http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, 2014; 65(5): 589–593 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.886181
IN VITRO AND ANIMAL STUDIES
Antimicrobial activities of various fractions of longan (Dimocarpus longan Lour. Fen Ke) seed extract Huang-Chung Tseng1,2, Wan-Ting Wu2, Ho-Shin Huang1, and Ming-Chang Wu2 1
Laboratory of Antrodia Certification Center, Joben Bio-medical Co, Ltd., Pingtung, Taiwan and 2Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan Abstract
Keywords
The antimicrobial activities of longan (Dimocarpus longan Lour. Fen ke) seed extracts were investigated using a disc diffusion method and also determining the minimal inhibitory concentration. The DL-P01-SI01 fraction showed that the strongest activity against Staphylococcus aureus and methicillin-resistant S. aureus at MIC 64 mg/mL, which was found to be due to the phenolic compounds. The HPLC analysis showed that the major phenolic compounds are gallic acid, corilagin, ethyl gallate and ellagic acid.
Antimicrobial activity, corilagin, ellagic acid, ethyl gallate, longan seed
Introduction Longan (Dimocarpus longan Lour.), which is a subtropical fruit of the Sapindaceae family, has been widely planted in South-East Asia including Taiwan and China for many years (Huang, 1995; Tindall, 1994). Among numerous longans, the Fen ke cultivar is the most commonly cultivated in Taiwan representing over 98% of the longan area because of its high yield. Owing to its delicious taste, longan has been gradually accepted by consumers and has enjoyed great popularity in the international market. Regardless of being consumed as fresh fruits or as processed products, longan seeds have become agricultural wastes and created environmental problems on a large scale. Fruit seeds are known to contain many phenolic compounds capable of protecting them from oxidative damage and defending them against yeast, fungi and bacteria that might inhibit their germination. In addition, longan seeds contain large amounts of other bioactive compounds, such as phenolic acids, flavonoids and polysaccharides (Jiang et al., 2009; Zheng et al., 2009), and exhibit antimicrobial, antioxidant and inflammatory properties (De Assis et al., 2009; Prasad et al., 2010; Wen et al., 2010). Rangkadliok et al. (2005) further suggested that the seed extract of longan could be a potential source of natural dietary antioxidants and skin whitening agents. Recently toxicological studies revealed no toxic effects of oral administration of longan seed extract during acute (Worasuutayangkurn et al., 2012). The water extract of longan fruit was found to contain high levels of polyphenolic compounds such as corilagin, gallic acid and ellagic acid (Soong & Barlow, 2005). The analysis of longan
History Received 30 July 2013 Revised 30 September 2013 Accepted 13 January 2014 Published online 17 February 2014
fruit from Thailand showed that there was a large variation in the contents of gallic acid, corilagin and ellagic acid ranging from 9.18 to 23.04, from 0 to 50.64 and from 8.13 to 12.65 mg/g respectively, in different longan tissues among cultivars (Rangkadilok et al., 2007). Corilagin is a member of the tannin family and has strong antioxidant (Kinoshita et al., 2007), antiHIV (Xu et al., 2000) and antimicrobial (Fogliani et al., 2005) activities. Gallic acid and ellagic acid are reported to be potent antioxidants and anticarcinogenic agents (Yilmaz & Toledo, 2004). Although the seed of D. longan has shown some physiological effects, limited studies have focused on the antimicrobial activity of Fen Ke Longan. In this study, we evaluated the antimicrobial activity of Fen Ke longan seed extracts and investigated the relationship between the phenolic acids and the antimicrobial activities in different fractions.
Materials and methods Plant material and chemicals Plant materials (Dimocarpus longan Lour. Fen ke) were obtained from the Taichung District Agricultural Improvement Station at commercial maturation season. Methanol (HPLC grade) and acetic acid was obtained from Merck. Gallic acid and ethyl gallate was purchased from Sigma (St. Louis, MO). Ellagic acid was purchased from Aldrich. Corilagin was purchased from PIN chemical Ltd (Ponpadi R.S., Pin-631213, Tamil Nadu, India). Ultrapure water purified using a Milli-Q system (Millipore Inc., Billerica, MA) was used. Extraction and fractionation
Correspondence: Ming-Chang Wu, Department of Food Science, National Pingtung University of Science and Technology, No. 1 Hseuhfu Rd., Pingtung, Taiwan. E-mail: [email protected]
The dried longan seed 3 kg was ground into powder and extracted with 12 kg water and 12 kg ethanol for 24 h. The whole mixture was filtered with filter paper. The residue was extracted two more times with water and ethanol as before. The total extracts were
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filtered, and the obtained filtrates were concentrated under reduced pressure to dryness, yielding the crude extract of 644 g. The resulting crude extract was stored at 4 C until future process. The 640 g crude extract then suspended in 3000 ml distilled water, and was partitioned with ethyl acetate (see the flow chart for Longan seed extraction). After 24 h settlement, the upper ethyl acetate layer was collected and lyophilized under reduced pressure to yield a powder of 38.21 g (DI-P01). The remaining aqueous portion was extracted with 3000 ml n-butanol. After 24 h settlement, the upper n-butanol layer was collected and lyophilized under reduced pressure to yield DL-P02 of 68.16 g. The remaining aqueous layer was lyophilized under reduced pressure to yield DL-P03 of 506.88 g. Since the ethyl acetate fraction, DL-P01, was found later to exhibit the highest antimicrobial activity, this fraction was further separated by a silica gel column chromatographic method flashed with ethyl acetate and methanol gradient to yield 10 sub-fractions. DL-P01 of 1.2 g was sonicated to dissolve in ethyl acetate and methanol and 40 g silica gel 60 was then added. The whole mixture was dried under reduced pressure and loaded onto a flash column. The column was auto-flashed with ethyl acetate for 30 min and later with decreasing concentration of ethyl acetate and an increasing concentration of methanol. Each 100 ml was collected and total of 10 tubes were collected. After lyophilized under reduced pressure, their proportions were as follows: DL-P01-SI0 765.3 mg, DL-P01-SI02 100.4 mg, DL-P01-SI03 37.0 mg, DL-P01-SI04 21.7 mg, DL-P01-SI05 140.1 mg, DL-P01-SI06 57.3 mg, DL-P01-SI07 28.4 mg, DL-P01-SI08 17.0 mg, DL-P01-SI09 6.4 mg and DL-P01-SI10 7.7 mg. The obtained fractions (Figure 1) were further subjected to the antimicrobial assay.
Int J Food Sci Nutr, 2014; 65(5): 589–593
Determination of minimal inhibitory concentration (MIC) The minimal inhibitory concentration values were studied for the fractions. The fractions were first solved in water and then diluted to the highest concentration to be tested, after that serial dilutions were made in a concentration range from 0.5 to 1024 mg/mL (1024, 512, 256, 128, 64, 32, 16, 8,4, 2, 1 and 0.5 mg/mL). The target micro-organisms were cultured in Mueller–Hinton broth (MHB) at 37 C for 24 h. After that, the suspensions were adjusted to approximately 108 CFU/mL with 0.5 McFarland standard turbidity and Mueller–Hinton broth. To 24-well microliters plates, 1.6 mL of Mueller–Hinton broth containing diluted bacteria and 409.6 mL of aliquot from the solutions of the fractions were added. A positive control (containing inoculums but no extracts) and negative control (containing extracts but on inoculums) were included on each micro-plates. The contents of the wells were mixed and the micro-plates were incubated at 37 C for 24 h. The MIC was defined as the lowest concentration at which no visible growth was observed. The results were expressed in mg/mL (Martins et al., 2013). Cell viability assay HaCaT, a well-known immortalized human keratinocyte cell line, was growth in DMEM medium (high glucose) supplemented with 2 mM L-glutamine and 10% fetal calf serum at 37 C in 5% CO2/ atmosphere humidified incubator. Subcultures were obtained by disaggregating the cells with 0.05% trypsin/EDTA solution and replating at 1:5–1:10. The viability of HaCaT was detected with reagents of MTS/PMF (2 mg/mL MTS reagent and 0.9 mg/mL PMS reagent; Sigma). After colorized at room temperature for 90 min, the intensity was read at 490 nm. The positive control used was curcumin.
Antimicrobial activity assays Microbial strains The antimicrobial activities were assayed against six different strains of microbes: Propionibacterium acne (BCRC10723); Streptococcus mutans (BCRC10793); Staphylococcus aureus (BCRC80277); methicillin-resistant S. aureus (BCRC15211); Acinetobacter baumannii (BCRC 80276); Salmonella gallinarum (BCRC10830), all of which were supplied by Bioresource Collection and Research Center of Taiwan. The extract was first dissolved in DMSO and then diluted to the desired concentrations but the final DMSO concentration was less than 0.5%, which was found to have no antimicrobial activity. The disc diffusion method The disc diffusion method was employed for screening the extract and its fractions for their antimicrobial activities. Briefly, a suspension of the tested micro-organism was spread on the Mueller–Hinton Agar (MHA). Filter paper discs (7 mm in diameter) were soaked with 10 mL of the extracts and placed on the inoculated plates, which were incubated at 37 C for 24 h. The antibacterial activity was determined by measuring diameters (d in mm) of inhibition zone with the following criteria: d510 mm, less active; 115d515 mm, middle active; d416 mm, very active. Preparation of inoculums The microbial suspension were prepared by making a saline suspension of isolated colonies selected from Mueller–Hinton agar, and the agar plates were grown for 18–24 h. The suspension was adjusted to match the tube of 0.5 McFarland turbidity standard using the spectrophotometer of 595 nm, which equals to 1 108 colony-forming units (CFU)/mL.
Bioactive compounds quantification Gallic acid, corilagin, ellagic acid and ethyl gallate were determined by high performance liquid chromatography with C18 5 mm (XBridge, 250 4.6 mm) column at 30 C, and UV detector at 280 nm. The mobile phase consisted of 1% acetic acid in water (solvent A) and methanol (solvent B) under the gradient profile. The gradient system started with 0% A at 0 min to 55% B in 35 min. The total run time was 50 min. The mobile phase was eluted in a flow rate of 1 mL/min, and samples of 10 mL were injected into the column. All the extracts were filtered through 0.22 mm membrane filters.
Results Gallic acid, corilagin, ethyl gallate and ellagic acid were the major polyphenolic compounds present in Fen Ke longan seed fractions. HPLC profile of fractions was shown in Figure 2. Retention times of gallic acid, corilagin, ethyl gallate and ellagic acid were found to be 6.8, 18.6, 21.6 and 27.9 min, respectively. Table 1 shows the contents of these acids in different extractions and fractions. Among the fractions, DL-P01-SI-01 exhibited the highest content than other fractions. The antimicrobial activities were tested against six pathogenic micro-organisms following the disc diffusion method. The results are shown in Table 2. In this case, the bacteria in the cultures with the clear zones more than 16 mm diameters were considered as highly susceptible, those between 11–15 mm as moderately susceptible and those with less than 10 mm as resistant. The DL-P01-SI-01 fraction was found to exhibit the highest antimicrobial activity, being most potent against S. aureus (with zone of inhibition of 30 mm). From the above results, it is evident that the DL-P01-SI-01 fraction exhibits stronger antimicrobial activity than other extract and fractions.
Antimicrobial activities of Dimocarpus longan Lour. Fen Ke
DOI: 10.3109/09637486.2014.886181
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Figure 1. Flow chart for extraction of Logan seed.
Table 1. Content of marker substance in Longan seed extract and fraction. Samplesa
Gallic acid (mg/g)
Corilagin (mg/g)
Ethyl gallate (mg/g)
Ellagic acid (mg/g)
4.9 5.7 72.8 112.2
10.5 20.8 159.2 245.2
– 2.4 24.8 34.3
5.7 8.9 73.6 63.0
DW DL DL-P01 DL-P01-SI-01 a
The tested samples were DW: crude water extract; DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; DL-P01-SI-01: ethyl acetate subfraction.
Table 2. Antimicrobial activity of Longan seed extract and fractions. Tested micro-organismsb Tested samples
a
DL (4 mg/disc) DLP01 (2 mg/disc) DLP02 (2 mg/disc) DLP03 (2 mg/disc) DL-P01-SI01 (1 mg/disc) DL-P01-SI02 (1 mg/disc) DL-P01-SI03 (1 mg/disc) DL-P01-SI04 (1 mg/disc) DL-P01-SI05 (1 mg/disc) DL-P01-SI06 (1 mg/disc) DL-P01-SI07(1 mg/disc) DL-P01-SI08 (1 mg/disc) DL-P01-SI09 (1 mg/disc) DL-P01-SI10 (1 mg/disc)
Inhibition zone diameter (mm)
PA
SM
SA
MRSA
AB
SG
13 13 – – 14 – – – 14 – – – – –
– 13 – – 15 – – – – – – – – –
– 16 – – 30 28 28 – 23 20 20 – – –
– 15 – – 15 12 12 13 12 15 11 10 – –
13 15 13 – 15 12 12 12 12 – – 10 – –
– 14 – – 15 15 14 14 15 13 12 – – –
a
The tested samples were DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; DL-P02: n-butanol; DLP03: Aquation; ethyl acetate subfractions: DL-P01-SI01- DL-P01-SI10. b Micro-organisms: PA: Propionibacterium acne; SM: Streptococcus mutans; SA: Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus; AB: Acinetobacter baumannii; SG: Salmonella gallinarum.
Discussion According to Michielin et al. (2009), plant materials can be classified as antimicrobial agents based on the MIC values of their extracts. Duarte et al. (2007) and Wang et al. (2008) classified the extracts as: strong inhibitors for MIC value below 500 mg/mL; moderate inhibitors for MIC between 600 and 1500 mg/mL; weak
inhibitors for MIC above 1600 mg/mL. This classification is very useful in detecting the potential biological activity of various plant materials. The DL-P01-SI-01 fraction showed the strongest activity on S. aureus and methicillin-resistant S. aureus at MIC 64 mg/mL (Table 3). Besides, the relationship between extract and fractions of Fen Ke longan seed and cell viability (%) after 24 h exposures using
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Int J Food Sci Nutr, 2014; 65(5): 589–593
Figure 2. HPLC chromatograms of Longan seed DL-P01-SI-01 fraction.
Table 3. Minimum inhibitory concentration (MIC, mg/mL) of extract and fractions obtained from Longan seed on growth of different micro-organisms. Tested samples Tested micro-organismsa Propionibacterium acne Streptococcus mutans Staphylococcus aureus Methicillin-resistant Staphylococcus aureus Acinetobacter baumannii Salmonella gallinarum
DL
DL-P01
DL-P01-SI-01
DL-P01-SI-05
DL-P01-S-I06
1024 – – – 41024 –
512 128 256 64 256 256
256 128 64 64 256 256
256 – – – – 256
– – – 128 – –
a
The tested samples were DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; subfractions: DL-P01-SI01, DLP01-SI05, DL-P01-SI06.
Figure 3. Effects of cell viability (%) of one fraction and one pure compound with LPS (lipopolysaccharide; 0.25 mg/mL) stimulated on cultured HaCaT cells for 24 h using MTS assay. PC: Positive Control (curcumin). Data are mean ± SD (2–3).
DOI: 10.3109/09637486.2014.886181
the MTS assay are presented in Figure 3, respectively. The cell viability of each sample was greater than 90% using the MTS assay, indicating an excellent margin of safety. Considering the results obtained, the activity fraction of Fen Ke longan seed has good antimicrobial activities, and its bioactivity may be partly due to the phenolic compounds. Higher contents of ethyl gallate, gallic acid, ellegic and corilagin present in DL-P01-SI-01 fraction may result in higher inhibitory effect than other extract and fractions. Gallic acid and ellagic acid are widely studied for their pharmacological activities such as antimicrobial and antioxidant (Mahoney & Molyneux, 2004; Panizzi et al., 2002). The ability of tannins to form chelates with metal ions, particularly iron, which lead to the disruption of the S. aureus membrane, could be one of the possible factors that contribute to it antimicrobial activity (Akiyama et al., 2001). In conclusion, this study therefore strongly suggested that the potential use of longan seed extract and its polyphenolic compounds will be applied antibacterial ointment and developed to treat acne ointment or cleanser product.
Conclusion This study successfully isolated the fraction of Fen Ke seed extract, which exhibited strong antimicrobial activity, which was found to be due to the phenolic compounds. The HPLC analysis showed that the major phenolic compounds are gallic acid, corilagin, ethyl gallate and ellagic acid.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Akiyama H, Fujii K, Yamasaki O, Oono T, Iwatsuki K. 2001. Antibacterial action of several tannins against Staphylococcus aureus. J Antimicrob Chemother 48:487–491. De Assis SA, Vellosa JCR, Bruunetti IL, Khalil NM, Leite KMSC, Martins ABG, Oliverira OMMD. 2009. Antioxidant activity, ascorbic acid and total phenol of exotic fruits occurring in Brazil. Int J Food Sci Nutr 62:439–448. Duarte MCT, Leme EE, Delarmelina C, Soares AA, Figueira GM, Sartoratto A. 2007. Activity of essential oils from Brazilian medicinal plants on Escherichia coli. J Ethnopharmacol 111:197–201. Fogliani B, Raharivelomanana P, Bianchini JP, Bouraima-Madjebi S, Hnawia E. 2005. Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia. Phytochemistry 66: 241–247. Huang HB. 1995. Advances in fruit physiology of the arillate fruits of litchi and longan. Annu Rev Hortic Sci 1:101–120.
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Jiang GX, Jiang YM, Yang B, Yu CY, Tsao R, Zhang HY, Chen F. 2009. Structural characteristics and antioxidant activities of oligosaccharides from longan fruit pericarp. J Agric Food Chem 57:9293–9298. Kinoshita S, Inoue Y, Nakama S, Ichiba T, Aniya Y. 2007. Antioxidant and hepatoprotective actions of medicinal herb, Terminalia catappa L. from Okinawa Island and its tannin corilagin. Phytomedicine 14: 755–762. Mahoney N, Molyneux RJ. 2004. Phytochemical inhibition of aflatoxigenicity in Aspergillus flavus by constituents of walnut (Juglans regia). J Agric Food Chem 52:1882–1889. Martins S, Amorim ELC, Sobrinho TJSP, Saraiva AM, Pisciottano MNC, Aguilar CN, Teixeiraa JA, Mussatto SI. 2013. Antibacterial activity of crude methanolic extract and fractions obtained from Larrea tridentata leaves. Ind Crop Prod 36:306–311. Michielin EMZ, Salvador AA, Riehl CAS, Smaˆnia JRA, Smaˆnia EFA, Ferreira SRS. 2009. Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods. Bioresource Technol 100:6615–6623. Panizzi L, Caponi C, Catalano S, Cioni PL, Morelli I. 2002. In vitro antimicrobial activity of extracts and isolated constituents of Rubus ulmifolius. J Ethnopharmacol 79:165–168. Prasad KN, Yang B, Shi J, Yu C, Zhao M, Xue S, Jiang Y. 2010. Enhanced antioxidant and antityrosinase activities of longan fruit pericarp by ultrahigh pressure-assisted extraction. J Pharm Biomed Anal 51:471–477. Rangkadilok N, Worasuutayangkurn L, Bennet RN, Satayavivad J. 2005. Identification and quantification of polyphenolic compounds in longan fruit. J Agric Food Chem 53:1387–1392. Rangkadilok N, Worasuutayangkurn L, Mahidol C, Ruchirawat M, Satayavivad J. 2007. Evaluation of free radical scavenging and antitryosinase activities of standardized longan fruit extract. Food Chem Toxicol 45:328–336. Soong YY, Barlow PJ. 2005. Isolation and structure elucidation of phenolic compounds from longan seed by high performance liquid chromatography electrospray ionization mass spectrometry. J Chromatogr A 1085:270–277. Tindall HD. 1994. Sapindaceous fruits: botany and horticulture. Hortic Rev 16:143–196. Wang YS, He HP, Yang JH, Di YT, Hao XJ. 2008. New monoterpenoid coumarins from Clausena anisumolens. Molecules 13:931–937. Worasuutayangkurn L, Watcharasit P, Rangkadilok N, Suntararuks S, Khamkong P, Satayavivad J. 2012. Safety evaluation of longan seed extract: acute and repeated oral administration. Food Chem Toxicol 50: 3949–3955. Wen LJ, Li YJ, Mao HJ, Zhang YC. 2010. Nutrients and antioxidant activity of longan seeds. Food Sci 31:243–246. Xu HX, Wan M, Dong H, But PP, Foo LY. 2000. Inhibitory activity of flavonoids and tannins against HIV-1 protease. Biol Pharm Bull 23: 1072–1076. Yilmaz Y, Toledo RT. 2004. Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid. J Agric Food Chem 52:255–260. Zheng GM, Xu LX, Wu P, Xie HHH, Jiang YM, Chen F, Wei XY. 2009. Polyphenols from longan seeds and their radical scavenging activity. Food Chem 116:433–436.
IN VITRO AND ANIMAL STUDIES
Antimicrobial activities of various fractions of longan (Dimocarpus longan Lour. Fen Ke) seed extract Huang-Chung Tseng1,2, Wan-Ting Wu2, Ho-Shin Huang1, and Ming-Chang Wu2 1
Laboratory of Antrodia Certification Center, Joben Bio-medical Co, Ltd., Pingtung, Taiwan and 2Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan Abstract
Keywords
The antimicrobial activities of longan (Dimocarpus longan Lour. Fen ke) seed extracts were investigated using a disc diffusion method and also determining the minimal inhibitory concentration. The DL-P01-SI01 fraction showed that the strongest activity against Staphylococcus aureus and methicillin-resistant S. aureus at MIC 64 mg/mL, which was found to be due to the phenolic compounds. The HPLC analysis showed that the major phenolic compounds are gallic acid, corilagin, ethyl gallate and ellagic acid.
Antimicrobial activity, corilagin, ellagic acid, ethyl gallate, longan seed
Introduction Longan (Dimocarpus longan Lour.), which is a subtropical fruit of the Sapindaceae family, has been widely planted in South-East Asia including Taiwan and China for many years (Huang, 1995; Tindall, 1994). Among numerous longans, the Fen ke cultivar is the most commonly cultivated in Taiwan representing over 98% of the longan area because of its high yield. Owing to its delicious taste, longan has been gradually accepted by consumers and has enjoyed great popularity in the international market. Regardless of being consumed as fresh fruits or as processed products, longan seeds have become agricultural wastes and created environmental problems on a large scale. Fruit seeds are known to contain many phenolic compounds capable of protecting them from oxidative damage and defending them against yeast, fungi and bacteria that might inhibit their germination. In addition, longan seeds contain large amounts of other bioactive compounds, such as phenolic acids, flavonoids and polysaccharides (Jiang et al., 2009; Zheng et al., 2009), and exhibit antimicrobial, antioxidant and inflammatory properties (De Assis et al., 2009; Prasad et al., 2010; Wen et al., 2010). Rangkadliok et al. (2005) further suggested that the seed extract of longan could be a potential source of natural dietary antioxidants and skin whitening agents. Recently toxicological studies revealed no toxic effects of oral administration of longan seed extract during acute (Worasuutayangkurn et al., 2012). The water extract of longan fruit was found to contain high levels of polyphenolic compounds such as corilagin, gallic acid and ellagic acid (Soong & Barlow, 2005). The analysis of longan
History Received 30 July 2013 Revised 30 September 2013 Accepted 13 January 2014 Published online 17 February 2014
fruit from Thailand showed that there was a large variation in the contents of gallic acid, corilagin and ellagic acid ranging from 9.18 to 23.04, from 0 to 50.64 and from 8.13 to 12.65 mg/g respectively, in different longan tissues among cultivars (Rangkadilok et al., 2007). Corilagin is a member of the tannin family and has strong antioxidant (Kinoshita et al., 2007), antiHIV (Xu et al., 2000) and antimicrobial (Fogliani et al., 2005) activities. Gallic acid and ellagic acid are reported to be potent antioxidants and anticarcinogenic agents (Yilmaz & Toledo, 2004). Although the seed of D. longan has shown some physiological effects, limited studies have focused on the antimicrobial activity of Fen Ke Longan. In this study, we evaluated the antimicrobial activity of Fen Ke longan seed extracts and investigated the relationship between the phenolic acids and the antimicrobial activities in different fractions.
Materials and methods Plant material and chemicals Plant materials (Dimocarpus longan Lour. Fen ke) were obtained from the Taichung District Agricultural Improvement Station at commercial maturation season. Methanol (HPLC grade) and acetic acid was obtained from Merck. Gallic acid and ethyl gallate was purchased from Sigma (St. Louis, MO). Ellagic acid was purchased from Aldrich. Corilagin was purchased from PIN chemical Ltd (Ponpadi R.S., Pin-631213, Tamil Nadu, India). Ultrapure water purified using a Milli-Q system (Millipore Inc., Billerica, MA) was used. Extraction and fractionation
Correspondence: Ming-Chang Wu, Department of Food Science, National Pingtung University of Science and Technology, No. 1 Hseuhfu Rd., Pingtung, Taiwan. E-mail: [email protected]
The dried longan seed 3 kg was ground into powder and extracted with 12 kg water and 12 kg ethanol for 24 h. The whole mixture was filtered with filter paper. The residue was extracted two more times with water and ethanol as before. The total extracts were
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filtered, and the obtained filtrates were concentrated under reduced pressure to dryness, yielding the crude extract of 644 g. The resulting crude extract was stored at 4 C until future process. The 640 g crude extract then suspended in 3000 ml distilled water, and was partitioned with ethyl acetate (see the flow chart for Longan seed extraction). After 24 h settlement, the upper ethyl acetate layer was collected and lyophilized under reduced pressure to yield a powder of 38.21 g (DI-P01). The remaining aqueous portion was extracted with 3000 ml n-butanol. After 24 h settlement, the upper n-butanol layer was collected and lyophilized under reduced pressure to yield DL-P02 of 68.16 g. The remaining aqueous layer was lyophilized under reduced pressure to yield DL-P03 of 506.88 g. Since the ethyl acetate fraction, DL-P01, was found later to exhibit the highest antimicrobial activity, this fraction was further separated by a silica gel column chromatographic method flashed with ethyl acetate and methanol gradient to yield 10 sub-fractions. DL-P01 of 1.2 g was sonicated to dissolve in ethyl acetate and methanol and 40 g silica gel 60 was then added. The whole mixture was dried under reduced pressure and loaded onto a flash column. The column was auto-flashed with ethyl acetate for 30 min and later with decreasing concentration of ethyl acetate and an increasing concentration of methanol. Each 100 ml was collected and total of 10 tubes were collected. After lyophilized under reduced pressure, their proportions were as follows: DL-P01-SI0 765.3 mg, DL-P01-SI02 100.4 mg, DL-P01-SI03 37.0 mg, DL-P01-SI04 21.7 mg, DL-P01-SI05 140.1 mg, DL-P01-SI06 57.3 mg, DL-P01-SI07 28.4 mg, DL-P01-SI08 17.0 mg, DL-P01-SI09 6.4 mg and DL-P01-SI10 7.7 mg. The obtained fractions (Figure 1) were further subjected to the antimicrobial assay.
Int J Food Sci Nutr, 2014; 65(5): 589–593
Determination of minimal inhibitory concentration (MIC) The minimal inhibitory concentration values were studied for the fractions. The fractions were first solved in water and then diluted to the highest concentration to be tested, after that serial dilutions were made in a concentration range from 0.5 to 1024 mg/mL (1024, 512, 256, 128, 64, 32, 16, 8,4, 2, 1 and 0.5 mg/mL). The target micro-organisms were cultured in Mueller–Hinton broth (MHB) at 37 C for 24 h. After that, the suspensions were adjusted to approximately 108 CFU/mL with 0.5 McFarland standard turbidity and Mueller–Hinton broth. To 24-well microliters plates, 1.6 mL of Mueller–Hinton broth containing diluted bacteria and 409.6 mL of aliquot from the solutions of the fractions were added. A positive control (containing inoculums but no extracts) and negative control (containing extracts but on inoculums) were included on each micro-plates. The contents of the wells were mixed and the micro-plates were incubated at 37 C for 24 h. The MIC was defined as the lowest concentration at which no visible growth was observed. The results were expressed in mg/mL (Martins et al., 2013). Cell viability assay HaCaT, a well-known immortalized human keratinocyte cell line, was growth in DMEM medium (high glucose) supplemented with 2 mM L-glutamine and 10% fetal calf serum at 37 C in 5% CO2/ atmosphere humidified incubator. Subcultures were obtained by disaggregating the cells with 0.05% trypsin/EDTA solution and replating at 1:5–1:10. The viability of HaCaT was detected with reagents of MTS/PMF (2 mg/mL MTS reagent and 0.9 mg/mL PMS reagent; Sigma). After colorized at room temperature for 90 min, the intensity was read at 490 nm. The positive control used was curcumin.
Antimicrobial activity assays Microbial strains The antimicrobial activities were assayed against six different strains of microbes: Propionibacterium acne (BCRC10723); Streptococcus mutans (BCRC10793); Staphylococcus aureus (BCRC80277); methicillin-resistant S. aureus (BCRC15211); Acinetobacter baumannii (BCRC 80276); Salmonella gallinarum (BCRC10830), all of which were supplied by Bioresource Collection and Research Center of Taiwan. The extract was first dissolved in DMSO and then diluted to the desired concentrations but the final DMSO concentration was less than 0.5%, which was found to have no antimicrobial activity. The disc diffusion method The disc diffusion method was employed for screening the extract and its fractions for their antimicrobial activities. Briefly, a suspension of the tested micro-organism was spread on the Mueller–Hinton Agar (MHA). Filter paper discs (7 mm in diameter) were soaked with 10 mL of the extracts and placed on the inoculated plates, which were incubated at 37 C for 24 h. The antibacterial activity was determined by measuring diameters (d in mm) of inhibition zone with the following criteria: d510 mm, less active; 115d515 mm, middle active; d416 mm, very active. Preparation of inoculums The microbial suspension were prepared by making a saline suspension of isolated colonies selected from Mueller–Hinton agar, and the agar plates were grown for 18–24 h. The suspension was adjusted to match the tube of 0.5 McFarland turbidity standard using the spectrophotometer of 595 nm, which equals to 1 108 colony-forming units (CFU)/mL.
Bioactive compounds quantification Gallic acid, corilagin, ellagic acid and ethyl gallate were determined by high performance liquid chromatography with C18 5 mm (XBridge, 250 4.6 mm) column at 30 C, and UV detector at 280 nm. The mobile phase consisted of 1% acetic acid in water (solvent A) and methanol (solvent B) under the gradient profile. The gradient system started with 0% A at 0 min to 55% B in 35 min. The total run time was 50 min. The mobile phase was eluted in a flow rate of 1 mL/min, and samples of 10 mL were injected into the column. All the extracts were filtered through 0.22 mm membrane filters.
Results Gallic acid, corilagin, ethyl gallate and ellagic acid were the major polyphenolic compounds present in Fen Ke longan seed fractions. HPLC profile of fractions was shown in Figure 2. Retention times of gallic acid, corilagin, ethyl gallate and ellagic acid were found to be 6.8, 18.6, 21.6 and 27.9 min, respectively. Table 1 shows the contents of these acids in different extractions and fractions. Among the fractions, DL-P01-SI-01 exhibited the highest content than other fractions. The antimicrobial activities were tested against six pathogenic micro-organisms following the disc diffusion method. The results are shown in Table 2. In this case, the bacteria in the cultures with the clear zones more than 16 mm diameters were considered as highly susceptible, those between 11–15 mm as moderately susceptible and those with less than 10 mm as resistant. The DL-P01-SI-01 fraction was found to exhibit the highest antimicrobial activity, being most potent against S. aureus (with zone of inhibition of 30 mm). From the above results, it is evident that the DL-P01-SI-01 fraction exhibits stronger antimicrobial activity than other extract and fractions.
Antimicrobial activities of Dimocarpus longan Lour. Fen Ke
DOI: 10.3109/09637486.2014.886181
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Figure 1. Flow chart for extraction of Logan seed.
Table 1. Content of marker substance in Longan seed extract and fraction. Samplesa
Gallic acid (mg/g)
Corilagin (mg/g)
Ethyl gallate (mg/g)
Ellagic acid (mg/g)
4.9 5.7 72.8 112.2
10.5 20.8 159.2 245.2
– 2.4 24.8 34.3
5.7 8.9 73.6 63.0
DW DL DL-P01 DL-P01-SI-01 a
The tested samples were DW: crude water extract; DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; DL-P01-SI-01: ethyl acetate subfraction.
Table 2. Antimicrobial activity of Longan seed extract and fractions. Tested micro-organismsb Tested samples
a
DL (4 mg/disc) DLP01 (2 mg/disc) DLP02 (2 mg/disc) DLP03 (2 mg/disc) DL-P01-SI01 (1 mg/disc) DL-P01-SI02 (1 mg/disc) DL-P01-SI03 (1 mg/disc) DL-P01-SI04 (1 mg/disc) DL-P01-SI05 (1 mg/disc) DL-P01-SI06 (1 mg/disc) DL-P01-SI07(1 mg/disc) DL-P01-SI08 (1 mg/disc) DL-P01-SI09 (1 mg/disc) DL-P01-SI10 (1 mg/disc)
Inhibition zone diameter (mm)
PA
SM
SA
MRSA
AB
SG
13 13 – – 14 – – – 14 – – – – –
– 13 – – 15 – – – – – – – – –
– 16 – – 30 28 28 – 23 20 20 – – –
– 15 – – 15 12 12 13 12 15 11 10 – –
13 15 13 – 15 12 12 12 12 – – 10 – –
– 14 – – 15 15 14 14 15 13 12 – – –
a
The tested samples were DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; DL-P02: n-butanol; DLP03: Aquation; ethyl acetate subfractions: DL-P01-SI01- DL-P01-SI10. b Micro-organisms: PA: Propionibacterium acne; SM: Streptococcus mutans; SA: Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus; AB: Acinetobacter baumannii; SG: Salmonella gallinarum.
Discussion According to Michielin et al. (2009), plant materials can be classified as antimicrobial agents based on the MIC values of their extracts. Duarte et al. (2007) and Wang et al. (2008) classified the extracts as: strong inhibitors for MIC value below 500 mg/mL; moderate inhibitors for MIC between 600 and 1500 mg/mL; weak
inhibitors for MIC above 1600 mg/mL. This classification is very useful in detecting the potential biological activity of various plant materials. The DL-P01-SI-01 fraction showed the strongest activity on S. aureus and methicillin-resistant S. aureus at MIC 64 mg/mL (Table 3). Besides, the relationship between extract and fractions of Fen Ke longan seed and cell viability (%) after 24 h exposures using
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Int J Food Sci Nutr, 2014; 65(5): 589–593
Figure 2. HPLC chromatograms of Longan seed DL-P01-SI-01 fraction.
Table 3. Minimum inhibitory concentration (MIC, mg/mL) of extract and fractions obtained from Longan seed on growth of different micro-organisms. Tested samples Tested micro-organismsa Propionibacterium acne Streptococcus mutans Staphylococcus aureus Methicillin-resistant Staphylococcus aureus Acinetobacter baumannii Salmonella gallinarum
DL
DL-P01
DL-P01-SI-01
DL-P01-SI-05
DL-P01-S-I06
1024 – – – 41024 –
512 128 256 64 256 256
256 128 64 64 256 256
256 – – – – 256
– – – 128 – –
a
The tested samples were DL: crude methanolic extract; fractions: DL-P01: ethyl acetate; subfractions: DL-P01-SI01, DLP01-SI05, DL-P01-SI06.
Figure 3. Effects of cell viability (%) of one fraction and one pure compound with LPS (lipopolysaccharide; 0.25 mg/mL) stimulated on cultured HaCaT cells for 24 h using MTS assay. PC: Positive Control (curcumin). Data are mean ± SD (2–3).
DOI: 10.3109/09637486.2014.886181
the MTS assay are presented in Figure 3, respectively. The cell viability of each sample was greater than 90% using the MTS assay, indicating an excellent margin of safety. Considering the results obtained, the activity fraction of Fen Ke longan seed has good antimicrobial activities, and its bioactivity may be partly due to the phenolic compounds. Higher contents of ethyl gallate, gallic acid, ellegic and corilagin present in DL-P01-SI-01 fraction may result in higher inhibitory effect than other extract and fractions. Gallic acid and ellagic acid are widely studied for their pharmacological activities such as antimicrobial and antioxidant (Mahoney & Molyneux, 2004; Panizzi et al., 2002). The ability of tannins to form chelates with metal ions, particularly iron, which lead to the disruption of the S. aureus membrane, could be one of the possible factors that contribute to it antimicrobial activity (Akiyama et al., 2001). In conclusion, this study therefore strongly suggested that the potential use of longan seed extract and its polyphenolic compounds will be applied antibacterial ointment and developed to treat acne ointment or cleanser product.
Conclusion This study successfully isolated the fraction of Fen Ke seed extract, which exhibited strong antimicrobial activity, which was found to be due to the phenolic compounds. The HPLC analysis showed that the major phenolic compounds are gallic acid, corilagin, ethyl gallate and ellagic acid.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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