Bioactive compounds content, free radical scavenging and anti-acne inducing bacteria activities of some fruit peels extract

1 1, 1 1 Udomlak Sukatta1, Prapassorn Rugthaworn1, Methiga Leebunyanon1 and Potechaman Pitpiangchan 1

, , , , , (54.45 %w/w) (345.49, 250.85 206.90 GAE/ ) 75.04, 68.92 62.92 CE/) 3 Staphylococcus aureus, S. epidermidis Propionibacterium acnes S. aureus S. epidermidis P. acnes

ABSTRACT

The main aim of this study was to determine bioactive compounds content, free radical scavenging and anti-acne inducing bacteria activities of ethanoic extracts of banana, pomelo, litchi, longan, mangosteen, pomegranate and rambutan. Rambutan peel gave highest yield of crude extract (54.45 %w/w). The high content of total phenolics were found in rambutan, pomegranate and mangosteen crude extracts (345.49, 250.85 and 206.90 mg GAE/g crude extract, respectively) while high content of total flavonoids were found in rambutan, mangosteen, longan crude extracts (75.04, 68.92 and 62.92 mgCE/g crude extract, respectively). Rambutan peel extract was the highest effective radical scavenging activity followed by pomegranate and litchi, respectively. For antibacterial test, pomegranate fruit peel extract exhibited the greatest antimicrobial activity against Staphylococcus aureus, S. epidermidis while Propionibacterium acnes was highly sensitive to mangosteen crude extract. The strong antibacterial, antioxidant activity of these fruit peels extract suggests its potential as antioxidant and a therapeutic agent for acne vulgaris. Keyword : phenolics, flavonoids, free radical scavenging, anti-acne inducing bacteria, fruit peel. Email-address : aapuls@ku.ac.th

1 , , Kasetsart Agricultural and Agro-Industrial Product Improvement Institute , Kasetsart University, Bangkok 10900, Thailand.

INTRODUCTION

Acne vulgaris is one of the most common skin diseases, involving abnormalities in sebum production, follicular epithelial desquamation, bacterial proliferation, and inflammation affect by dermatotype (Leyden, 2003). The proliferation of bacteria such as Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Kocuria rhizophila and Staphylococcus pyogenes causing primary or secondary skin infections. P. acnes and S. epidermidis are pus-forming organisms that trigger inflammation in acne. S.pyogenes, K. rhizophila and S. aureus are often isolated from patients with clinical symptoms of acne. (Chomnawang, et al., 2005; Yamaguchi et al., 2009). Antibiotic, retinoids and other agents including benzoyl peroxide, salicylic acid, azelaic acid, and alpha-hydroxy acids were generally recommended for mind to moderate acne treatments (Leyden, 2003). Although antibiotic and chemical substance give a high potential for acne treatment but they had some adverse effects such as generated free radicals in the skin; its effect is similar to that of unprotected exposure to the sun of benzoyl peroxide (Kennedy et al., 1995) and increasing in antibiotic resistance (Leyden, 2003). To overcome the potential risk of adverse effects and antibiotic resistance from synthetic drugs, plant extract have been extensively studied as alternative treatments for many diseases caused by their antimicrobial and antioxidant activity. Among various plant fruits contain many antimicrobial and antioxidant compounds, including tannins, flavonoids and phenolics. (Chanwitheesuk et al., 2005). Some indigenous fruits such as litchi, longan, pomegranate, pomelo, rambutan and mangosteen were consumed directly as fresh arils or were used in the food industry in the manufacture of concentrates, juices, and caned. These fruit peels are often the waste and effect to the environment they are gradually fermented and released off odors. Recently peel and seed of fruit were growing interest for their biological activity and the current study focuses on the possibility of using peel and seed waste as source of low-cost natural antioxidant and antimicrobial. This study aim at investigating and comparing the total phenolic, total flovonoid, free radical scavenging and anti-acne inducing bacteria activities of fruit peels commonly consumed and grown in Thailand.

MATERIALS AND METHODS

Plant materials

Fresh fruits of Musa sapientum L. (banana), Citrus maxima (Burm.) Merrill (pomelo), Chinenesis Sonn (litchi), Dimocarpus Longan Lour. (longan), Garcinia mangostana L. (mangosteen), Punica granatum (pomegranate) and Nephelium lappaceum (rambutan) were purchased from a local grocery store in Bangkok, Thailand. The fresh fruits were washed to

remove impurities and peeled. The fruit peels cut into small piece and dried at 45 C for 48 hours in a try dryer. They were then ground with a blender to make a powder.

Preparation of the ethanol crude extract

One hundred gram of each sample was extracted with 95% ethanol (Analytical grade) thrice, at room temperature by using maceration method for three days. The filtrates were pooled and concentrated by rotary evaporator at 40 C. %yield of the extract was calculated using the following equation: %yield = (Wcrude extract/Wdried plant) x 100 where: W crude extract = weight of crude extract, Wdried plant = weight of dried plant material. The obtained semisolid extracts were kept in a desiccators at 4 C until further used. Determination of total phenolic content

The total phenolic content of the fruit peels samples were measured using a modified colorimetric Folin-Ciocalteu method (Wolfe, 2003) with slight modification. Briefly a volume of 0.125 ml of 0.01g/ml of ethanolic solution of each fruit peel samples and 0.5 ml of deionized water was added to a test tube, 0.125 ml of Folin-Ciocalteu reagent was added to the solution and allowed to react for 6 min. Then, 1.25 ml of 7% sodium carbonate solution was aliquoted into the mixture, and diluted to 3 ml with deionized water. The color developed for 90 min, and the absorbance was read at 760 nm using a spectrophotometer. The measurement was compared to a standard curve of prepared gallic acid solutions and expressed as milligrams of gallic acid equivalents (GAE)/g dry basis. All experiments were performed in triplicate.

Determination of Flavonoid Content

The flavonoid content of the fruit peel samples were measured using a modified colorimetric method (Wolfe, 2003). Briefly a volume of 0.25 ml of 0.01g/ml of ethanolic solution of each fruit peel samples was added to a test tube containing 1.25 ml of distilled water, 0.075 ml of 5% sodium nitrite solution was added to the mixture, and allowed to stand for 5 min. Then, 0.15 ml of 10% aluminum chloride was added. After 6 min, 0.5 ml of 1 M sodium hydroxide was added, and the mixture was diluted with another 0.275 ml of distilled water. The absorbance of the mixture at 510 nm was measured immediately using a spectrophotometer and compared to a standard curve of prepared catechin solutions. The flavonoid content was expressed as milligrams of catechin equivalents/g crude extract. The experiments were performed in triplicate.

Determination of DPPH radical scavenging activity

The free radical scavenging activity was determined by the method of Karagozler et al., (2008) with some modifications. Briefly the ethanolic fruit peel crude extracts were dissolved with ethanol to prepare various sample solutions. 3 ml of each extract solution was mixed with 1ml of 0.1mM of DPPH ethanolic solution, the mixture was shaken vigorously and maintained for 30 min in dark. The absorbance of the mixture at 517 nm was measured immediately using a spectrophotometer. The absorbance of the control was obtained by replacing the sample with ethanol. BHA were used as standard reference. The inhibition of DPPH radicals by the samples was calculated according to the following equation: DPPH radical scavenging activity (%) = [(A0 A 1)/ A 1] x 100. Where A0 is the absorbance of the control reaction, and A1 is the absorbance of the test compound. Crude extract concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against fruit peels crude extract concentration.

Bacterial cultures

Three acne-inducing bacteria ; Staphylococcus aureus DMST 8840, Staphylococcus epidermidis DMST 15505, Propionibacterium acnes DMST 14916 were obtained from Thailand National Institutes of Health, Thailand S. aureus and S. epidermidis were maintained in TSA and in BHI with 1 % glucose for P.acnes under anaerobic conditions at 4 C. Antibacterial activity

The agar disc diffusion method was employed for screening of the antimicrobial activities of the extracts. (NCCLS, 2008) with slightly modification. Briefly, a suspension of the tested microorganism (0.1 ml of 108 CFU/ml) was spread on the TSA medium for S. aureus and S. epidermidis and BHI medium with 1 % glucose for P. acnes. The ethanol crude extracts were dissolved in dimethylsulfoxide (DMSO) to a final concentration of 200 mg/ml and sterilized by filtration by 0.45 m millipore filters. The discs (6 mm in diameter) were impregnated with the 200 mg/ml extracts (10 l/disc) and placed on the inoculated plates. Negative controls were prepared using DMSO. Erythomycin (15 g/disc) was used as positive reference standards to determine the sensitivity of each bacterial species tested. The inoculated plates of P. acnes were incubated at 37 C for 72 h under anaerobic conditions while S. aureus and S. epidermidis were incubated at 37 C for 24 h. Antibacterial activity was evaluated by measuring the diameter of clear zone of the tested bacteria expressed in millimeters and the antimicrobial index was computed as: (diameter of clear zone diameter of disc)/diameter of disc (Adapted from Barre et al. (1997)). All tests were performed in triplicate.

RESULTS AND DISCUSSION

The yield of crude extract obtained by ethanoic extraction from banana, litchi, longan, mangosteen, pomegranate, pomelo and rambutan peels were 16.23, 16.57, 8.32, 18.17, 18.17, 33.09, 20.51 and 54.45 (%w/w), respectively, (Table1). Rambutan gave the higest of extraction yield while the lowest yield was observed in the longan peel. This finding were supported by the report of Okonogi, et al. (2007). Although all of plant materials were extracted by the same method, solvent and the same temperature, the yields of the extracts are usually different. This may be due to the difference in plant compositions such as tannin, cellulose, chlorophyll, protein and lipid in the materials (Bullangpoti et al., 2004). More over variations in the yield of extracts from different plant materials might be attributed to the availability of different extractable components, defined by the chemical composition of the plant, nature of the soil and agro-climatic conditions Among other parameters, effectiveness of the extracting solvent to dissolve endogenous compounds might also be a contributing factor. (Sultana et al., 2007). Table 1 Percent yield of extracts from different kinds of fruit peels.

Plant name % yield (w/w) Banana 16.23 +1.24d Litchi 16.57 +1.18d Longan 8.32 +0.99e Mangosteen 18.17 +0.70d Pomegranate 33.09 +1.41b Pomelo 20.51 +1.13c Rambutan 54.45 +1.10a

Values are the mean + standard deviation (n = 3). Mean values followed by different superscripts within a column are significantly different using Duncans multiple range test (P < 0.05).

The amounts of total phenolics, flavonoids in fruit peel extracts were determined.

The result revealed that these extracts were found to have various phenolic levels (P < 0.05), ranging from 34.49 to 345.49 mg GAE/g crude extract with a decreasing order: rambutan> pomegranate> mangosteen> longan> litchi> pomelo> banana. (Figure 1) The flavonoid contents of these fruit peel extracts showed in Figure 1 as cathechin equivalents (CE)/g crude extract. The peels of rambutan had the highest flavonoid content (75.04 mg CE/g crude extract) while pomelo gave the lowest quantity of flavonoid (14.094 mg CE/ g crude extract). This is consistent with the data reported by Tomas-Barberan et al. (2001) who found that peel tissues usually contained larger amount of phenolics, anthocyanins and

flavonols than did flesh tissues in nectarines, peaches and plums. However, flavonoids or proanthocyanidins account for only a small part of total phenolics present in the peel extract. The variation of phenolics compounds content in the fruit depends on many factor. In the red color fruit varieties phenolics compounds were increased during the last ripening stage due to the maximal accumulation of anthocyanines and flavonoid (Marinova, 2005).

Antioxidant properties, especially radical scavenging activities are very important due to the deleterious role of free radicals in foods and in biological system. The free radical scavenging capacity of the extracts against DPPH free radicals in vitro were further determined. The results indicated that the peel extracts exhibited a potential free radical scavenging activity. The half inhibition concentration (IC50) of the radical scavenging activity of the peel extracts were calculated and are illustrated in Figure 1. The results exhibited that the extract with the highest effective radical scavenging activity was the rambutan fruit peel (3.80+ 0.68 g/ml) while lower activities were found in the pomelo extract (511.99+ 1.24 g/ml). BHT revealed less effective activities (P< 0.05) than the all of fruit peel extract except polemo crude extract. These finding were supported by the previous research, Mokbel and Hashinaga, (2005) ; Okonogi et al., (2007); Li et al., (2005); Thitilertdecha et al., (2009); Chaverri,et al., (2008); Prasad et al., (2009), Prasad et al., (2010).

34.49

95.21132.14

206.90250.85

83.03

345.49

19.6256.57 62.92 68.92 39.22

14.09

75.0437.66

7.12 20.28 11.34 4.13

511.99

3.800

100

200

300

400

500

600

Banana Litchi Longan Mangosteen Pomegranate Pomelo Rambutan

Total phenolics (mg GAE/g extract)Total flavonoids (mg CE/g extract)IC50 value (ug/ml)

Figue 1 Total phenolics, total flavonoids and free radical scavenging activities of fruit peels extracted.

Phenolic compounds have been proved to be responsible for the antioxidant activity of

various fruit (Kumar et al., 2006). The contributions of phenolic compounds to antioxidant activity were much greater than those of vitamin C and carotenoids (Gil et al., 2002). The significant correlation between the phenolic content and the antioxidant activity of rambutan fruit peels has been previously observed (Palanisamy et al., 2008; Thitilertdecha et al., 2009). Phenolic

compound possess a high potential to scavenge radicals can be explained by their ability to donate a hydrogen atom from their phenolic hydroxyl groups (Sawa et al, 1999). So that high amount of phenolics contained in peel extract causing peel extract displays strong activity in scavenging or preventive capacity against free radicals (Li et al., 2006). Mousavinejad et al. (2009) reported that ellagic acid are the major phenolic compounds of pomegranate are directly related with the antioxidant activities of pomegranate juices. Li and Jiang (2007) reported that litchi flavanols and anthocyanins exhibit good potential antioxidant activity, their procyanidin B2 exhibited strong hydroxyl radical and superoxide anion scavenging activities while epicatechin has the highest (DPPH) scavenging activity. Table 2 Antibacterial activities of the studied extracts against acne-inducing bacteria.

Plant name Clear Zone (mm.) Antimicrobial index***

S. aureus S. epidermidis P .acnes S. aureus S. epidermidis P. acnes Pomelo 9.00 + 0.71d ND 7.50+0.55e 0.50+0.12 d 0.00+0.00f 0.25+0.09e Banana 7.00 +0.00e 7.00 + 0.00e ND 0.17+0.00 e 0.17+0.00 e 0.00+0.00f Longan 9.00 +0.00 d 11.83 +0.41d 9.00 +0.00d 0.50+0.00 d 0.97+0.07 d 0.50+0.00d Rambutan 13.33 +0.52b 20.50 +0.55b 13.83 +0.98b 1.22+0.09 b 2.42+0.09 b 1.31+0.16b Mangosteen 12.83 +0.41c 13.17 +0.41c 20.83 +0.98a 1.14+0.07 c 1.19+0.07c 2.47+0.16a Litchi 7.00 +0.00e ND 7.50 +0.55e 0.17+0.00 e 0.00+0.00f 0.25+0.16e Pomegranate 20.67 +0.52a 24.50 +0.84a 12.00 +0.63c 2.44+0.09 a 3.08+0.14a 1.00+0.11c DMSO* ND ND ND 0.00+0.00 0.00+0.00 0.00+0.00 Erythomycin** 39.33+0.52 10.50+0.77 60.83+0.98 5.56+0.09 0.75+0.13 9.14+0.16

Mean values followed by different superscripts within a column are significantly different using Duncans multiple range test (P < 0.05). ND, No activity detected., * are negative control 10l/disc, ** are positive control 15g/disc. *** are Antimicrobial index = (diameter of clearing zone - diameter of disc)/diameter of disc.

The antimicrobial efficacy of fruit peel extracts against 3 strains acne inducing bacteria; S. aureus, S. epidermidis and P. acnes was evaluated by the disc diffusion method via determination of the surrounding zones of inhibition. The result showed that the mean values antimicrobial index of all fruit peel crude extract produced against S. aureus, S. epidermidis and P. acnes ranged from 0.17+0.00 to 2.44+0.09 from 0.00+0.00 to 3.08+0.14 and from 0.00+0.00 to 2.47+0.16, respectively. Pomegranate crude extract gave highest efficacy against S. aureus and S. epidermidis while it showed moderate efficacy to inhibit the growth of P. acnes. On the other hand P. acnes was more sensitive to mangosteen crude extract which presented moderate antibacterial activity against S. aureus and S. epidermidis. Moreover rambutan crude extract represented good activity against S. epidermidis and had moderate inhibitory activity against S. aureus and P. acnes. Differences in the antimicrobial activity of fruit peel extracts among studies could be partially explained by variations in phenolic content of extracts. This was in

complete agreement with the finding of Voravuthikunchai et al., 2004; Reddy et al.,2007 Shan et al.,2007) regarding the correlation between total phenolics and the antibacterial activity of various plant extracts, including pomegranate peels. The mechanism responsible for phenolic toxicity to microorganisms was related to reaction with sulfhydryl groups of proteins and unavailability of substrates to microorganism (Naz et al., 2007). Pomegranate extracts interfered with bacterial protein secretions. In addition this extract were able to inhibit not only growth of bacterial cell but also the production of enterotoxins (Braga et al., 2005). The efficacy of mangosteen crude extract against acne inducing bacteria extract due to its contained higher contents of -mangostin, a xanthone derivative that gave higher anti-acne producing bacteria activity especially P. acnes. (Pothitirat et al., 2009). Present data showed that the extract of fruit peels of pemegranate, mangosteen and rambutan contained high content of phenolic compounds and flovonoids promoting its good potential as a source for natural antioxidants. Moreover these extract fruit peels appeared to exhibit significant antibacterial activity against acne inducing bacteria. The strong antibacterial, antioxidant activity of these fruit peels extract suggests its potential as a therapeutic agent for acne vulgaris.

CONCLUSION

The results obtained from this study demonstrated the wide range of total phenolics and total flovonoids content among different fruit peel crude extracts. Rambutan, pomegranate and mangosteen crude extracts contain high content of total phenolics (345.49, 250.85 and 206.90 mg GAE/g crude extract, respectively) while high content of total flavonoids were found in rambutan, mangosteen and longan crude extracts (75.04, 68.92 and 62.92 mgCE/g crude extract, respectively). The result indicated that pomegranate mangosteen and rambutan exhibited high free radical scavenging activity with IC(50) values 3.80-7.12 mg/ml and showed great potent antibacterial activity against acne inducing bacteria. Moreover total phenolics and flovonoids in fruit peels are highly related to their antioxidant and antibacterial capacity. Pomegranate mangosteen and rambutan fruit peels may serve as a very good source of potential antioxidant and antibacterial agents used for acne vulgaris treatment.

ACKNOWLEDGEMENTS

The authors would like to gratefully thank Kasetsart University Research and Development Institute, Kasetsart University, Bangkok, Thailand for their research grant.

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