis and withCentella antimicrobial paniculata asiatica) reported with activity antimicrobial in the literature, activity it was found that When the activity of the herbal ball extract found in this study was compared with its herb components [18,19]. Centella asiatica[18,19]. and Centella the Benchalokawichianthe asiatica herbal and ball the extract Benchalokawichian remedy had better would activity. synergize remedy The itswould MIC activity. of synergize the The water activity its extract activity. of An Thedrographis activity paniculata and (Andrographis paniculata and Centella asiatica) reported in the literature, it was found that the herbal ball of the extract fromof the herbalextract ballCentellafrom was the asiatica thereforeherbal against ball much was P. acnesgreater therefore were than 0.625much the mg/mL activitygreater and thanfrom 5 mg/mL, theits herbalactivity respectively from its [4]. herbal The combination of extract had better activity. The MIC of the water extract of Andrographis paniculata and Centella asiatica components. The samecomponents. amount Theofnatural MIC same and substances am MBCount obtainedof withMIC andsimilar from MBC the biological obtainedherbal ball activity from extract the that herbalagainst could ball P. provide acnes extract additive against P. activity acnes against acne‐ against P. acnes were 0.625 mg/mL and 5 mg/mL, respectively [4]. The combination of natural suggested that thesuggested herbal ball that extract causingthe herbalcould bacteria possiblyball extract was act also couldas a foundbactericidal possibly in the act agent study as a bactericidalfor by this Julianti microorganism. agent et al. for[17]. this The microorganism. Benchalokawichian remedy substances with similar biological activity that could provide additive activity against acne-causing was reported to be effective against a variety of bacteria including both gram positive and negative bacteria3.3. Physicochemical was also found Properties in the study of Gel by Formulation Julianti et al.Containing [17]. The the Benchalokawichian Herbal Ball Extract remedy and Stability was reported 3.3. Physicochemicalbacteria Properties [10], however,of Gel Formulation for antimicrobial Containing activity the Herbal against Ball Extract P. acnes and, itStability has not been reported. The to be effective against a variety of bacteria including both gram positive and negative bacteria [10], Carbomer gel formulationsBenchalokawichian containing the herbal remedy ball might extract also in be thre beneficiale concentrations for acne treatment were caused by bacteria. The main however, for antimicrobialCarbomer activity gel against formulationsP. acnes, it containing has not been the reported. herbal Theball Benchalokawichianextract in three concentrations were successfully prepared. The lowestanti (0.1% P. wacnes/w) and activity highest of (5%the wherbal /w) concentrations ball extract shouldwere about come 32 ‐fromfold Andrographis paniculata, which remedy might alsosuccessfully be beneﬁcial prepared. for acne treatmentThe lowest caused (0.1% byw/w bacteria.) and highest The main (5% w anti/w) P.concentrations acnes activity were about 32‐fold and 1600‐fold higher in concentrationcontains than andrographolide, the MIC, respectively. an active The gelprinciple formulations of Andrographis were clear paniculata and with antimicrobial activity of the herbal ball extractand 1600 should‐fold come higher from in concentrationAndrographis than paniculata the MIC,, which respectively. contains andrographolide,The gel formulations were clear and translucent with pale yellow, yellow[18,19].‐brown Centella and asiatica dark and brown the Benchalokawichian colors for the 0.1, remedy1 and would5% w/ wsynergize its activity. The activity an active principletranslucent of Andrographis with paniculata pale yellow,with yellow antimicrobial‐brown activityand dark [18 ,brown19]. Centella colors asiatica for thande 0.1, 1 and 5% w/w formulations, respectively (Tableof 1). the The extract 5% w/ wfrom formulation the herbal had ball a mild was odor therefore while muchthe others greater were than the activity from its herbal the Benchalokawichianformulations, remedy would respectively synergize (Table its activity.1). The 5% The w activity/w formulation of the extract had a from mild the odor herbal while the others were odorless. A visual observation revealedcomponents. that The all sameformulations amount ofwere MIC homogeneous. and MBC obtained The pHfrom of the the herbal ball extract against P. acnes ball was therefore muchodorless. greater A visual than theobservation activity from revealed its herbal that all components. formulations The were same homogeneous. amount of The pH of the formulations was between 5.5 andsuggested 7.2. This that was the due herbal to the ball ne utralizationextract could of possibly the carbomer act as a(acid) bactericidal and agent for this microorganism. MIC and MBC obtainedformulations from the was herbal between ball 5.5 extract and 7.2. against ThisP. was acnes duesuggested to the neutralization that the herbal of the ball carbomer (acid) and triethanolamine (base) and the gel was formed at a pH of around 6 [20]. A pH value of 5.5 in extract could possiblytriethanolamine act as a bactericidal (base) agentand the for gel this was microorganism. formed at a pH of around 6 [20]. A pH value of 5.5 in formulations for skinformulations use has been for3.3. skin recommended Physicochemical use has been [21]. Properties recommended Therefore, of Gel the Formulation [21]. prepared Therefore, Containing gel wasthe preparedsuitable the Herbal for gel Ball was Extract suitable and for Stability 3.3.skin Physicochemical use. skin Properties use. of Gel FormulationCarbomer Containing gel formulations the Herbal containing Ball Extract the and herbal Stability ball extract in three concentrations were Carbomer gel formulationssuccessfully containing prepared. the herbal The ball lowest extract (0.1% in w three/w) and concentrations highest (5% w were/w) concentrations were about 32‐fold Table 1. PhysicochemicalTable 1.properties Physicochemical of gel formulations properties of containing gel formulations the herbal containing ball extract the atherbal the ball extract at the successfully prepared. The lowestand (0.1% 1600‐ w/w)fold higher and highest in concentration (5% w/w) than concentrations the MIC, respectively. were about The gel formulations were clear and concentration of 0.1,concentration 1 and 5% w/w of. 0.1, 1 and 5% w/w. 32-fold and 1600-fold higher in concentrationtranslucent with than thepale MIC, yellow, respectively. yellow‐brown The gel and formulations dark brown were colors for the 0.1, 1 and 5% w/w clear and translucentProperties with pale yellow,formulations,Properties yellow-brown respectivelyFormulations and (Table dark brown 1). TheFormulations colors 5% w/ forw formulation the 0.1, 1 and had 5% a mild odor while the others were w/w formulations, respectively (Tableodorless.0.1).1% The wA/ 5%wvisual w/w observation formulation0.1%1% w w/w/w revealed had a5% mild1% that w w/ odorw/ wall formulations while the5% othersw/ wwere homogeneous. The pH of the were odorless. A visual observationformulations revealed that was all between formulations 5.5 and were 7.2. homogeneous. This was due to The the pH ne ofutralization the of the carbomer (acid) and formulations was between 5.5 andtriethanolamine 7.2. This was due (base) to the and neutralization the gel was of formed the carbomer at a pH (acid) of around and 6 [20]. A pH value of 5.5 in Appearance Appearance triethanolamine (base) and the gel wasformulations formed at afor pH skin of around use has 6 [been20]. Arecommended pH value of 5.5 [21]. in formulations Therefore, the prepared gel was suitable for for skin use has been recommendedskin [21 use.]. Therefore, the prepared gel was suitable for skin use. Color PaleColor yellow YellowPale yellow‐brown YellowDark brown‐brown Dark brown Table 1. PhysicochemicalOdor propertiesTable ofOdor‐ gel 1. formulations Physicochemical containing‐‐ properties theMild herbalof gel odor‐ ballformulations extractMild at containing the odor the herbal ball extract at the concentration of 0.1, 1 and 5% w/w. concentration ofHomogeneity 0.1, 1 and 5% w/w. Homogeneity+++ ++++++ ++++++ +++ pH 5.58 pH± 0.02 Properties6.615.58 ± ± 0.02 0.02 7.246.61 ± ± 0.01 0.02 Formulations7.24 ± 0.01 Properties Formulations Note: homogeneity Note:was graded homogeneity as +++ good, was graded ++ fair andas0. 1%+++ + poor.wgood,/w ++ fair and1% +w poor./w 5% w/w 0.1% w/w 1% w/w 5% w/w

Appearance Appearance Color Pale yellow Yellow‐brown Dark brown Odor ‐ ‐ Mild odor Color Pale yellow Yellow-brown Dark brown Odor -Homogeneity - +++ Mild odor+++ +++ Homogeneity +++pH +++5.58 ± 0.02 +++6.61 ± 0.02 7.24 ± 0.01 pH 5.58 ± 0.02Note: 6.61 homogeneity± 0.02 was graded 7.24 as +++± 0.01 good, ++ fair and + poor. Note: homogeneity was graded as +++ good, ++ fair and + poor. Sci. Pharm. 2018, 86, 8 7 of 9 Sci. Pharm. 2018, x, x FOR PEER REVIEW 7 of 9

AfterAfter aa freeze-thaw freeze‐thaw cycle cycle for for six six cycles, cycles, the the physicochemical physicochemical properties properties of of the the gel gel formulations formulations in termin term of appearance,of appearance, color, color, odor, odor, homogeneity homogeneity and and pH pH were were unchanged. unchanged. Therefore, Therefore, the preparedthe prepared gel formulationsgel formulations were were physically physically stable stable in accelerated in accelerated conditions. conditions. 3.4. Anti Propionibacterium acnes Activity of Gel Formulation Containing the Herbal Ball Extract 3.4. Anti Propionibacterium acnes Activity of Gel Formulation Containing the Herbal Ball Extract The gel formulation containing the herbal ball extract at a concentration of 5% w/w only exhibited The gel formulation containing the herbal ball extract at a concentration of 5% w/w only exhibited antimicrobial activity against P. acnes with a clear zone diameter 10.00 ± 1.00 mm (Figure3), while the antimicrobial activity against P. acnes with a clear zone diameter 10.00 ± 1.00 mm (Figure 3), while the lower concentration gel formulations (0.1 and 1% w/w) could not inhibit the growth of P. acnes. lower concentration gel formulations (0.1 and 1% w/w) could not inhibit the growth of P. acnes. The The placebo had no activity to inhibit the growth of P. acnes while the Clindalin® gel (positive control) placebo had no activity to inhibit the growth of P. acnes while the Clindalin® gel (positive control) could effectively inhibit the growth of P. acnes as no P. acnes was observed on the whole surface of the could effectively inhibit the growth of P. acnes as no P. acnes was observed on the whole surface of agar plate. The anti P. acnes activity of the gel formulations was relatively low although the herbal ball the agar plate. The anti P. acnes activity of the gel formulations was relatively low although the herbal extract at high concentrations of about 1000-fold of MIC was used. This might be due to the effect of the ball extract at high concentrations of about 1000‐fold of MIC was used. This might be due to the effect gel base, which retarded the diffusion of the herbal ball extract into the agar. The agar well diffusion of the gel base, which retarded the diffusion of the herbal ball extract into the agar. The agar well method could be used for only screening the antimicrobial activity of the gel formulation. In order diffusion method could be used for only screening the antimicrobial activity of the gel formulation. to effectively use this formulation for acne treatment in practice, the improvement of its activity and In order to effectively use this formulation for acne treatment in practice, the improvement of its testing more P. acnes strains should be further investigated. activity and testing more P. acnes strains should be further investigated.

FigureFigure 3.3.Photographs Photographs showing showing the the inhibition inhibition zone zone of gel of formulation gel formulation containing containing the herbal the ballherbal extract ball atextract (a) 0.1% at (a w/w,) 0.1% (b w)/ 1%w, ( w/wb) 1% and w/w ( cand) 5% (c w/w) 5% w against/w againstP. acnes P. acneswhen when compared compared with with that that of (d of) the (d) placebothe placebo and and (e) the (e) positive the positive control. control.

4.4. ConclusionsConclusions GelGel formulationsformulations containing containing the the herbal herbal ball ball extract extract with with various various amounts amounts of the of herbal the ballherbal extract ball wereextract prepared were prepared in an attempt in an attempt for use for instead use instead of the traditionalof the traditional method method of the of herbal the herbal ball for ball acne for treatment.acne treatment. The gel The formulations gel formulations were subjectedwere subjected for antimicrobial for antimicrobial activity activity against againstP. acnes P.. The acnes herbal. The compositionherbal composition of the herbal of ballthe includedherbal ballAndrographis included paniculata Andrographis, Centella paniculata asiatica,, the Centella Benchalokawichian asiatica, the remedyBenchalokawichian and the stem remedy bark power and oftheHesperethusa stem bark power crenulata of Hesperethusa(Thanaka), which crenulata have (Thanaka), different mechanisms which have fordifferent treating mechanisms acne. The herbal for treating ball extract acne. had The good herbal activities ball extract for antioxidants had good andactivities anti P. for acnes antioxidantsaccording toand the anti DPPH P. acnes assay according and broth dilutionto the DPPH study. assay The gel and formulation broth dilution containing study. the The herbal gel formulation ball extract withcontaining the concentration the herbal ofball 5% extract w/w showedwith the antimicrobial concentration activity of 5% against w/w showedP. acnes antimicrobialaccording to the activity agar wellagainst diffusion P. acnes study. according The obtained to the results agar well suggest diffusion that gel study. formulation The obtained containing results an herbal suggest ball that extract gel formulation containing an herbal ball extract of at least 5% w/w could be effectively used for acne treatment. Importantly, the prepared gel was more convenient to use in comparison to the traditional method of herbal ball usage.

Sci. Pharm. 2018, 86, 8 8 of 9 of at least 5% w/w could be effectively used for acne treatment. Importantly, the prepared gel was more convenient to use in comparison to the traditional method of herbal ball usage.

Acknowledgments: This work was part of a special problem in the pharmacy of Atthaphon Konpian, Gorawit Sukkua and Prutthicha Wongprasert. Financial support from Walailak University is gratefully acknowledged. Author Contributions: C.J., P.S. and T.C. conceived and designed the experiments; A.K., G.S. and P.W. performed the experiments; C.J. and T.C. analyzed the data; and C.J. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Institute of Dermatology, Department of Medical Sciences, Ministry of Public Health, Thailand. Available online: http://inderm.go.th/inderm/inderm_pan.php (accessed on 4 January 2018). 2. Zaenglein, A.L.; Pathy, A.L.; Schlosser, B.J.; Alikhan, A.; Baldwin, H.E.; Berson, D.S.; Bowe, W.P.; Graber, E.M.; Harper, J.C.; Kang, S.; et al. Guidelines of care for the management of acne vulgaris. J. Am. Acad. Dermatol. 2016, 74, 945–973. [CrossRef][PubMed] 3. Patel, M.; Bowe, W.P.; Heuqhebaert, C.; Shalita, A.R. The development of antimicrobial resistance due to the antibiotic treatment of acne vulgaris: A review. J. Drugs Dermatol. 2010, 9, 655–664. [PubMed] 4. Chomnawang, M.T.; Surassmo, S.; Nukoolkarn, V.S.; Gritsanapan, W. Antimicrobial effects of Thai medicinal plants against acne-inducing bacteria. J. Ethnopharmacol. 2005, 101, 330–333. [CrossRef][PubMed] 5. Neamsuvan, O.; Kama, A.; Salaemae, A.; Leesen, S. A survey of herbal formulas for skin diseases from Thailand’s three southern border provinces. J. Herb. Med. 2015, 5, 190–198. [CrossRef] 6. Azimi, H.; Fallah-Tafti, M.; Khakshur, A.A.; Abdollahi, M. A review of phytotherapy of acne vulgaris: Perspective of new pharmacological treatments. Fitoterapia 2012, 83, 1306–1317. [CrossRef][PubMed] 7. National List of Essential Medicines, 2018—Thailand. Available online: http://www.ratchakitcha.soc.go.th/ DATA/PDF/2561/E/014/4.PDF (accessed on 19 February 2018). 8. Juckmeta, T.; Thongdeeying, P.; Itharat, A. Inhibitory effect on β-hexosaminidase release from RBL-2H3 cells of extracts and some pure constituents of Benchalokawichian, a Thai herbal remedy, used for allergic disorders. Evid. Based Complement. Altern. Med. 2014, 2014, 828760. [CrossRef][PubMed] 9. Lertlop, W. The appropriate temperature of the Thai herbal ball compress for relaxing effected. Procedia Soc. Behav. Sci. 2015, 197, 1653–1660. [CrossRef] 10. Nuaeissara, S.; Kondo, S.; Itharat, A. Antimicrobial activity of the extracts from Benchalokawichian remedy and its components. J. Med. Assoc. Thai. 2011, 94 (Suppl. 7), S172–S177. [PubMed] 11. Wangthong, S.; Palaga, T.; Rengpipat, S.; Wanichwecharungruang, S.P.; Chanchaisak, P.; Heinrich, M. Biological activities and safety of Thanaka (Hesperethusa crenulata) stem bark. J. Ethnopharmacol. 2010, 132, 466–472. [CrossRef][PubMed] 12. Sharma, R.; Kishore, N.; Hussein, A.; Lall, N. The potential of Leucosidea sericea against Propionibacterium acnes. Phytochem. Lett. 2014, 7, 124–129. [CrossRef] · 13. Chen, Z.; Bertin, R.; Froldi, G. EC50 estimation of antioxidant activity in DPPH assay using several statistical programs. Food Chem. 2013, 138, 414–420. [CrossRef][PubMed] 14. Rao, P.R.; Rathod, V.K. Rapid extraction of andrographolide from Andrographis paniculata Nees by three phase partitioning and determination of its antioxidant activity. Biocatal. Agric. Biotechnol. 2015, 4, 586–593. [CrossRef] 15. Anand, T.; Mahadeva, N.; Phani, K.G.; Farhath, K. Antioxidant and DNA damage preventive properties of Centella asiatica (L.) Urb. Phcog. J. 2010, 2, 53–58. [CrossRef] 16. Kuete, V. Potential of Cameroonian plants and derived-products against microbial infections: A review. Planta Med. 2010, 76, 1479–1491. [CrossRef][PubMed] 17. Julianti, E.; Rajah, K.K.; Fidrianny, I. Antibacterial activity of ethanolic extract of cinnamon bark, honey and their combination effects against acne-causing bacteria. Sci. Pharm. 2017, 85, 19. [CrossRef][PubMed] 18. Singha, P.K.; Roy, S.; Dey, S. Antimicrobial activity of Andrographis paniculata. Fitoterapia 2003, 74, 692–694. [CrossRef] Sci. Pharm. 2018, 86, 8 9 of 9

19. Arifullah, M.; Namsa, N.D.; Mandal, M.; Chiruvella, K.K.; Vikrama, P.; Gopal, G.R. Evaluation of anti-bacterial and anti-oxidant potential of andrographolide and echiodinin isolated from callus culture of Andrographis paniculata Nees. Asian Pac. J. Trop. Biomed. 2013, 3, 609–610. [CrossRef] 20. Islam, M.T.; Rodriquez-Hornedo, N.; Ciotti, S.; Ackermann, C. Rheological characterization of topical carbomer gels neutralized to different pH. Pharm. Res. 2004, 21, 1192–1199. [CrossRef][PubMed] 21. Lambers, H.; Piessens, S.; Bloem, A.; Pronk, H.; Finkel, P. Natural skin surface pH is on average below 5, which is beneﬁcial for its resident ﬂora. Int. J. Cosmet. Sci. 2006, 28, 359–370. [CrossRef][PubMed]

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