Extraction, Characterization and Biological

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Chiang Mai J. Sci. 2014; 41(1) Chiang Mai J. Sci. 2014; 41(1) : 14-26 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Extraction, Characterization and Biological Activities of Extracts from Freshwater Macroalga [Rhizoclonium hieroglyphicum (C.Agardh) K tzing] Cultivated in Northern Thailand Lapatrada Mungmai [a], Supat Jiranusornkul [a], Yuwadee Peerapornpisal [b], Busabun Sirithunyalug [a] and Pimporn Leelapornpisid*[a] [a] Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University 50200, Thailand. [b] Division of Microbiology, Department of Biology, Faculty of Science, Chiang Mai University 50200, Thailand. *Author for correspondence; e-mail: [email protected] Received: 3 May 2012 Accepted: 1 May 2013

ABSTRACT In this study, Rhizoclonium hieroglyphicum (C.Agardh) K tzing from the Nan River in northern Thailand was selected for investigation. The dried macroalga was extracted by water and 95% (v/v) ethanol to obtain an aqueous extract (RW) and ethanolic extract (RE), respectively. Each extract was examined for antimicrobial activity against Staphylococcus aureus ATCC 29213, methicillin resistant S. aureus and Propionibacterium acne ATCC 6919 by agar well diffusion method. Their in vitro antioxidant activity was determined by three different methods: 2, 2’-diphenyl-1picrylhydrazyl (DPPH), 2, 2’-azino-bis3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and thiobarbituric acid reaction substances (TBARS) assays. The total phenolic content was further evaluated. The polysaccharide analysis and physicochemical characterizations of the RW extract were examined. The results revealed that both of the extracts had no effective antibacterial activity against the test of microorganism. Interestingly, RW exhibited antioxidant activity twice that of Trolox, a vitamin E analog, and showed a higher Gallic acid Equivalent Antioxidant Capacity (GEAC) value than the RE extract. The Fourier Transform infrared (FT-IR) spectrum assay of RW presented the absorption characteristics of sulfated polysaccharides and Thin Layer Chromatography (TLC) analysis showed that it consisted of rhamnose, xylose, arabinose and galactose as sugar units. The 10% of RW gel showed 52.68±2.56 g/cm2 of gel strength while the gelling and melting temperature of this extract ranged from 50-55°C and 70-85°C, respectively. These gelling properties resembled that of carrageenan. This finding suggested that R. hieroglyphicum could be used for nutritional, pharmaceutical and cosmetic products. Keywords: Rhizoclonium, anti-microbial, antioxidant, gelling property 1. INTRODUCTION The freshwater green algae Rhizoclonium spp., Cladophora spp. and Aegagropila spp. are macroalgae found in Britain, India, China

and Australia. Among other places, they are also found in northern and northeastern Thailand, and locally referred to as “Kai”.

Chiang Mai J. Sci. 2014; 41(1)

Kai are abundant during the dry season in the Nan River, Nan Province and the Mekong River, Chiang Rai Province, Thailand. Locals in the area traditionally consumed these algae as a health food [1]. Three genera of Kai; Rhizoclonium spp., Cladophora spp. and Aegagropila spp. are classified in Division Chlorophyta, Class Chlorophyceae, Order Cladoporales, Family Cladophoraceae [2]. This research focuses on Rhizoclonium hieroglyphicum (C.Agardh) K tzing, the edible algae in the Nan River and the one richest in cell-wall polysaccharides. It exhibits maximum growth during the dry season (November-March), when the temperature and velocity of water are low [1]. Many edible algae have been shown to have important nutritional and health benefits. Spirulina platensis, a blue-green alga is a famous food supplement found in soil, freshwater, brackish water and seawater. It has been proven to have a high nutritional value (containing protein and amino acids) and offers health benefits, including antioxidative and antimicrobial properties stemming from its polyunsaturated fatty acids, phycobiliprotein, phycocyanin and phenolics [3, 4, 5]. Many studies also exist on a wide variety of edible freshwater macroalgae, looking at a range of issues including diversity, ecology, nutritional value, toxicity, and pharmaceutical and medicinal properties. For example, Cladophora glomerata and Nostochopsis lobatus exhibit anti-gastriculcer, anti-inflammatory, analgesic and antioxidant activities, offering the potential to play an important role in pharmacological activities [6]. The location, morphology and biochemical contents of R. hieroglyphicum have been studied in Thailand [1], but its biological activities have yet to be investigated. Given R. hieroglyphicum’s traditional uses, it shows promise for potentially beneficial uses, and has been selected for further analysis here,

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particularly its potential antioxidative role. One particularly interesting feature of R. hieroglyphicum is its polysaccharide richness. Polysaccharides are used as a natural polymer in various applications, including as thickening or gelling agents in food manufacturing and pharmaceutical production [7]. For many years, researchers have been interested in the uses of polysaccharides from algae. Most of this attention has been devoted to agars and carrageenans from red seaweeds; and alginates from brown seaweeds [8, 9, 10, 11]. Some polysaccharides from seaweeds have been reported to possess many biological activities and have been used as natural bioactive compounds in the food and pharmaceutical industries [10]. In addition, cosmetic ingredients such as alginic acid, which is used as a consistency agent and as a moisture retaining surface film, are produced from brown algae; and carrageenan, a gelling agent, is produced from red algae [12]. Several studies have verified the antioxidant properties of polysaccharides from seaweeds [7, 10, 13]. The antioxidant activity of the polysaccharides depends on several structural parameters such as the molecular weight, type of sugar and glycosidic branching, the degree of sulfation (DS) and the sulfation position [13]. In contrast to seaweeds, the properties of polysaccharides from freshwater algae and their potential health and cosmetic applications have not been widely investigated. In particular, polysaccharides from R. hieroglyphicum, whether from Thailand or elsewhere, have yet to be investigated. This study examines some of the biological activities of R. hieroglyphicum extracts, in particular its antioxidative and antimicrobial activity. The physical characteristics and chemical composition of its polysaccharides were also determined. The results from this study could provide fundamental data for developing potentially beneficial applications

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of polysaccharides from R. hieroglyphicum. 2. MATERIALS AND METHODS

2.1 Sample Collection R. hieroglyphicum was collected from the Nan River, Nan Province, Thailand (latitude 19°05′12.12′′N and longitude 100° 47′14.91′′E) between November 2010 and March 2011. Samples were identified using the morphological features of its macroscopic, microscopic structures [14, 15, 16] and molecular analysis [17]. The fresh alga were washed and dried at 60°C for 48 hours, ground into powder and stored in a vacuum desiccator at room temperature for further study. Spirulina platensis was purchased from Boonsom Farm, Chiang Mai, Thailand and used as a positive control for the antimicrobial and antioxidant activities assays. 2.2 Extraction Extraction was performed on 50 g dry weight of the alga. Extracts from the dried powder were produced in 1 liter each of two different solvents, distilled water and 95% (v/v) ethanol, at 50°C for 1 hour. The solutions were separated from the residual alga by filtration using a No.1 Whatman filter and then dried to obtain aqueous extract (RW) and ethanolic extract (RE), respectively. These were done in triplicate. In the drying process, the aqueous extracts were freeze-dried while the ethanolic extracts were evaporated under vacuum until dryness. The percentage yield of the dry weight of each extract was then calculated. 2.3 Determination of Antimicrobial Activity The extracts of algae were examined for their antimicrobial activity by Agar diffusion method [18]. Trypticase soy agar (TSA) was inoculated with the test organisms


-Staphylococcus aureus ATCC 29213, methicillin resistant S. aureus (MRSA) and Propionibacterium acne ATCC 6919 (obtained from the Microbiology Laboratory, Faculty of Medicine, Chiang Mai University) (0.5 McFarland turbidity standards) and allowed to solidify. Twelve millimeter diameter wells were punched into the agar and then filled with 200 μl of the extract. Extracts of 2 and 5 % (w/v) were used. Distilled water was used as a negative control and two standard antimicrobial agents (gentamicin and benzoyl peroxide) were used as positive controls. S. aureus and MRSA plates were incubated at 37°C for 24 hours and the P. acne plate was incubated at 37°C for 72 hours under anaerobic conditions. The antimicrobial activity was determined by observation and measuring the inhibition zone diameter. These were compared with the positive control, S. platensis extract. 2.4 Determination of Antioxidant Activities The antioxidant activities were compared with the well-known edible alga, S. platensis. 2.4.1 Determination of antioxidant activity by free radical scavenging activity on 2, 2’-diphenyl-1-picrylhydrazyl (DPPH•) The antioxidant activity of the algae extracts in terms of their radical scavenging ability based on hydrogen donating capacity was measured using the stable DPPH method adapted from Brem et al. [19]. Twenty microlitres of each extract with different dilutions was added to 180 μl of 167 μM DPPH•/ethanol solution in a 96-well microtiter plate. After incubation at room temperature in the dark for 30 minutes, the absorbance of the mixtures was measured spectrophotometrically at 540 nm with a multimode detector (DTX 880, Beckman


Coulter). Gallic acid served as the reference standard. The antioxidant activity of each sample was then calculated in terms of its Gallic Acid Equivalent Antioxidant Capacity (GEAC) and IC50 value (mg/ml). 2.4.2 Determination of antioxidant activity by free radical scavenging activity on 2, 2’-azino-bis3-ethylbenzothiazoline6-sulfonic acid (ABTS•+) The antioxidant activity of the algae extracts in terms of their radical scavenging ability by electron donation was measured using a modified version of the method described by Re et al. [20]. The stock solutions included 7 mM ABTS•+ solution and 2.45 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 hours at room temperature in the dark. The solution was diluted by mixing 1 ml ABTS•+ solution with 30 ml of absolute ethanol to obtain an absorbance of 0.7-0.9 units at 734 nm using a spectrophotometer. Fresh ABTS•+ solutions were prepared for each assay. The extracts (70 μl) were allowed to react with 630 μl of the ABTS•+ solution for 5 minutes in the dark. The absorbance was then measured at 734 nm using a spectrophotometer UV-2450, shimadzu. Trolox was used as the reference standard. The antioxidant activity of each sample was then in terms of its Trolox Equivalents Antioxidant Capacity (TEAC) and IC50 value (mg/ml). 2.4.3 Determination of antioxidant activity by inhibition of lipid peroxidation using the thiobarbituric acid reaction substances (TBARS) assay The antioxidant activity of the crude extracts in terms of their inhibition on lipid peroxidation was measured using a modified TBARS assay [21,22]. The liposome

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suspension (600 μ l) and 100 μ l of the algal extracts were mixed with 60 μl of 0.07 M AAPH (2 ,2′-azobis-2-methylpropanimidamide dihydrochloride) into the tube and incubated at 60°C for 24 hours. Then 80 μl of the mixture was mixed with 24 μl of 0.02% w/v BHT, 100 μl of 3% v/v Triton X-100, 500 μl of 20% v/v acetic acid pH 3.5 and 250 μl of 0.2% w/v TBA in a microtiter plate. The reaction mixture was incubated for 30 minutes at 90°C and then cooled to room temperature. The absorption of the mixture was measured at 540 nm with a multimode detector (DTX 880, Beckman Coulter). Quercetin was used as the positive control. The inhibition of lipid peroxidation was expressed as IC50 value (mg/ml) 2.4.4 Determination of total phenolic content The total phenolic content of the extracts was determined using the Folin-Ciocalteu reagent method [23]. Each sample was mixed with Folin-Ciocalteu reagent for 3 minutes and followed by addition of 7.5% (w/v) sodium carbonate. The solutions were mixed with a vortex mixer. The reaction mixture was incubated for 30 minutes in the dark. The absorption of the mixture was measured at 765 nm using a UV-2450 Spectrophotometer (Shimadzu Corporation). The concentration of total phenolic compounds in all samples was expressed as micrograms of Gallic Acid Equivalents (GAE) per gram of the extract. 2.5 Content Analysis 2.5.1 Protein analysis 2.5.1.1 Bradford protein assay The protein content was determined by Bradford assay [24]. One hundred μl of extract were added to 1 ml of Coomassie reagent and incubated for 2 minutes at room temperature. Absorbance was

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measured at 595 nm with spectrophotometer (Beckman DU-62, Backman Coulter). Protein determination was made by comparison to protein assay standards, usually prepared as a series of known dilutions of bovine serum albumin (BSA). 2.5.1.2 Amino acid analysis Amino acid residues were analyzed as described by AOAC [25]. Approximately 2-3 mg of the algal extract was added to 6 N HCl 15 ml, then heated at 100-110°C for 72 hours, followed by methyl chloroformate derivatization, which could be identied and quantitatively determined by gas chromatography equipped with a mass spectroscopic detector (GC-MS). 2.5.2 Polysaccharide analysis 2.5.2.1 Fourier transform infrared (FT-IR) spectroscopy A FT-IR spectrum of the alga extract was recorded in the 4,000-400 cm-1 range using a Nicolet Nexus Model 470 FT-IR instrument with KBr pellet technique. 2.5.2.2 Determination of the molecular weight by size exclusion chromatography (SEC) mass/molar The RW extract was separated on a size exclusion chromatography system consisting of two columns filled with Sephacryl S200 (100 × 1.5 cm) and Biogel P2 (120 × 1.5 cm) using bidest water as an eluent. Each fraction was freeze-dried and stored in a vacuum desiccator at room temperature for further study. The molecular weight distributions were obtained from the refractive index detection according to Praznik and Huber [26]. For each fraction, 3 mg of the sample was dissolved in 1 ml of 0.05 M NaCl. The flow rate was set to 0.6 ml/min by a LKB2150 HPLC-pump.


2.5.2.3 Sugar analysis Approximately 2-3 mg of each SEC fraction was mixed with 0.5-1.0 ml of a 2M TFA solution in a glass vial sealed with a screw cap and left for hydrolysis to be completed for two hours at 100-110°C. The solution was dried under a stream of nitrogen at 60°C until the TFA was totally evaporated. One ml of absolute methanol was added to the solution and dried under nitrogen. This process was repeated until the solution was neutral and then re-dissolved in 1 ml of distilled water. The hydrolyzed solutions were stored in a refrigerator (2-8°C). Approximately 30 μl of the standard solutions (Merck Chemical Co.) and the fractions were applied to the bottom of the TLC plates using Linomat 4 with a syringe. The spots on the TLC plates were detected by spraying Thymol reagent. The TLC plates were dried using a hair dryer and then heated in an oven for 5-10 minutes at 105-110°C. 2.6 Physicochemical Properties of Alga Extracts 2.6.1 The textural properties Textural properties were determined with a Texture Analyzer equipped with a 10 mm in diameter cylindrical plunger operating at a cross-head speed of 1.0 mm.s-1 [11,27]. The measurements were performed on a 10% aqueous solution of R. hieroglyphicum gel after stabilization for 24 hours at room temperature. The gel strength, rupture force, deformation, cohesiveness and flexibility of the gels were determined. These were compared with k-carrageenan, given its similar gelling qualities. 2.6.2 The gelling and melting temperature The gelling and melting temperatures were determined by a modified method of Rodriguez et al. [27]. The gelling temperature


was measured on 10 ml of a 10% (w/v) hot solution of the agar. The gels were placed in glass test tubes at a cooling rate of 0.5°C minutes -1. A glass rod was periodically introduced in the agar solution and the temperature at which a permanent hole developed was considered as the gelling temperature. The melting temperature was determined by heating the 10% w/v gel with a temperature increase of about 0.5°C minutes-1. The temperature at which a glass bead (on the surface of the gel) dropped to the bottom was recorded as the melting temperature. 3. RESULTS AND DISCUSSION

3.1 Extraction of the Alga From the extraction of R. hieroglyphicum with different solvents, the RW showed a higher extraction yield (21.56 ±0.28%) than the RE (12.58 ± 0.44%). This indicated that most components of the algae dissolved in high polarity solvents and that more polar compounds were found. Matanjun et al. [28] also reported that the extraction of some marine algae such as Caulerpa racemosa, Sargassum polycystum and Padina sp. exhibited an increasing percentage yield when the polarity of the solvents was increased. Similarly, Boonchum et al. [29] reported that the extraction of marine algae with water yielded a higher quantity than ethanol. 3.2 Biological Activities 3.2.1 Determination of antimicrobial activity Regarding the application for bacterialinfected skin, the antimicrobial activity of the extracts (RW and RE) against three gram-positive bacteria, viz. S. aureus ATCC

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29213, MRSA and P. acne ATCC 6919 showed that both the 2% and 5 % (w/v) extracts could not inhibit the growth of these bacteria. The same result was observed from the extract from S. platensis. Peerapornpisal et al. [1] also reported that extracts of “Kai” algae (species not specified) did not react against gram-positive bacteria Bacillus subtilis and Micrococcus luteus and also gram-negative Escherichia coli, Klebsiella pneumonia and Proteus valgaris. Our results indicate that R. hieroglyphicum extracts did not contained active antimicrobial compounds. Bhowmik et al. [30] reported that S. platensis at various concentrations of 1, 5 and 10 mg/ml showed antimicrobial activity against three gram negative (Escherichia coli MTCC443, Pseudomonas aeruginosa MTCC424 and Proteus vulgaris MTCC426) and three gram positive bacteria (S. aureus MTCC96, Bacillus subtilis MTCC619 and B. pumulis MTCC1456). In contrast, Pradhan et al. [5] had shown that the aqueous extract of S. platensis had no effective antimicrobial activity against the test microorganism (Pseudomonas putida, P. aeruginosa, P. fluorescens, Aeromonas hydrophila, Vibrio alginolyticus, V. anguillarum, V. fluvialis and Escherichia coli). These results indicated that S. platensis possessed antimicrobial ability depended on the strain of microbial, method of extraction, different solvents of the extract, source of the extracts and conditions of the culture. 3.2.2 Determination of antioxidant activities The antioxidant activities of the extracts (RW and RE) were compared with each other and the ethanolic extracts of Spirulina platensis (SE) (Table 1).

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Table 1. IC50, standard equivalent of antioxidant activities and total phenolic content of Rhizoclonium hieroglyphicum (C.Agardh) K tzing and Spirulina platensis. Extracts

DPPH IC50 (mg/ml)

ABTS

TBARS

IC50 (mg/ml) TEAC IC50 (mg/ml) QEAC GEAC mg trolox /g mg quercetin mg gallic acid/g extract /g extract extract

Total Phenolic content mg GAE /g extract

RW a a c c a b b extract 23.883±1.123 0.921±0.042 13.949±0.027 2.079±0.031 55.208±0.269 0.09276±0.002 0.1505±0.003 c c b b c c 0.755±0.034 c 81.810±3.041 0.354±0.013 47.439±0.843 0.11211±0.000 0.7349±0.004 RE extract 29.144±1.330 SE extract 15.481±0.621a 1.349±0.052a 67.450±0.028b 0.430±0.000b 28.293±0.697a 0.16268±0.001a 0.5188±0.001b Data shown are mean ± standard error (SD) of three replicates. a, b and c are statistical comparison between groups using ANOVA post hoc Tukey’s b Test (p