Nine Propionibacterium acnes (P. acnes) strains resistant to both clindamycin and erythromycin were selected to investigate the inhibitory ability of extracts and isolated compounds from leaves of Cassia grandis L.f to P. acnes. Ethyl acetate extract inhibited all P. acnes strains, with an aseptic ring diameter of 15-25 mm at a concentration of 200 mg/mL and a MIC value of 30 mg/mL.
AGU International Journal of Sciences – 2019, Vol (4), – 11 INHIBITORY ABILITY OF EXTRACTS AND COMPOUNDS FROM LEAVES OF CASSIA GRANDIS L.F TO PROPIONIBACTERIUM ACNES Ngo Quoc Luan1,2, Phung Thi Yen Thanh2, Tran Nhan Dung3, Huynh Van Ba4 Vietnam Academy of Science and Technology School of Education, Can Tho University Biotechnology Research & Development Institute, Can Tho University Can Tho Dermatology Hospital Information: Received: 070/8/2018 Accepted: 03/01/2019 Published: 11/2019 Keywords: P acnes, Cassia grandis L.f, quercitrin ABSTRACT Nine Propionibacterium acnes (P acnes) strains resistant to both clindamycin and erythromycin were selected to investigate the inhibitory ability of extracts and isolated compounds from leaves of Cassia grandis L.f to P acnes Ethyl acetate extract inhibited all P acnes strains, with an aseptic ring diameter of 15-25 mm at a concentration of 200 mg/mL and a MIC value of 30 mg/mL The fraction H:E 1:1 showed the strongest antibacterial activity in the three investigated fractions (H:E 1:1, H:E 1:3 and E 100%) with an aseptic ring diameter of 19.3-23.7 mm at a concentration of 200 mg/mL (tested on three strongest resistance strains) and the MIC value was determined to be 0.3 mg/mL In three pure compounds isolated from ethyl acetate extract, quercitrin strongest inhibited for P acnes with a MIC value of 0.2 mg/mL INTRODUCTION al., 2014; Fu Y et al., 2007) but no remarkable published announcement shows potential applications Propionibacterium acnes (P acnes) is considered to be an important cause of acne However, the treatment with synthetic antibiotics in the long term also causes many unwanted side effects On the other hand, the resistance of these bacterial strains to antibiotics is increasingly trending This increase is due to the mutation of the resistance gene (Vu Thi Phuong Thao & Van The Trung, 2014) Therefore, the search for new sources of antibiotics to replace these synthetic antibiotics is essential Some authors have studied the antibacterial effects of P acnes of some essential oils and herbal extracts (Charde YM et Traditionally, it is said that leaves of Cassia grandis L.f can cure a number of skin diseases such as Ringworm and Pityriasis Versicolor (Do Huy Bich et al., 2003) whose scientific basis can be the antimicrobial compounds found in leaves of Cassia grandis L.f, probably among the isolated compounds such as quercitrin, isoquercetin, afzelin, aloe-emodin, kaempferol, rutin, nicotiflorin (Ngo Quoc Luan et al., 2012; 2013) Therefore, this study aims to explore the resistance to P acnes strains that are resistant to the current common antibiotics AGU International Journal of Sciences – 2019, Vol (4), – 11 of extracting fragments and natural compounds from leaves of Cassia grandis L.f 2.3 Equipment, instruments and chemicals Main equipment in the laboratory for natural compound study such as rotary evaporator under low pressure, extraction equipment, chromatography column etc were supplied by Department of Chemistry Education, Can Tho University MATERIALS AND METHODS 2.1 Materials The plant sample used was the Cassia grandis L.f collected in Vinh Thanh district, Can Tho city in January 2015 The sample was identified by Dr Dang Minh Quan and was stored at the Department of Biology - School of Education Can Tho University The samples were treated by washing, removing damaged parts, cutting, drying, grinding into fine powder as raw materials for extracting and isolating compounds Main equipment in the laboratory for microbiological research was supplied by the Biotechnology Research and Development Institute, Can Tho University Thin layer chromatography (TLC) was carried out on pre-coasted silica gel 60F254 (0.25 mm) alumium sheet (Merck) For common phase column chromatography (CP-CC), silica gel 60 (0.040-0.063 mm, Merck) with increasing polarity solvent systems including n-hexane (H), EtOAc (E), CHCl3 (C), MeOH (M) and H2O (W) were used Source of P acnes (24 strains) was isolated and identified from 40 medical waste samples which were collected from skin of patients with common acne at Can Tho Dermatology Hospital in February 2015 Culture medium for TYEG agar (TrypticaseYeast Extract-Heart Extract-Glycerol Agar) prepared according to Kishishita M et al., 1980 Kovacs reagent used is produced by Merck Antibiotic plates, gram-staining kit and other chemicals were provided by Nam Khoa Company 2.2 Methods Method of extracting high modulation: marceration, liquid-liquid distributing extract (Nguyen Kim Phi Phung, 2007) Isolation of bacteria was followed by the method of Kishishita M et al., 1980; Hans B.L and Kilian M., 2010: Identifying P acnes strains of bacteria by biochemical tests and 16S-rARN sequencing (Rajakaruna L et al., 2009) EXPERIMENTAL PROCEDURE 3.1 Extraction products and isolation of plant The leaves of Cassia grandis L.f were exhausted with 96% methanol The sample extract was evaporated to remove solvent and gain total extract The methanol extract was dissolved in warm water and liquid-liquid extracted with single solvents in order to increase polarity, resulting in n-hexane (H) and ethyl acetate (E) extracts The E extract continued to be processed by column chromatography and distributed into fractions corresponding to the elution solvent systems H:E 1:1, H:E 1:3 and E 100% Antibiogramme based on the diffusion of antibiotics on agar plates of Bauer A.W et al, 1966; Nguyen Thanh Ha, 1991 Testing P acnes’ antibacterial activity by diffusion method on agar plates of Murugan T and Saranraj P., 2011; Identifying the minimum inhibitory concentration (MIC) by dilution according to Saising S and Voravuthikunchai S.P., 2012 Statistical data processing using software: Statgraphic centurion XVI.II, SPSS and Microsoft Excel 2007 AGU International Journal of Sciences – 2019, Vol (4), – 11 The process of isolation and structural identification of quercitrin, rutin and aloeemodin was reported in previous publications (Ngo Quoc Luan et al., 2012; 2013) All 24 isolated strains of P acnes were fully identified forms, biochemical and gene expression 16S-rRNA, signed from Pa01 to Pa24 3.2 Isolation of P acnes 3.3 Antibiogramming P acnes strains Dilute the solution containing medical waste samples in distilled water; extract 10 μL from test tubes of 10-2, 10-3, 10-4 dilution; drip on medium TYEG agar with bromocresol purple 0.002%, drops on each concentration; anaerobically incubate in a desiccator at 37°C for 5-7 days (Kishishita M et al., 1980) Antibiogramme of the P acnes strain was investigated to identify its resistance to antibiotics Based on this result, the resistant strains were selected to conduct further steps The bacterial suspension of strains one with a density of about 108 cells/mL (equivalent to Mc Farland turbidity 0.5) was placed on the agar plates (TYEG agar, mm thick); place them the antibiotics in turn so that the sides of the paper are close to the medium, the antibacterial wells are 20 mm apart; anaerobically incubate for 48 hours at 37°C The diameter of aseptic ring was measured and followed according to standards of Clinical and Laboratory Standards Institute (2009) (Table 1) to assess the antibiotic sensitivity of the isolated bacterial strains Yellow colonies forming and emerging over agar plates were selected, and once the medium color was changed from purple into yellow, streaking in the plate of TYEG agar for strain separation Observe under microscopic and select the rod-shape strain and store in TYEG broth with supplement of 20% glycerol at -40 °C as the source of material for further experiments (Hans B.L and Kilian M., 2010) Table Standard for sensitivity evaluation of P acnes to some synthetic antibiotics Diameter of aseptic ring (mm) No Antibiotic type Sign Concentration/well (μg) Sensitive Medium Resistant Tetracycline Te 30 ≥31 24-30 ≤23 Cefuroxime Cu 30 ≥36 27-35 ≤26 Levofloxacin Lv ≥31 25-30 ≤24 Erythromycin Er 15 ≥31 22-30 ≤21 Clindamycin cL ≥31 24-30 ≤23 Then the results were counted according to the number of strains at levels of susceptibility: Sensitive, Medium, Resistant extracts (or compound) with the highest antibacterial activity and resistant was selected to conduct the next experiment 3.4 Investigating the inhibitory ability of extracts, fractions and compounds to P Acnes After spreading the bacterial suspension on the agar plate (TYEG agar); drain; wells with a diameter of about mm was created so that each well is about 2-3 cm apart 30 µL of the agar was added into each well of compounds, negative control and positive control The extracts (or compound) from the leaves of Cassia grandis L.f was identified to inhibit P acnes From the experimental results, the AGU International Journal of Sciences – 2019, Vol (4), – 11 respectively Clindamycin (2 μg/mL) was used for the positive control and DMSO 1% was used for the negative control Proceed in turn at different concentrations; Anaerobically incubate them at 37 °C for 48 hours Measure aseptic ring diameter compounds: Cassia grandis L.f extracted in types of solvents: n-hexane, ethyl acetate and methanol at concentrations of 200 (Figure 1), 100 and 50 mg/mL; ethyl acetate extract in E 100%, H:E 1:3 and H:E 1:1 fractions at concentrations of 200, 100 and 50 mg/mL; quercitrin, rutin and aloe-emodin compounds at concentrations of 10, and 2.5 mg/mL Conduct an experiment to test the antibacterial activity of the following extracts and Pa01 Pa22 4 3 1 Figure The ability to inhibit P acnes of leaves of Cassia grandis L.f extracts at a concentration of 200 mg/mL Notes: (1) Ethyl acetate extract, (2) n-hexane extract, (3) Methanol extract, (4) Control (-) DMSO 3.5 Determining the minimum inhibitory concentration (MIC) of extracts, fractions and compounds hours at 37 °C The negative control was DMSO 1% Each concentration of the survey was diluted and spread to TYEG agar, anaerobically incubate them at 37 °C for 48 hours, then see the results The MIC value was defined as the minimum concentration of extracts (or compounds) where no P acnes colonies appeared Based on the results obtained from the experiment on inhibitory ability of the bacteria strains of the extracts (or compounds), select one extract (or compound) with the highest resistance to P acnes and concentration range containing the MIC value for the experiment RESULTS AND DISCUSSION Add 100 μL of extract (or compound) to eppendorf tube with different concentrations, with a difference of 5-10 mg/mL in each concentration; add 500 μL of P acnes suspension (density 108 cells/mL) and 400 μL of medium; Anaerobically incubate them for 24 4.1 Antibiogramme results Investigate the susceptibility of 24 strains of P acnes to synthetic antibiotics Antibiogramme results of P acnes for common antibiotics were presented in Table AGU International Journal of Sciences – 2019, Vol (4), – 11 Table Antibiogramme results of P acnes for some common antibiotics Susceptible Medium Resistant Number of Strains Rate (%) Number of Strains Tetracycline 10 41,7 14 58,3 0,0 Cefuroxime 13 54,2 11 45,8 0,0 Levofloxacin 13 54,2 11 45,8 0,0 Erythromycin 0,0 13 54,2 11 45,8 Clindamycin 0,0 20,8 19 79,2 37,5% Antibiotics Erythromycin & Clindamycin 24 investigated strains of P acnes had the rate of resistance to antibiotics as follows: Clindamycin 79.2%; erythromycin 45.8%; tetracycline 0%; levofloxacin 0%; cefuroxime 0% and 9/24 P acnes lines were resistant to both clindamycin and erythromycin antibiotics (accounting for 37.5%) Rate (%) Number of Strains Rate (%) produced in many forms: topical application in single or combined form in acne treatment Very few tetracycline group topical products are currently used in the treatment of acne due to many side effects From this result, strains of P acnes resistant to both clindamycin and erythromycin antibiotics were selected to test the inhibitory ability of extracts from leaves of Cassia grandis L.f (Table 3) This result was consistent with studies of Tan et al., 2007 in Singapore; Zandi et al., 2011 in Iran; Luk et al., 2013 in Hong Kong; Nguyen Thanh Hung and Nguyen Tat Thang, 2013 in Vietnam 4.2 The results of the survey on the inhibitory ability to P acnes of extracts from the leaves of Cassia grandis L.f Thus, the resistance to antibiotics such as clindamycin and erythromycin of P acnes, which caused acne, was very high This increasing resistance may be for the prescription used a combination of oral and topical antibiotics that were the same types of drugs rather than different from each other In Vietnam as well as Asian countries, many clindamycin and erythromycin products are Experimental results (Table 3) showed that: Leaves of Cassia grandis L.f extracted in ethyl acetate solvent at concentrations of 200, 100 and 50 mg/mL inhibited all examined strains of P acnes, while n-hexane and methanol extracts did not show antibacterial activity at tested concentrations AGU International Journal of Sciences – 2019, Vol (4), – 11 Table The ability of extracts to inhibiting P acnes by diffusion method on agar plates Diameter of aseptic ring (mm) Extract (mg/mL) Strains n-Hexane Control (+) Ethyl acetate Methanol Clindamycin 200 200 100 50 200 2µg/well Pa01 0a 15,3de 11,7d 6,0de 0a 1,0d Pa05 0a 23,3b 17,7a 12,3ab 0a 14,0b Pa08 0a 15,0e 11,0d 5,0e 0a 5,0c Pa11 0a 20,3c 16,3ab 13,0a 0a 14,0b Pa15 0a 20,7c 15,7bc 10,7bc 0a 14,0b Pa17 0a 19,3c 14,3c 10,3c 0a 14,0b Pa19 0a 22,7b 17,3ab 10,7bc 0a 18,0a Pa22 0a 17,0d 12,3d 7,7d 0a 2,0d Pa23 0a 25,0a 17,3ab 11,3abc 0a 8,0b Note: The data of aseptic ring diameter was the average value of the three replicates In the same column, numbers with at least same digit following were not different in the 5% sense Control (-): DMSO resulted in zero as the diameter of resistance ring At a concentration of 200 mg/mL, ethyl acetate extract strongly inhibited P acnes to create an aseptic ring with a diameter of 15-25 mm This result showed that the inhibitory ability to P acnes strain of ethyl acetate extract at this concentration was higher than clindamycin (positive control) resistance ring: 11.3-13 mm) but the resistance activity decreased significantly, having the smallest diameter of the resistance ring, especially in strain Pa08 (5 mm) At this concentration, the antibacterial activity of ethyl acetate extract was higher than that of the positive control on Pa01, Pa08 and Pa22 strains but lower than the others At a concentration of 100 mg/mL, ethyl acetate extract created a diameter of P acnes-resistance rings from 11.0 to 17.7 mm The diameter of the aseptic ring reached the largest (16.3-17.7 mm) on Pa11, Pa19, Pa23, Pa05 strains and it had the smallest size (11-12.3 mm) for strains Pa08, Pa01 and Pa22 At this concentration, the antibacterial activity of ethyl acetate extract was generally higher than that of the positive control Based on the results of the above experiment, it showed that the Pa08 strain has the smallest antibacterial ring diameter, which means that this bacterium has a higher activity and the inhibitory ability of ethyl acetate extract to Pa08 was lower than that of other bacteria strains Therefore, the Pa08 strain was chosen to determine the minimum inhibitory concentration of ethyl acetate extract (because if it can inhibit the strongest strain, all it can also inhibit the other strains), the results were presented in Table At a concentration of 50 mg/mL, the ethyl acetate extract was the strongest inhibitory with strains Pa11, Pa05 and Pa23 (diameter of AGU International Journal of Sciences – 2019, Vol (4), – 11 Table Results of determining the MIC value of ethyl acetate extract on P acnes by dilution method No Concentration (mg/mL) P acnes Colony 50 - 40 - 30 - 20 + 10 + + 0,9 + 0,8 + 0,7 + 10 0,6 + 11 Control (-) DMSO 1% + Note: This was the average value of the three replicates; (-) no colonies appear; (+) colonies appear Experimental results showed that 30 mg/mL is the lowest concentration of ethyl acetate extract capable of inhibiting P acnes (Pa08 strain) Statistical result (Table 5) showed the ability to inhibit P acnes strains of ethyl acetate extract and its fractions: the lowest antibacterial activity was ethyl acetate extract, up to fractions E 100% and H:E 1:3 (non-significant difference) and the highest was the H:E 1:1 fraction Highly ethyl acetate effectively inhibiting P acnes bacteria was divided into segments by solvent polarity to examine in more detail which segment contains active compounds 4.3 Results of the investigation of the ability to inhibit P acnes of ethyl acetate extract and its fractions Table The ability of ethyl acetate extract and fractions to inhibiting P acnes strains by the diffusion method on agar plates Diameter of aseptic ring (mm) Extracts & fractions 200 mg/mL 100 mg/mL 50 mg/mL Pa22 Pa01 Pa08 Pa22 Pa01 Pa08 Pa22 Pa01 Pa08 Ethyl acetate 17,0d 15,3c 15,0c 12,3c 11,7d 11,0c 7,7b 6,0b 5,0c H:E 1:3 19,3c 17,7b 17,3b 14,3c 14,0c 13,0b 8,0b 7,3b 6,3bc E 100% 21,7b 18,3b 17,7b 16,7b 15,3b 14,7b 8,0b 7,7b 6,7b H:E 1:1 23,7a 22,0a 19,3a 19,0a 18,3a 17,7a 13,3a 13,0a 12,7a Note: The data of aseptic ring diameter was the average of the three replicates In the same column, numbers with at least similar letter following are not different in the 5% sense Control (-): DMSO resulted in zero as the resistance diameter AGU International Journal of Sciences – 2019, Vol (4), – 11 In general, at the three concentrations tested, the difference in the diameter of antibacterial rings between ethyl acetate extract and its fractions had a significant difference of 5% The difference in antibacterial activity of H:E 1:1 fraction compared with ethyl acetate extract and the remaining fractions increased with decreasing concentration The H:E 1:1 fraction continued to be investigated for MIC value, the results were shown in Table Table Results of determining MIC value of H:E 1:1 fraction on P acnes by dilution method Extract concentration No H:E (1:1) (mg/mL) P acnes Colony 50 - 40 - 30 - 20 - 10 - - 0,9 - 0,8 - 0,7 - 10 0,6 - 11 0,5 - 12 0,4 - 13 0,3 - 14 0,2 + 15 Control (-) DMSO 1% + Note: This is the result of three repetitions; (-) no colonies appearing; (+) colonies appearing The results above showed that 0.3 mg/mL is the lowest concentration of the extract from leaves of Cassia grandis L.f in ethyl acetate in H: E 1: fraction capable of inhibiting P acnes bacteria of Combretaceae family) showed MIC value of 0.315 mg/mL on P acnes bacteria 4.4 Results of investigating the resistance of pure compounds to P acnes From the experimental results (Table 7), the quercitrin showed a higher ability to inhibit P acnes (Pa08) than rutin and aloe-emodin, the difference was significant at 5% The results of this experiment can be compared with that of Vijayalakshmi et al., 2011 that the flavonoid segments from ethyl acetate extract from Terminalia arjuna bark (a flowering plant AGU International Journal of Sciences – 2019, Vol (4), – 11 Table The ability of quercitrin, rutin and aloe-emodin to inhibit P acnes strains by diffusion method on agar plates Diameter of aseptic ring (mm) No Compounds 10 mg/mL mg/mL 2,5 mg/mL Quercitrin 16,3a 13,7a 5,7a Rutin 7,0b 0b 0b Aloe-emodin 5c 0b 0b Note: The data of aseptic ring diameter is the average value of the three replicates In the same column, the numbers with at least similar letter following are not different in the 5% sense Control (-): DMSO resulted in zero as the resistance diameter At a concentration of 10 mg/mL, the quercitrin compound created a resistance diameter of 16.3 mm that was significantly different from the other two compounds on the P acnes strains studied Diameter of resistance ring made by rutin and aloe-emodin were and mm, respectively (Table 7) Based on this result, the quercitrin compound with the highest antimicrobial activity to P acnes (Pa08 strain) was selected for the experiment to determine the minimum inhibitory concentration Table Results of determining the MIC value of quercitrin on P acnes by dilution method No Quercitrin concentration (mg/mL) P acnes colony - - 0,9 - 0,8 - 0,7 - 0,6 - 0,5 - 0,45 - 0,4 - 10 0,35 - 11 0,3 - 12 0,25 - 13 0,2 - 14 0,15 + 15 0,1 + 16 0,05 + 17 Control (-) DMSO 1% + Note: This was the result of three replicates; (-) no colonies appeared; (+) colonies appeared AGU International Journal of Sciences – 2019, Vol (4), – 11 Experimental results showed that 0.2 mg/mL was the lowest concentration of quercitrin compound capable of inhibiting P acnes (Table 8) Medicinal plants and animals in Vietnam, Part II Science and Technology Publisher: 495-496 (in Vietnamese) Fu Y., Zu Y., Chen L., Efferth T., Liang H and Liu Z (2007) Investigation of antibacterial activity of rosemary essential oil against Propionibacterium acnes with atomic force microscopy Planta Medica, 73(12), 12751280 Quercitrin belongs to the flavonoid group isolated from H:E 1:1 fraction of ethyl acetate extract In the H:E 1:1 fraction, the MIC value for P acnes was 0.3 mg/mL, then the pure quercitrin compound had a lower MIC value of 0.2 mg/mL From this result, it can be deduced that quercitrin was a very important compound contributing to the inhibitory ability to P acnes of ethyl acetate extract, namely H:E 1:1 fraction Hans B.L and Kilian M (2010) Population genetic analysis of Propionibacterium acnes identifies a subpopulation and epidemic clones associated with acne PLoS One, 5(8), e12277 CONCLUSION Kishishita M., Ushijima T., Ozaki Y and Ito Y (1980) New Medium for Isolating Propionibacteria and its application to assay of normal flora of human facial skin Applied and environment Microbiology, 40(6), 1100-1105 The study investigated the inhibitory ability of extracts and compounds from leaves of Cassia grandis L.f on strains of P acnes The H:E 1:1 fraction and quercitrin from ethyl acetate extract have antimicrobial effect against P acnes with MIC values of 0.3 mg/mL and 0.2 mg/mL, respectively This result can be reference for subsequent studies aimed at applying natural products from leaves of Cassia grandis L.f for acne treatment Luk N.M., Hui M., Lee H.C., Fu L.H., Liu Z.H., Lam L.Y., Eastel M., Chan Y.K., Tang L.S., Cheng T.S., Siu F.Y., Ng S.C., Lai Y.K and Ho K.M (2013) Antibioticresistant propionibacterium acnes among acne patients in a regional skin centre in Hong Kong European Academy of Dermatology and Venereology, 27(1), 3136 REFERENCE Bauer A.W., Kirby W.M.M., Sherris J.C and Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method The American Journal of Clinical pathology, 45(4), 493-496 Murugan T and Saranraj P (2011) Antibacterial activity of various solvent extracts of the Indian herbal plant acalypha indica against human pathogens causing nosocomial infection International Journal of Pharmaceutical & Biological Archives, 2(5),1473-1478 Charde Y.M., Sharma P.H., Choudhary N.G and Avari J.G (2014) Development and evaluation of herbal formulation for the treatment 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The study investigated the inhibitory ability of extracts and compounds from leaves of Cassia grandis L.f on strains of P acnes The H:E 1:1 fraction and quercitrin from ethyl acetate extract... Thanh Hung and Nguyen Tat Thang, 2013 in Vietnam 4.2 The results of the survey on the inhibitory ability to P acnes of extracts from the leaves of Cassia grandis L.f Thus, the resistance to antibiotics... were selected to test the inhibitory ability of extracts from leaves of Cassia grandis L.f (Table 3) This result was consistent with studies of Tan et al., 2007 in Singapore; Zandi et al., 2011