Chemical Composition, Properties, and Antimicrobial Activity of the

Article pubs.acs.org/JAFC

Chemical Composition, Properties, and Antimicrobial Activity of the Water-Soluble Pigments from Castanea mollissima Shells Ting-Ting You,† Su-Kun Zhou,† Jia-Long Wen,† Chao Ma,‡ and Feng Xu*,† †

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, China National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, China

‡

ABSTRACT: Agricultural residues Castanea mollissima shells represent a promising resource for natural pigments for the food industry. This study provides a comprehensive and systematic evaluation of water-soluble pigments (CSP) from C. mollissima shells, which were obtained by 50% ethanol with microwave-assisted extraction. Spectroscopic techniques (UV, FT-IR, 13C NMR), elemental analysis, and chromatographic techniques (HPAEC, GPC) revealed that the main components in the CSP were flavonoids procyanidin B3 (condensed tannin), quercetin-3-O-glycoside, and steroidal sapogenins. As a consequence, CSP was water-soluble and presented significant DPPH scavenge capacity (EC50 value was 0.057 mg/mL). Specially, CSP gave excellent antibacterial activity, and even better than 5% aqueous phenol in some case. Moreover, CSP was practically nontoxic and exhibited good stability with temperature, natural light, and metal ions. These outstanding properties will enlarge the application of CSP for natural food additives production. KEYWORDS: Castanea mollissima shells, natural water-soluble pigments, chemical characterization of natural pigments, antimicrobial activity of natural pigments

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consumer demands for food security.7−10 Compared with synthetic ones, natural pigments are indeed healthier and have positive biological effects, such as antioxidant and antimicrobial activities. The biological effects of natural pigments are a consequence of the presence of bioactive components, such as procyanidin and quercetin-3-O-glycoside.11 In addition, the chemical components and properties of the pigments depend on the raw material and pigment extraction procedures. Commonly, the water-soluble pigments, such as tetrapyrrols and flavonoids, can be extracted by water or lower alcohols, while the water insoluble pigments can be obtained by alkaline extraction.12−14 Therefore, although the complex and irregular structure of pigments has been extensively investigated, it is still necessary to broaden the knowledge of components and properties of new pigments extracted from different raw materials. In the past decades, various methods have been developed to extract pigments from plant species. Some modern pigment preparations have been developed recently for fast extraction, such as microwave-assisted extraction, ultrasound-assisted extraction, and supercritical fluid extraction.14−16 Among them, microwave-assisted extraction (MAE) has been considered as the simplest and the most economical technique for natural compounds extraction. Based upon polarity of the extraction solvent, MAE technology makes the sample selective and rapidly localized heated.14 As a consequence, it reduces solvent amount, decreases the extraction time, and thus enhances the overall extraction efficiency. Although MAE is a

INTRODUCTION Castanea mollissima Blume, commonly known as Chinese chestnut, is a chestnut variety of considerable economic importance.1,2 Compared with European and American species, C. mollissima has evolved higher degrees of resistance to chestnut blight fungus after centuries of seed selection, meanwhile increasing the fruit production. According to 2011 statistical data from Food and Agriculture Organization of the United Nations, China accounted for about 84% (1 700 000 MT/year) of the total world chestnut production.3 In addition to the high productivity, C. mollissima fruit is rich in carbohydrate but low in protein and fat.4 The beneficial effects on human health of C. mollissima fruit make it widely consumed both as fresh fruit and as several derivates. Therefore, C. mollissima is considered a nutritional, cultural, and economical resource for many civilizations across China, and even eastern North America. C. mollissima shells are generated from chestnut processing as the main byproduct and currently used as fuels. Potential industrial application of chestnut shells includes their use as a source of natural pigments for food industry. A few research studies have focused on investigating the extraction, physicochemical properties, and dyeing characteristics of pigments in C. mollissima shells, especially the water-soluble pigments.5,6 However, a comprehensive and systematic evaluation of the water-soluble pigments from C. mollissima shell is quite scarce. The valorization of chestnut shells agricultural residues would improve the industrial process both economically and environmentally. Natural pigments are chemical compounds occurring in plants, animals, and microorganisms, and absorb light in the visible wavelength region of 400−800 nm. In recent years such pigments have attracted increasing research to satisfy rising © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1936

October 9, 2013 January 22, 2014 January 22, 2014 January 22, 2014 dx.doi.org/10.1021/jf4045414 | J. Agric. Food Chem. 2014, 62, 1936−1944

Journal of Agricultural and Food Chemistry

Article

pressure (−0.09 MPa) until the final volume was 50 mL. The concentrated solution was then purified by a two-step precipitation method. First, the concentrated solution was poured into 3 volumes of 95% ethanol to remove hemicelluloses by precipitation. Second, the above solution was concentrated again and then the solubilized lignin could be removed by precipitating the solution into 10 volumes of deionized water.15,16 After the two-step precipitation, the pigments were redissolved in the solution of ethanol/water (1:1, v/v), and successively extracted by 3 volumes of organic solvents (ethyl acetates, petroleum ether) for three times to remove lipids.17 Finally, the water layer was freeze-dried overnight. Chemical Composition and Structural Analysis. To analyze the associated sugars in pigments, sugar analyses were conducted by acid hydrolysis according to the procedures described in a previous paper.18 Then the hydrolysate analyses were performed on a highperformance anion chromatography system (ICS3000, Dionex, Sunnyvale, CA) with pulsed amperometric detector and an ion exchange Carbopac PA-1 column (40 × 250 mm). The neutral sugars were separated in 18 mM NaOH (carbonate free and purged with nitrogen) with post column addition of 0.3 M NaOH at a rate of 0.5 mL/min. Run time was 45 min, followed by a 10-min elution with 0.2 M NaOH to wash the column and then a 15-min elution with 18 mM NaOH to reequilibrate the column. Calibration was performed with a standard solution of L-rhamnose, L-arabinose, L-glucose, L-galactose, Dmannose, and D-xylose. Measurements were conducted with two parallels, and reproducibility of the values was found within the range of 5%. The elemental composition of the pigments before and after acid hydrolysis was recorded on a Vario EL analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Infrared spectra of pigments were collected on a Thermo Scientific Nicolet iN 10 FTIR Microscopy instrument (Thermo Nicolet Corp., Madison, WI) equipped with a liquid nitrogen-cooled MCT detector. The weightaverage (Mw) and number-average (Mn) molecular weights of the pigments were determined by Agilent1200 gel permeation chromatography (Agilent, Santa Clara, CSA) on a 300 mm × 7.7 mm i.d., 10 μm, PL aquagel−OH column of the same material (Agilent, England), calibrated with PL pullulan polysaccharide standards (peak average molecular weights of 783, 12 200, 100 000, 1 600 000 g/mol, Polymer Laboratories Ltd.). 13C NMR spectra of the pigments were recorded on a Bruker AVIII 400 MHz spectrometer (Germany) instrument at 25 °C and chemical shifts were referenced to solvent signal. The preparation of 100 mg of CSP was carried out using 0.5 mL of DMSOd6 (δC 39.5). 13C NMR spectra were obtained in the pulse FT mode (100.6 MHz). A 30° pulse angle, a 9.2-μs pulse width, 1.89-s delay time, and 1.36-s acquired time between scans were used. All chemical shifts were expressed in ppm (δC) from TMS. Physical Properties Determination. The solubility and stability studies of the pigments were based on the previous literature with minor alterations.19 The solubility in water and other common solvents (benzene, chloroform, diethyl ether, acetone, ethyl acetate, petroleum ether, hexane, methanol, ethanol, acetic acid, DMSO, and alkaline solutions) was investigated by adding 0.05 g of the pigments in 5 mL of solvents with stirring at 25 °C for 1 h, and standing for 1 h. As for the stability, 0.005 g/100 mL water/ethanol (1:1, v/v) solution was used for studying the pigment stability of temperature (25, 50, 75, and 100 °C), light (natural light, ultraviolet light, and dark), and different kinds of metal ions (Ca2+, Cu2+, Na+, Zn2+, Al3+, Mg2+, Fe3+, and K+). All absorption coefficients of samples were determined at λmax by a UV 2300 spectrophotometer (Techcomp, Shanghai, China). All the determinations were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). Analysis of variance (ANOVA) was performed by using the data analysis tools in Microsoft Excel 2007, and a least significant difference (LSD) test was used to determine the differences of means. P values less than 0.05 were considered to be statistically significant. Antioxidant Activity of the Pigments. The pigments were reacted with a stable free radical, 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), to evaluate antioxidant activity.20 The DPPH radical scavenging capacity was estimated using the reported method.21 The antioxidant activity was expressed as a percentage of reduction of the

promising method for natural compounds extraction, there are still some limitations (security, appropriate reactors, and craft determination, et al.) for its industrial applications. Therefore, further fundamental study on natural compounds extraction by MAE method is needed for large scale production. In the present work, water-soluble pigments were quickly obtained from C. mollissima shells by water/ethanol (1:1, v/v) with microwave-assisted extraction, and purified by a two-step precipitation for the first time. The chemical composition and physical properties of the new pigments were subsequently determined by a combination method of spectroscopic techniques (UV, FT-IR, 13C NMR), elemental analysis, and chromatographic techniques (HPAEC, GPC). Furthermore, the physical properties, DPPH radical scavenging capacity, antimicrobial activity, and the acute oral toxicity test were also investigated. The comprehensive and systematic knowledge of the new pigments from C. mollissima shells will help to maximize the exploitation of these agricultural residues for possible use in food additives production.

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MATERIALS AND METHODS

Materials. Ten kg of C. mollissima fruit was collected from Qianxi, Hebei province, China. After peeling, about 1.47 kg of C. mollissima shells was obtained. The shells were washed extensively with distilled water. After drying at 55 °C for 16 h in an oven, the shells were smashed in an FZ120 plant shredder (Truelab, Shanghai, China), then sieved to 20−40 mesh. The resulting powder was stored in sealed bags (under N2 atmosphere) at room temperature. All chemicals used were of analytical grade and purchased from Sigma Chemical Co. (Beijing, China). Extraction and Purification of Pigments. The extraction and purification procedures of pigments are shown in Figure 1. MAE experiments were performed on an Ethos T microwave extractor (Milestone, Sorisole, Italy) with a maximum delivered power of 600 W at 70 °C for 30 min. Briefly, C. mollissima shell powder was submitted to aqueous ethanol/water (1:1, v/v) in a 2-L round-bottom flask with a solid to liquid ratio of 1:20 (g/mL) under magnetic stirring at 50% of nominal power. After centrifugation, 1500 mL of crude pigments supernatant was obtained and evaporated at 60 °C under reduced

Figure 1. Extraction and purification procedures of CSP. 1937

dx.doi.org/10.1021/jf4045414 | J. Agric. Food Chem. 2014, 62, 1936−1944


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DPPH radical: radical scavenging rate (SC%) = [1 − (absorbance of sample)/(absorbance of control)] × 100%. For comparison, the DPPH radical scavenging capacity of synthetic antioxidant butylated hydroxytoluene (BHT, 0.5 mg/mL) and butylated hydroxyanisole (BHA, 0.5 mg/mL) was also tested. The DPPH solution with no CSP added was used as control. The EC50 value (mg extract/mL), an effective concentration at which 50% radical was scavenging, was also calculated. All tests were performed in triplicate. Antimicrobial Activity of the Pigments. To evaluate the antimicrobial activity of CSP, disc-diffusion assay was carried out using 6-mm filter disc.22 Gram-negative bacteria strain Escherichia coli (CMCC (B) 44103) and gram-positive bacteria strains Streptomyces somaliensis (NBRC 12916 (T)), Staphylococcus aureus (ATCC 25923), Bacillus subtilis (CGMCC 3376), and fungus Penicillium oxalicum (CGMCC 13428), and Aspergillus japonicus (ATCC 204480) used were kindly given by the Institute of Microbiology, Beijing Forestry University, Beijing, China. After culturing, the microorganisms were adjusted with sterile saline to a concentration of 1.0 × 107 to 109 cfu/ mL. The suspension was added to the top of the agar plates and dissolved in Petri dishes (20 μL/agar plate) with solid agar. The filter paper discs with pigment solution (concentrations of 0.05, 0.10, and 0.15 g/mL, 20 μL/disc) were placed on agar plates. After 24 h of incubation for bacteria and 3−4 days of incubation for fungus at 28 °C, the diameters of the growth inhibition zones were measured. Additionally, the 5% aqueous phenol as a positive control and 50% aqueous ethanol and water as negative controls were used. All tests were done in triplicate. Acute Oral Toxicity Test of the Pigments. The acute toxicity of the pigments was determined according to the U.S. Food and Drug Administration (FDA) guidelines for toxicity tests IV C 2.23 Briefly, the animals were divided into four groups (two groups of male and two groups of female), and same-sex animals were housed together in groups of three at room temperature (20 ± 2 °C), 30−60% relative humidity, and 12 h light−dark cycle. Prior to testing, the animals were starved for 4 h. After fasting, 200 mg/mL of pigments suspension (single dose of 5 g/kg body weight) was administered orally to the experimental groups, while the vehicle control groups were given distilled water (10 mL/kg). Food and water were provided 2 h later. Symptoms of toxicity and mortality were observed for 14 days after the administration. On day 14, all mice were sacrificed and subjected to necropsies. Kunming mice (17.5−20.0 g) used in the tests were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) and allowed to acclimate for 1 week prior to beginning experiment. All procedures used in this experiment were compliant with the local ethics committee.

Table 2. Elemental Composition and Molar Ratio of CSP elemental composition (wt%) a

P1 P2b a

Table 1. Average Neutral Sugar Contents (%) (± 3%)a of CSP ara gal glu xyl total a

average (%)

4.35 1.69 16.23 0.99 23.26

4.23 1.75 15.58 0.38 21.94

4.29 1.72 15.9 0.68 22.6

O

N

N/C

H/C

O/C

46.00 56.15

5.43 4.17

47.62 39.68

0.95 0

0.018 0

1.42 0.89

0.78 0.53

The original CSP. bCSP after acid hydrolysis.

chestnut shells. A two-step (by 95% ethanol, and water subsequently) precipitation method was first applied to remove hemicelluloses (0.3645 g/75 g dry weight) and lignin (0.6437 g/75 g) from the pigments. After purification, the total yield of pigments was 3.8625 g/75 g, which was a little higher than traditional extraction for 36 h by aqueous alkaline (Fr. 3, a yield of 2.7 g/100 g from chestnut shells), but much lower than that from tea leaves under the same condition (about 29%).5,25 This phenomenon might be the result of severe purification and the high level of lignification of chestnut shells. The sugar analysis of CSP was applied to estimate the associated neutral sugars. It is well-known that pigments are readily bound to proteins, polysaccharides, and lipids. To figure out the associated polysaccharides, CSP was hydrolyzed by acid and analyzed through HPAEC. The results of sugar analysis are listed in Table 1. Results showed that the total amount of the neutral sugars in CSP was 22.6%. The sugars in the isolated pigments demonstrated a predominance of glucose (70.35%), followed by arabinose, galactose, and xylose. This suggested that the main associated monosaccharide in CSP was glucose and it might attach to pigments to some extent. Elemental Composition. The average elemental compositions for the pigments before (P1) and after (P2) acid hydrolysis are presented in Table 2. As noted, the original elemental composition of P1 was C = 46.00%, N = 0.95%, H = 5.43%, O = 47.62%, and the C element content increased while N element was in too trace a concentration to be detected after acid hydrolysis (P2). The H/C ratio (1.42) in P1 was high, which reflected a relatively high content of aliphatic chains. The low content of N element and N/C ratio in P1 suggested that the content of bounding protein was low and was comparable with the deproteinated samples extracted by alkaline.5 In addition, a high ratio of O/C showed a high degree of oxygen substitution in the pigments, especially the bounding glycosides. This was in line with the sugar analysis aforementioned. After acid hydrolysis, both H/C and O/C ratios were decreased, suggesting that the linked sugars were almost removed during the hydrolysis. As the ether bonds in

RESULTS AND DISCUSSION Yield and Sugar Analysis. Conventionally, pigments can be obtained by extracting for several hours or overnight using

CSP-2 (%)

H

Figure 2. Actual gel permeation chromatograms of CSP.

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CSP-1 (%)

molar ratio

C

Pooled standard error.

hot water, bases, acids, or some organic solvents. Compared with traditional extractive methods, MAE has been considered as an efficient and rapid process to extract pigments or antioxidants from plants with higher yield.24 In this study, the polar ethanol/water (1:1, v/v) and the temperature of 70 °C accelerated the mass transfer of water-soluble pigments from 1938



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Figure 3. FT-IR spectrum of CSP.

Figure 4. 13C NMR spectrum (in DMSO-d6) of CSP.

FT-IR Analysis. FT-IR spectroscopy provides information on the main functional groups in the pigment structure. The FT-IR spectrum of the pigments is illustrated in Figure 3. As shown, a broad band at 3450 cm−1, which is attributed to stretching vibrations of phenolic OH group, was evidently distinguished. The peak at 1442 cm−1 was indicative of a strong aliphatic character.26 The 1607 cm−1 band was assigned to aromatic CC stretching. The 1047 cm−1 band attributing to C−O stretching of polysaccharides suggested a certain amount of glycosidic bonds in the pigments. Moreover, the absorption peak at 1280 cm−1 represents bending vibration of hydroxyl.27 The results of FT-IR spectrum demonstrated a certain amount of aliphatic chains in the pigments structure, accompanying OH

compounds are apt to cleave by acid, this probably inferred that the associated carbohydrates were attached to pigments via ether bonds. Furthermore, the disappearance of N element in P2 suggested the extracted pigment was lacking chlorophyll a, betacyanin, phycocyanobilin, or rubropunctamine, which are relatively rich in N element.12 Molecular Weight Distributions. The values of the weight-average (Mw) and number-average (Mn) molecular weights, estimated from GPC curves, and polydispersity indexes (Mw/Mn, PI) of the pigments extracted from chestnut’s shells are shown in Figure 2. The Mw of pigments from chestnut shells was 1240 g/mol with a PI of 8.25, indicating an inhomogeneous molecular weight distribution of CSP. 1939



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Table 3. Main Assignmentsa of CSP in the 13C NMR Spectra Shown in Figure 4 chemical shift (ppm) 156.8 154.9 144.8 131.2 117.9 115.3 104.2 102.1 98.1 97 91.9 82.6/82.0 81.1 79.9 77.3 76.8 75.9 75.5 74.5 73/72.9 71.8 70.7 70.4 70 63.8 63.2 62.3 61.3 60.7 a

assignment C-5 in procyanidin B3; C-2 in querctin-3-O-glycoside C-7 in procyanidin B3 C-3′ in procyanidin B3; C-3′ in querctin-3-O-glycoside C-1′ in procyanidin B3 C-8 in flavanol C-2′ of the H ring of flavanol moiety in flavylium; C-2′ in querctin-3-O-glycoside gl-1 in luteolin-5-O-glycoside and querctin-3-O-glycoside C-4a in procyanidin B3 C-8 in luteolin-5-O-glycoside; C-6 in kaempferol and quercetin C-6 in procyanidin B3 flavonoid glycoside C-2 in procyanidin B3 C-17 in 5β-steroidal sapogenins C-2′ in phenolic glycoside gl-5 in querctin-3-O-glycoside gl-3 in querctin-3-O-glycoside C-4 in arabinoxylans C-2 in β-glucose gl-2 in querctin-3-O-glycoside C-3 in procyanidin B3 flavonoid glycoside C-4 in β-glucose C-3,4 in α-galactose C-4 in α,β-xylose C-5 in the arabinoxylans C-17 in 5β-steroidal sapogenins C-5 in xylose C-6 in β-glucose flavonoid glycoside

Signals were assigned by comparison with the literature.20,23,36−40

Figure 5. Main components of CSP, involving procyanidin B3, flavanol, kaempferol, quercetin, luteolin, and steroidal sapogenins identified by 13C NMR.

and C−O groups. This was supported by the data of the elemental analysis. 13 C NMR Analysis. Application of 13C NMR technique allows quick qualitative determination of different components in pigments. To further investigate the structural features and the main components in pigments, the pigments were analyzed by solution-state 13C NMR. The 13C NMR spectrum of the pigments is shown in Figure 4. The spectrum can be divided into aliphatic (δC 20−90) and aromatic (δC 90−160) regions.28 The main signals in the 13C NMR spectrum were assigned by comparison with the published literatures, and are listed in Table 3; the main components are depicted in Figure 5.28−35 The upfield signals ranging from δC 20 to 90 were due to the aliphatic carbons in pigments, which mainly resulted from the associated sugars and some micromolecule substances. As can be seen, Figure 4 apparently showed the spectral characteristic of sugars in this region. Based on the previous publications, the signals at δC 75.5, 76.8, 70.7, 77.3, 61.3 were assigned to carbon atoms 2, 3, 4, 5, and 6 in β-glucose, respectively.30 The signals for atoms 5 and atoms 4 of xylose were discovered at δC 62.3 and 70.0. Signals resulted from α-galactose could be detected at δC 70.4.31 Among these, signals from the β-glucose could be seen at higher contour levels, which indicates a higher amount of β-glucose in CSP. Weak signals at 102.1, 74.5, 75.9, and 63.8 ppm, which are assigned respectively to C-1, C-3, C-4, and C-5 positions of the arabinoxylan, suggested that the associated

arabinose in CSP appeared in the form of arabinoxylan. Above all, β-glucose was the most predominant in CSP, followed by similar amounts of arabinose, xylose, and galactose, which was in agreement with the results obtained by sugar analysis. In addition, some interglycosidic linkages such as phenolic glycosides were also recognized in the sugar region of the spectrum. It has been reported that two signals at δC 79.9 and 74.5 are assigned to phenolic glycosides.32 The most important glycosides found in the CSP might be flavonoids-O-glycosides, such as quercetin-3-O-glycoside, which had strong signals at δC 77.3, 76.8, 74.5, 70.7, 70.4, 68, and 61.3 in the sugar region.33 Furthermore, some micromolecule substances were also distinguished. Interestingly, flavanoid procyanidin B3 (condensed tannin) was also revealed by the signals at 82.6, 82.0, 73.0, 72.9, and 68.0 ppm in this region.28 It could be concluded that the condensed tannins in C. mollissima shells could be extracted by aqueous ethanol. Except for the flavonoids, signals at 81.1 and 63.2 ppm could be probably assigned to 5βsteroidal sapogenin.29 The aromatic region of the spectrum gave more useful information about different groups of flavonoids and some micromolecular substances present in the pigments. Most of the downfield signals at δC 156.8, 154.9, 144.8, 131.2, and 102.1, are caused by C-5, C-7, C-3′, C-1′, and C-4a positions of 1940



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the main components in CSP were flavonoids, steroidal sapogenins, and the ether linked sugars which may be flavonoid glycosides, such as quercetin-3-O-glycoside and luteolin-5-Oglycoside. Flavanol and procyanidin were observed as the major flavonoids in CSP, whereas signals of kaempferol, free quercetin, and luteolin appeared in a trace amount. Physical Properties Analysis. As the UV spectrum indicated, the absorption spectrum of the pigments exhibited strong optical absorbance in a spectral range from 270 to 290 nm, and relatively weak absorbance range from 290 to 650 nm. The wavelength of maximum absorption was 278.5 nm, which is a typical peak of flavonoids. Therefore, as the main components in pigments, the absorbance of flavonoids at 278.5 nm was selected as reference in the following experiments. Solubility of CSP. The solubility results indicated that the pigments were scarcely soluble in benzene, chloroform, diethyl ether, acetone, ethyl acetate, petroleum ether, and hexane. However, the pigments could be dissolved in methanol, ethanol, and acetic acid. Furthermore, they displayed a good solubility in water, DMSO, and alkaline solutions (such as Na2CO3, NaOH, and NH4OH). The solubility in water and DMSO may be due to the flavonids and water-soluble glycosides in CSP. Stability of CSP. The effect of temperature on the CSP stability was evaluated to investigate the potential use as an antioxidant. The absorbance of the pigment solution at λmax changed slightly from 25 to 50 °C after incubating in a thermostatically controlled bath for 3 h. On the contrary, the absorbance of CSP solution at 75 °C increased during the early incubation period, and decreased gradually to meet the initial value. This phenomenon was probably related to the thermal auto-oxidation and degradation of the flavonoids in CSP.36,37 Unfortunately, the CSP was extremely unstable at the temperature of 100 °C, which might be correlated with the polarity of the CSP.37 These results suggested that the pigments were stable when the temperature was below 75 °C. To study the effect of light on CSP, the pigment solutions were performed under ultraviolet light, indoor natural light, and stored in a dark place for 1−10 d. Only 0.03 and 0.01 of absorbance were increased under natural light and dark after 10 days’ treatment, respectively. However, the absorbance increased first and then decreased with the increasing irradiation time when the solution was irradiated under ultraviolet-light. After being treated for 3 days, about 0.07 of the absorbance was increased. These results indicated that the pigments were stable to the indoor natural light and under dark condition, while the ultraviolet light had a relatively strong effect on the light stability. This phenomenon might be explained by the degree of glucosylation in CSP, which has an important contribution to pigment stability for light.7

Figure 6. (a) Scavenging abilities of different concentrations of CSP, BHT (0.5 mg/mL), and BHA (0.5 mg/mL) on DPPH radical with increasing time. (b) DPPH radical scavenging rate of CSP with increasing concentration when the mixture was treated for 30 min. Error bars represent standard deviation of the means (n = 3); P ≤ 0.05.

the procyanidin B3, respectively.28 As for quercetin-3-Oglycoside, signals in this region were detected at δC 156.8, 144.8, 115.3, 104.2, 100.8, and 98.1. In addition, the luteolin-5O-glycoside showed a prominent signal for glucose carbon atom 1 at δC 104.2. A weak signal at 98.1 ppm may originate from C-8 of luteolin-5-O-glycoside, C-6 of kaempfero,l and quercetin. Moreover, the signal at δC 117.9 was attributed to C8 of flavonol.34 In short, the 13C NMR spectrum indicated that

Table 4. Antibacterial Activity of CSP by Diameter of Inhibition Zones (mm) CSP bacteria E. coli S. somaliensis S. aureus B. subtilis a

1 mg/disc 6.12 10.60 6.57 9.96

± ± ± ±

0.03 0.10 0.18 0.15

2 mg/disc 7.21 12.82 6.94 11.62

± ± ± ±

0.08 0.27 0.02 0.05

3 mg/disc 7.20 13.68 7.02 13.46

± ± ± ±

0.05 0.24 0.06 0.22

phenol

50% aqueous ethanol

sterile water

1 mg/disc

20 μL/disc

20 μL/disc

± ± ± ±

6.60 ± 0.04 n.a. 8.68 ± 0.11 7.78 ± 0.14

n.a.a n.a. n.a. n.a.

7.50 7.15 7.59 9.63

0.22 0.06 0.09 0.19

n.a. The pigment was not active at the tested concentration. 1941



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Figure 7. Results of antibacterial activity against Gram-positive bacteria strain Streptomyces somaliensis and Bacillus subtilis of CSP in disc-diffusion method. (1) a: 1 mg/disc CSP; b: 1 mg/disc phenol; c: 50% aqueous ethanol; d: sterile water. (2) a: 2 mg/disc CSP; b: 1 mg/disc phenol; c: 50% aqueous ethanol; d: sterile water. (3) a: 3 mg/disc CSP; b: 1 mg/disc phenol; c: 50% aqueous ethanol; d: sterile water.

Antimicrobial Activity of CSP. With respect to the antimicrobial activity of CSP, the disc-diffusion method was used to test the diameter of inhibition zones effect on bacteria and fungus and the results are listed in Table 4. As shown, the pigments (1, 2, and 3 mg/disc) indicated an increased antibacterial activity with the increasing pigments content per disc. However, the pigments were not sensitive to the fungus (P. oxalicum and A. japonicus), which exhibited considerable antibacterial activity, especially the Gram-positive bacteria. This was in agreement with the antimicrobial activities of extracts from some other plants.22 The highest inhibition zones could be obtained for S. somaliensis, which may refer to pathogens,42 ranging from 10.60 to 13.68 mm. The pigment solution was active against B. subtilis as well, with inhibition zones of 9.96− 13.46 mm (Figure 7). Moreover, the lowest inhibition zones (6.12−7.50 mm) were recorded for E. coli, while S. aureus possessed almost the same activity, with inhibition zones 6.57− 7.59 mm. The antimicrobial potential of pigments tested in the discdiffusion method can be presented as S. somaliensis > B. subtilis > S. aureus > E. coli > fungus. Compared with the commercial antibiotic phenol, it was remarkable that the pigments showed stronger antibacterial potential toward S. somaliensis, while almost the same with regard to B. subtilis. The excellent antibacterial activity of CSP might be a good proof of the presence of the natural antioxidants procyanidin and quercetin3-O-glycoside. The interesting results display the eminent antibacterial activity of CSP, which will broaden the potential applications of CSP as food additives. Acute Toxicity Effect of CSP. Acute toxicity of CSP was assessed at a maximum dose of 5.0 g/kg of body weight. No treatment-related mortality, body weights, or clinical signs of toxicity including hair loss, scabbing, soft or mucoid feces,

With regard to metal ions stability, it was apparent that the absorbance of the pigment solution diminished sharply when mixed with Zn2+ solution for 48 h, whereas the pigment solutions mixed with the other metal ions (Ca2+, Cu2+, Na+, Zn2+, Al3+, Mg2+, Fe3+, and K+) had no obvious change. Therefore, most of the metal ions aforementioned affected the pigments slightly. DPPH Radical Scavenging Capacity of CSP. DPPH is a stable free radical, gives rise to the deep violet solution in ethanol, and has a strong absorption at 515 nm. When the solution is added with an antioxidant, the free radical of DPPH can be paired and reduce the amount of the free radical, resulting in a decrease in the absorbance of DPPH at 515 nm.38 The scavenging ability of CSP, BHT, and BHA on DPPH radical is shown in Figure 6. As illustrated, the scavenging capacity of the pigments was increased with the increasing reaction time (Figure 6a) and concentration (Figure 6b). Compared with the synthetic antioxidant BHT, the CSP had a strong DPPH radical scavenging capacity, and was closed to BHA. At 0.2−1.2 mg/mL, CSP showed scavenging abilities of 22.6−72.6% on DPPH radicals in 60 min (Figure 6a), which was better than that of pigments from the seeds of Osmanthus f ragrant.21 Moreover, the EC50 value was 0.057 mg/mL (Figure 6b), which was significantly lower than that of polysaccharides isolated from Ganoderma tsugae and comparable to that of the flavonoid glycosides from Daphniphyllum calycinum, indicating a higher antioxidant activity of CSP.39,40 The significant DPPH radical scavenging capacity of CSP may reflect a certain amount of substances with an available OH group,41 such as tannins and procyanidin, which was verified by FT-IR and 13C NMR spectra. These results implied that the 50% ethanol extracted and a two-step precipitation purified pigments from C. mollissima shells processed excellent DPPH scavenge capacity. 1942



Article

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decreased defecation or feces smaller than normal, wet yellow material in urogenital area, or vocalization upon handling, were observed for 14 days following the oral administration of CSP. All animals gained weight and appeared active and normal. After sacrifice on the 14th day, macroscopic and gross pathology observations conducted at the necropsy examination revealed no visible lesions in any animals. Relative weight of organs in mice of control and experimental groups did not show significant difference. These phenomena indicated that the mice could tolerate maximum dose at 5.0 g/kg of body weight of CSP, and the LD50 should be greater than 5.0 g/kg in both male and female mice according to the guidance for the safety assessment of botanicals and botanical preparations for use in food and food supplements.43 Based on the toxicity classification proposed by Loomis and Hayes, substances with LD50 between 5000 and 15 000 mg/kg body weight are regarded as being practically nontoxic.44 The present results suggested that CSP could be regarded as being practically nontoxic, which might be related to the quercetin-3-O-glycoside in CSP.45 In summary, the water-soluble pigments from C. mollissima shells extracted by 50% ethanol with microwave assisted were purified by a two-step precipitation method for the first time. The comprehensive and systematic evaluation of the pigments provided valuable results in relation to the chemical composition and characteristics of these natural pigments. It demonstrated a predominance of flavonoids (procyanidin B3) and ether-linked sugars (quercetin-3-O-glycoside), followed by a small amount of steroidal sapogenins. As a consequence, CSP was water-soluble, practically nontoxic, and exhibited wonderful DPPH radical scavenging ability and antibacterial activity. With respect to physical properties, CSP revealed good stability with temperature, natural light, and metal ions. However, CSP was not resistant to UV light. The detailed study of pigments from C. mollissima shells will shed light on the utilization of these agricultural residues.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-62336387. Fax: +86-10-62336903. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from National Science Foundation for Distinguished Young Scholars of China (31225005), National Science and Technology Program of the Twelfth Five-Year Plan Period (2012BAD32B06), and Chinese Ministry of Education (113014A).

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REFERENCES

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Chemical Composition, Properties, and Antimicrobial Activity of the

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