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A novel process for isolation and purification of the bioactive polysaccharide TLH-3′ from Tricholoma lobayense
Process Biochemistry 50 (2015) 1146–1151
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
A novel process for isolation and purification of the bioactive polysaccharide TLH-3 from Tricholoma lobayense Liu Liu 1 , Yongming Lu 1 , Xuehui Li, Liyuan Zhou, Dan Yang, Liming Wang, Yan Chen ∗ School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei 230601, Anhui, PR China
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 10 April 2015 Accepted 12 April 2015 Available online 28 April 2015 Keywords: Tricholoma lobayense Heim Polysaccharides High-pressure homogenization Quaternary ammonium salt precipitation Ultrafiltration Antioxidant activity
a b s t r a c t Our previous studies have revealed that the Tricholoma lobayense polysaccharide TLH-3, which has an IC50 value for scavenging superoxide radicals comparable to that of Vitamin C (VC), could be prepared by hot water extraction and column chromatography. However, this method is tedious and inefficient. In this study, high-pressure homogenization (HPH) was used to extract polysaccharides from T. lobayense Heim. The yield of 17.3% using HPH was higher than the 12.3% yielded by traditional extraction. Moreover, the operation combined quaternary ammonium salt precipitation with ultrafiltration (QASP-UF) to purify the crude polysaccharides successfully, which provided a higher TLH-3 yield, a larger separation volume, and a shorter purification time than that of column chromatography. The whole process for TLH3 preparation presented here exhibited significant advantages of quicker processing, low consumption, high efficiency, and flexible compatibility with follow-up studies. Based on a characterization of molecular weight, Fourier Transform Infrared Spectroscopy (FT-IR) analysis, and monosaccharide composition analysis, it can be concluded that TLH-3 and TLH-3 are the same polysaccharide. Antioxidant activity was tested to prove that the TLH-3 prepared in this study could scavenge DPPH and superoxide radicals, with IC50 values of 111.7 g/mL and 160.0 g/mL, respectively. Its outstanding antioxidant ability was comparable to that of ascorbic acid. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Edible fungi, which are widely distributed in China and Japan [1] and are famous for their abundant nutritive and medicines value [2], have long been cultivated and studied. The fungi have been widely used as folk medicines and healthy foods [3]. Natural polysaccharides found in the edible fungi have attracted increasing attention due to their multiple bioactivities, pharmacological activities, and safety values [4]. Tricholoma lobayense Heim is a nutritious and valuable precious edible fungus under development for its pharmacological potential. Proteoglycans from T. lobayense Heim have been proven to exhibit excellent immune regulation and anti-tumor activities
Abbreviations: DPPH, 1,1-diphenl-2-picryldydrazyl; ELSD, evaporative light scattering detector; FT-IR, Fourier transform IR spectroscopy; CTAB, hexadecyl trimethyl ammonium bromide; HPLC, high-performance liquid chromatography; HPH, high-pressure homogenization; MWCO, molecular weight cut off; PMP, 1phenyl-3-methyl-5-pyrazolone; QASP, quaternary ammonium salt precipitation; TFA, trifluoroacetic acid; UF, ultrafiltration. ∗ Corresponding author. Tel.: +86 55163861751. E-mail address: [email protected] (Y. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.procbio.2015.04.011 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
[5]. Our previous study of in vitro antioxidant activities revealed that TLH-3 had the strongest antioxidant activity amongst the T. lobayense polysaccharides, with an IC50 value of 126 g/mL which is comparable to that of ascorbic acid [6]. TLH-3 has shown great potential for applications in various fields such as natural non-toxic anti-oxidants, foods, pharmaceuticals and cosmetics. However, a tedious process is usually needed to ultimately produce the polysaccharide. Hot water extraction has traditionally been used for the extraction of crude polysaccharides. Further purification of the polysaccharides can be accomplished by column chromatography [7]. Li et al. [8] demonstrated that Ganoderma capense polysaccharides could be extracted by boiled water and purified by column chromatography (DEAE Sepharose CL-6B). Hot water extraction at 60 ◦ C has also been used for Pleurotus cornucopiae polysaccharide extraction, with subsequent purification by column chromatography (DEAE-52 cellulose) [9]. Our previous study also showed that TLH-3 could be successfully prepared by hot water extraction and column chromatography. However, this method consumes a high level of starting material, involves an relatively long extraction time, and has a low extraction yield [10]. The development of a feasible, efficient, economic and easily handled protocol for separating bioactive TLH-3 is still greatly demanded.
L. Liu et al. / Process Biochemistry 50 (2015) 1146–1151
The major objective of this research was to isolate TLH-3 quickly and efficiently. In this study, according to a preliminary study on the characterization of the polysaccharides TLH-1, TLH-2 and TLH-3, new technologies such as the high-pressure homogenization (HPH) method and ultrafiltration (UF) were used to isolate the polysaccharide TLH-3 . Such a protocol not only made the extraction simple and efficient but also retained the bioactivities of the extracted polysaccharides. The polysaccharide TLH-3 is expected to exhibit excellent activity for a wide range of applications in numerous fields.
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Ion-change chromatography and gel filtration column chromatography were also used for the purification of the polysaccharide [15]. DEAE ion-exchange was first used to obtain TLH-1 and TLH-S. The column was first eluted with distilled water, followed by elution with a linear concentration gradient of NaCl from 0 to 1 mol/L. Further separation was performed to obtain TLH-2 and TLH-3 from polysaccharide TLH-S by gel Superdex-75 column chromatography (2.6 × 50 cm), eluted with deionized water at a flow rate of 0.5 mL/min. 2.4. Measurement of molecular weights of TLH-3 and TLH-3
2. Materials and methods 2.1. Materials T. lobayense Heim was obtained from the Panji Shenshan edible fungus cooperative (Huainan, Anhui Province, China). 1,1-Diphenl-2-picryldydrazyl (DPPH), vitamin C (VC), gallic acid and hexadecyl trimethyl ammonium bromide (CTAB) were obtained from China National Medicines Corporation, Ltd. All other chemicals and solvents used in this study were analytical grade. 2.2. Extraction of polysaccharides by HPH and conventional extraction The HPH [11] equipment was obtained from AH-PILOT (ATS Engineering Inc., Canada). The stalks of T. lobayense were dried to a constant weight, then crushed over an 80 mesh sieve. The dried T. lobayense powder (100 g) was mixed with 3500 mL of distilled water, and extracted two times by HPH at 80 MPa. Then, the supernatant was evaporated in vacuo, concentrated, and precipitated with 4 equivalents of ethanol [12]. The resulted precipitates were collected, dissolved in distilled water, and subjected to the Sevag method to remove protein [13]. After the protein was completely removed, the supernatant was concentrated, dialyzed, and lyophilized. The yield of the T. lobayense polysaccharide extract by HPH was found to be 17.3 ± 0.2%. The total saccharide content was 99.2%. The UV spectrum of the extract showed no absorption at 260 and 280 nm for nucleic acid and protein, respectively. Conventional extraction was carried out according to a previously reported method [14]. The dried T. lobayense powder (100 g) was mixed with 3500 mL of water at 83 ◦ C and incubated for 2.5 h. After centrifugation the supernatant was concentrated by evaporation in vacuo, precipitated by ethanol, re-dissolved, subjected to protein removal, dialyzed, and freeze-dried to obtain T. lobayense polysaccharides. The yield of T. lobayense polysaccharides by hot water extraction was found to be 12.3 ± 0.2%. The total saccharide content of the extract was 98.5%. The UV spectrum of the extract showed no absorption at 260 and 280 nm, suggesting no nucleic acid or protein content. 2.3. Isolation and purification of TLH-3 by QASP-UF and column chromatography The dried polysaccharide extract (100 g) was dissolved in 5000 mL distilled water, to which 5000 mL 10% hexadecyl trimethyl ammonium bromide (CTAB) solution was then added. After incubating the solution overnight, the precipitate was collected and re-dissolved with 10% NaCl. Then, 3 to 4 equivalents of ethanol were added to precipitate polysaccharides from the solution. The residue was lyophilized in a freeze-dryer to yield T. lobayense polysaccharides (TLH-S ). Finally, the dried TLH-S polysaccharides were dissolved and diluted to 2 mg/mL, which was then used for further purification of TLH-3 by ultrafiltration membranes with a MWCO of 10 kDa. The resulting solution was concentrated, dialyzed, and lyophilized to obtain TLH-3 powder.
High Performance Liquid Chromatography (HPLC) was used for determining the molecular weight of TLH-3 and TLH-3 [16]. Standard dextrans 2000, T-10, T-40, T-70 and T-100 were detected by ELSD-HPLC, and retention times were plotted against the logarithms of their respective molecular weights. TLH-3 and TLH-3 polysaccharides (4 mg each) were separately dissolved in distilled water (2 mL) and centrifuged. Then, each supernatant was subjected to ELSD–HPLC and eluted at a fixed flow rate (1 mL/min) by distilled water. The retention times of the polysaccharides were then plotted on the same graph as the standards, and their molecular weights could be determined. 2.5. Infrared spectroscopic analysis The IR spectra of the polysaccharides were determined using Fourier Transform IR Spectrophotometry (FT-IR). The purified polysaccharides were ground with KBr powder and then pressed into pellets for FT-IR measurement in the frequency range of 4000–400 cm−1 . 2.6. Analysis of monosaccharide composition The monosaccharide compositions of TLH-3 and TLH-3 were analyzed by HPLC. Each polysaccharide (10 mg) was dissolved in 5 mL 2 mol/L trifluoroacetic acid (TFA) at 110 ◦ C in a sealed-tube for 8 h. The resultant solution was concentrated using a rotary vacuum evaporator at 50 ◦ C and excess acid was removed until the obtained hydrolysate was neutral. After that the hydrolysate was added 1 mL distilled water, at which point it was considered ready for the following experiments. PMP derivatization of monosaccharides was carried out according to the method of Lv et al. [17] with proper modifications. Briefly, standard monosaccharides and the hydrolyzed samples were mixed with 50 L of a methanol solution of 1-phenyl-3methyl-5-pyrazolone (PMP, 0.5 mol/L) and 50 L NaOH(0.3 mol/L), and incubated at 70 ◦ C for 30 min. Then, the reaction mixture was neutralized with 50 L of 0.3 mol/L HCl. The resulting products were analyzed using HPLC coupled with UV detection. The operation conditions for the HPLC were as follows: the analytical column was an Agilent ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm, particle size 5 m). The flow rate, column temperature, and wavelength for UV detection were 1.0 mL/min, 25 ◦ C, and 245 nm, respectively. The mobile phase A was a mixture of phosphate buffer (50 mmol/L) – acetonitrile (85:15, v/v) and the mobile phase B was a mixture of phosphate buffer (50 mmol/L) – acetonitrile (60:40, v/v). The analysis was using a gradient elution of 15–23–15% phase B by a linear from 0–20–35 min. 2.7. Antioxidant activity 2.7.1. Assay of DPPH radical scavenging DPPH scavenging activity was measured according to the method of Yao et al. [18] with some modifications. TLH-3 and TLH-3
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solutions (2 mL) with different concentrations of the polysaccharides (50 g/mL–500 g/mL) were added to 2.5 mL of DPPH solution (1.3 × 10−4 mol/L). The reaction mixture was shaken vigorously and incubated for 30 min at room temperature. The absorbance was measured at 517 nm using a spectrophotometer (METASH, UV 5100B, Shanghai). VC was used as a positive control. The DPPH scavenging effect was calculated as follows: Scavenging effect =
1−
A1 A0
× 100%
where A0 is the absorbance value of the DPPH solution without sample and A1 is the absorbance value of a tested sample. 2.7.2. Assay of superoxide radical (O2 − ) scavenging Superoxide radical scavenging was measured according to the method of Wang et al. [6] with minor modifications. Briefly, 4.0 mL of Tris–HCl buffer (50 mM, PH 8.2), 4.2 mL of H2 O and 1 mL of sample at different concentrations (50 g/mL–500 g/mL) were incubated at 25 ◦ C for 20 min. Then, 0.3 mL of pyrogallol was added to the mixture, and the reaction was incubated at 25 ◦ C for 4 min. Finally, the absorbance of each mixture were measured at 320 nm using a spectrophotometer (METASH, UV 5100B, Shanghai). The scavenging effect on superoxide radical was calculated as follows:
Scavenging effect =
1−
A1 A0
× 100%
where A0 is the absorbance value of control without sample and A1 is the absorbance value of a tested sample. 2.8. Statistical analysis All experiments were performed at least three times with similar results obtained. Statistical analysis was conducted using the Statistical Product and Service Solutions (SPSS) software. Differences at P < 0.05 were considered significant.
3. Results and discussion 3.1. Extraction of polysaccharides Conventional extraction techniques such as hot water extraction have been widely used to extract polysaccharides. However, such techniques have some drawbacks, such as the long extraction times required, low extraction rates and high operating temperatures. In this study, HPH instead of hot water extraction (Fig. 1) was adopted for the extraction of polysaccharides. As shown in Table 1, the operating temperature for the hot water extraction was 83 ◦ C, whereas the temperature for HPH extraction was only 4 ◦ C. Therefore, energy consumption could be reduced with HPH. Moreover, the extraction time was greatly shortened from 2.5 h to 0.5 h using HPH. Clearly, the HPH technique significantly reduces extraction time and is therefore suitable for follow-up studies and large-scale production. In terms of extraction yield, cell walls could be broken easily and completely with the aid of HPH, and bioactive components could fully be released from the cells [19]. As a result, an increase in the extraction yield of the polysaccharides could be expected. As shown in Table 1, the extraction yields by HPH and the conventional method were 17.3% and 12.3%, respectively; a 41% increase in extraction yield was achieved by utilizing HPH. Thus, HPH shows a great potential for efficiently extracting bioactive polysaccharides. 3.2. Separation and purification of TLH-3 Our previous study found that T. lobayense polysaccharides consisted of three main components, the neutral polysaccharide TLH-1 and the acidic polysaccharides TLH-S (TLH-2, TLH-3). The molecular weights of these polysaccharides were 8.75 × 105 Da, 5.85 × 105 Da and 4.22 × 103 Da, respectively [6]. According to the acid–base properties of polysaccharides and the fact that quaternary ammonium salts can form insoluble precipitates with acidic polysaccharides, QASP instead of DEAE ion-exchange was utilized for separating TLH-1 from the crude polysaccharides; the resulting precipitates were TLH-S’ polysaccharides (Fig. 1b). Furthermore,
Fig. 1. Traditional and novel flow charts for the purification of TLH-3 (a) and TLH-3 (b) from T. lobayense.
L. Liu et al. / Process Biochemistry 50 (2015) 1146–1151 Table 1 Comparison of parameters for extraction of polysaccharides by the conventional method and high-pressure homogenization (HPH).
Extraction yield (%) Extraction time (h) Extraction temperature (◦ C)
Conventional method
HPH
12.3 2.5 83.0
17.3 0.5 4.0
Table 2 Comparison of effectiveness of purification of polysaccharides by column chromatography and quaternary ammonium salt precipitation with ultrafiltration (QASP-UF).
Cost ($) Amount of starting sample (g) Time (d) TLH-3 yield (%)
Column chromatography
QASP-UF
2000 0.01 8 3
350 100 2 5
taking into account the fact that the molecular weights of TLH-2 and TLH-3 are considerably different, UF instead of Superdex-75 chromatography could be used to separate and purify TLH-3 easily (Fig. 1b). A comparison of the effectiveness of purification of polysaccharides by QASP-UF and column chromatography is shown in Table 2. First, the price of the gel used in column chromatography was high. In addition, the time for required for separation of TLH-3 was up to 8 days, and the limit to the amount of starting sample that could be separated by column chromatography at a time was only 0.01 g. By utilizing QASP-UF, more than 100 g of a sample could be handled at a time, and the purification time could be reduced to 2
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days. Finally, the extraction yield of TLH-3 by QASP-UF was 66.7% higher than that by column chromatography. 3.3. Measurement of molecular weight of TLH-3 and TLH-3 The purities and molecular weights of TLH-3 and TLH-3 were determined by HPLC. As shown in Fig. 2, TLH-3 and TLH-3 both showed a single and symmetrical peak, indicating their homogeneity. The results of the ELSD-HPLC analysis showed that the average molecular weights of TLH-3 and TLH-3 were estimated to be 4.24 × 103 Da (Fig. 2a) and 4.23 × 103 Da (Fig. 2b), respectively. 3.4. Infrared spectroscopic analysis FT-IR has been widely used for the characterization of polysaccharides. The FT-IR spectra of TLH-3 and TLH-3 are shown in Fig. 3. Typical signals were clearly presented at 3394, 2931, 1643,1413 and 1037 cm−1 for TLH-3 and TLH-3 . A strong and broad absorption peak at 3394 cm−1 could be assigned for the stretching vibration of O H, and another weak absorption peak at 2931 cm−1 could attributed to the stretching vibration of C H. The band at 1643 cm−1 was attributed to the bending vibration of O H. Absorption at 1413 cm−1 was attributed to C H deformation vibration. The absorptions at 1037 cm−1 indicate a pyranose form of sugars. 3.5. Analysis of monosaccharide composition The monosaccharide compositions of TLH-3 and TLH3 are shown in Fig. 4. The polysaccharides were mainly composed of seven monosaccharides, and the molar ratio of monosaccharide compositions was basically the same between the two. The monosaccharide proportions in TLH-3
Fig. 2. Profiles of TLH-3 (a) and TLH-3 (b) in HPLC with Evaporative Light Scattering Detector (ELSD). Experimental conditions: the analytical column, flow rate, column temperature, and flow-rate were TSK gel G4000 PWXL (300 × 7.8 mm), 1.0 mL/min, 35 ◦ C, and distilled water, respectively.
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monosaccharide composition analysis, it can be concluded that TLH-3 and TLH-3 are the same polysaccharide.
3.6. Antioxidant activity
Fig. 3. IR spectra of the purified TLH-3 and TLH-3 .
and TLH-3 were rhamnose:mannose:glucuronic acid:galacturonic acid:glucose:galactose:arabinose = 0.05:0.16:0.03:0.02:1.58:1: 0.13 (Fig. 4a) and 0.07:0.23:0.02:0.02:1.57:1:0.11 (Fig. 4b), respectively. From the characterization of molecular weights, FT-IR, and
Different concentrations of TLH-3 and TLH-3 were tested for their DPPH radical scavenging activities, and the results are presented in Fig. 5a. The DPPH radical scavenging activities of TLH-3 and TLH-3 were both concentration dependent within the range of concentrations from 50 to 500 g/mL. The IC50 values of VC, TLH3 and TLH-3 were measured to be 113.3 g/mL, 123.5 g/mL and 111.7 g/mL, respectively. The scavenging activities of TLH-3 and TLH-3 were very close to that of VC. Superoxide radical scavenging activities of TLH-3 and TLH-3 were also tested, and the results are shown in Fig. 5b. The superoxide radical scavenging activities of TLH-3 and TLH-3 also varied in a concentration-dependent manner within the range of concentrations from 50 to 500 g/mL. The IC50 values of VC, TLH-3 and TLH-3 were measured to be 141.9 g/mL, 159.4 g/mL and 160.0 g/mL, respectively. The results demonstrate that TLH-3 and TLH-3 have appreciable scavenging activities on superoxide radicals. The antioxidant activity study shows that TLH-3 prepared by a traditional method and TLH-3 prepared by HPH and QASP-UF maintain their activities well throughout the purification process. HPH followed by QASP-UF treatment has the unique advantages of
Fig. 4. Monosaccharide compositions of TLH-3 (a) and TLH-3 (b). Experimental conditions: the analytical column, flow rate, column temperature, and wavelength for UV detection were Agilent ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm, particle size 5 m), 1.0 mL/min, 25 ◦ C, and 245 nm, respectively. (The asterisk indicates solvent peak, 1—rhamnose, 2—mannose, 3—glucuronic acid, 4—galacturonic acid, 5—glucose, 6—galactose, 7—arabinose).
L. Liu et al. / Process Biochemistry 50 (2015) 1146–1151
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Fig. 5. Scavenging effect of TLH-3, TLH-3 and VC at different concentrations on DPPH radicals (a) and superoxide radicals (b); values represent the mean ± SD, n = 5. VC abbreviation stands for Vitamin C as a positive control for antioxidant activity.
being able to efficiently extract polysaccharides with high bioactivities.
[6]
4. Conclusions [7]
In this paper, HPH technology was adopted to extract T. lobayense polysaccharides, and QASP-UF was used to separate and purify TLH-3 from the T. lobayense polysaccharides. The employed extraction protocol was of easily operated and highly efficient, which makes it more suitable for industrial application. Further study on the antioxidant activity of the polysaccharide (in terms of DPPH radical scavenging and superoxide radical (O2 − ) scavenging) shows that TLH-3 has activity for oxidation resistance comparable with that of VC. This work paves a new way forward for the large-scale and efficient extraction of bioactive polysaccharides. Acknowledgments We gratefully acknowledge the financial support for this study from the National Natural Science Foundation of China (31271817) and the Key Project of Science and Technology of Anhui (1501031099). References [1] Zhang M, Cui SW, Cheung PCK, Wang Q. Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci Technol 2007;18:4–19. [2] Wang HX, Ng TB, Ooi VEC. A protein with inhibitory activity on cell-free translation from cultured mycelia of the edible mushroom Tricholoma lobayense. Comp Biochem Physiol, B: Biochem Mol Biol 2000;125:247–53. [3] Kim BH, Choi D, Piao YL, Park S-S, Lee MK, Cha W-S, et al. Comparative study on the antioxidant and nitrite scavenging activity of fruiting body and mycelium extract from Pleurotus ferulae. Korean J Chem Eng 2012;29:1393–402. [4] Chen H, Wang Z, Qu Z, Fu L, Dong P, Zhang X. Physicochemical characterization and antioxidant activity of a polysaccharide isolated from oolong tea. Eur Food Res Technol 2009;229:629–35. [5] Liu F, Ooi VEC, Liu WK, Chang ST. Immunomodulation and antitumor activity of polysaccharide-protein complex from the culture filtrates of a local
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Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
A novel process for isolation and purification of the bioactive polysaccharide TLH-3 from Tricholoma lobayense Liu Liu 1 , Yongming Lu 1 , Xuehui Li, Liyuan Zhou, Dan Yang, Liming Wang, Yan Chen ∗ School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei 230601, Anhui, PR China
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 10 April 2015 Accepted 12 April 2015 Available online 28 April 2015 Keywords: Tricholoma lobayense Heim Polysaccharides High-pressure homogenization Quaternary ammonium salt precipitation Ultrafiltration Antioxidant activity
a b s t r a c t Our previous studies have revealed that the Tricholoma lobayense polysaccharide TLH-3, which has an IC50 value for scavenging superoxide radicals comparable to that of Vitamin C (VC), could be prepared by hot water extraction and column chromatography. However, this method is tedious and inefficient. In this study, high-pressure homogenization (HPH) was used to extract polysaccharides from T. lobayense Heim. The yield of 17.3% using HPH was higher than the 12.3% yielded by traditional extraction. Moreover, the operation combined quaternary ammonium salt precipitation with ultrafiltration (QASP-UF) to purify the crude polysaccharides successfully, which provided a higher TLH-3 yield, a larger separation volume, and a shorter purification time than that of column chromatography. The whole process for TLH3 preparation presented here exhibited significant advantages of quicker processing, low consumption, high efficiency, and flexible compatibility with follow-up studies. Based on a characterization of molecular weight, Fourier Transform Infrared Spectroscopy (FT-IR) analysis, and monosaccharide composition analysis, it can be concluded that TLH-3 and TLH-3 are the same polysaccharide. Antioxidant activity was tested to prove that the TLH-3 prepared in this study could scavenge DPPH and superoxide radicals, with IC50 values of 111.7 g/mL and 160.0 g/mL, respectively. Its outstanding antioxidant ability was comparable to that of ascorbic acid. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Edible fungi, which are widely distributed in China and Japan [1] and are famous for their abundant nutritive and medicines value [2], have long been cultivated and studied. The fungi have been widely used as folk medicines and healthy foods [3]. Natural polysaccharides found in the edible fungi have attracted increasing attention due to their multiple bioactivities, pharmacological activities, and safety values [4]. Tricholoma lobayense Heim is a nutritious and valuable precious edible fungus under development for its pharmacological potential. Proteoglycans from T. lobayense Heim have been proven to exhibit excellent immune regulation and anti-tumor activities
Abbreviations: DPPH, 1,1-diphenl-2-picryldydrazyl; ELSD, evaporative light scattering detector; FT-IR, Fourier transform IR spectroscopy; CTAB, hexadecyl trimethyl ammonium bromide; HPLC, high-performance liquid chromatography; HPH, high-pressure homogenization; MWCO, molecular weight cut off; PMP, 1phenyl-3-methyl-5-pyrazolone; QASP, quaternary ammonium salt precipitation; TFA, trifluoroacetic acid; UF, ultrafiltration. ∗ Corresponding author. Tel.: +86 55163861751. E-mail address: [email protected] (Y. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.procbio.2015.04.011 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
[5]. Our previous study of in vitro antioxidant activities revealed that TLH-3 had the strongest antioxidant activity amongst the T. lobayense polysaccharides, with an IC50 value of 126 g/mL which is comparable to that of ascorbic acid [6]. TLH-3 has shown great potential for applications in various fields such as natural non-toxic anti-oxidants, foods, pharmaceuticals and cosmetics. However, a tedious process is usually needed to ultimately produce the polysaccharide. Hot water extraction has traditionally been used for the extraction of crude polysaccharides. Further purification of the polysaccharides can be accomplished by column chromatography [7]. Li et al. [8] demonstrated that Ganoderma capense polysaccharides could be extracted by boiled water and purified by column chromatography (DEAE Sepharose CL-6B). Hot water extraction at 60 ◦ C has also been used for Pleurotus cornucopiae polysaccharide extraction, with subsequent purification by column chromatography (DEAE-52 cellulose) [9]. Our previous study also showed that TLH-3 could be successfully prepared by hot water extraction and column chromatography. However, this method consumes a high level of starting material, involves an relatively long extraction time, and has a low extraction yield [10]. The development of a feasible, efficient, economic and easily handled protocol for separating bioactive TLH-3 is still greatly demanded.
L. Liu et al. / Process Biochemistry 50 (2015) 1146–1151
The major objective of this research was to isolate TLH-3 quickly and efficiently. In this study, according to a preliminary study on the characterization of the polysaccharides TLH-1, TLH-2 and TLH-3, new technologies such as the high-pressure homogenization (HPH) method and ultrafiltration (UF) were used to isolate the polysaccharide TLH-3 . Such a protocol not only made the extraction simple and efficient but also retained the bioactivities of the extracted polysaccharides. The polysaccharide TLH-3 is expected to exhibit excellent activity for a wide range of applications in numerous fields.
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Ion-change chromatography and gel filtration column chromatography were also used for the purification of the polysaccharide [15]. DEAE ion-exchange was first used to obtain TLH-1 and TLH-S. The column was first eluted with distilled water, followed by elution with a linear concentration gradient of NaCl from 0 to 1 mol/L. Further separation was performed to obtain TLH-2 and TLH-3 from polysaccharide TLH-S by gel Superdex-75 column chromatography (2.6 × 50 cm), eluted with deionized water at a flow rate of 0.5 mL/min. 2.4. Measurement of molecular weights of TLH-3 and TLH-3
2. Materials and methods 2.1. Materials T. lobayense Heim was obtained from the Panji Shenshan edible fungus cooperative (Huainan, Anhui Province, China). 1,1-Diphenl-2-picryldydrazyl (DPPH), vitamin C (VC), gallic acid and hexadecyl trimethyl ammonium bromide (CTAB) were obtained from China National Medicines Corporation, Ltd. All other chemicals and solvents used in this study were analytical grade. 2.2. Extraction of polysaccharides by HPH and conventional extraction The HPH [11] equipment was obtained from AH-PILOT (ATS Engineering Inc., Canada). The stalks of T. lobayense were dried to a constant weight, then crushed over an 80 mesh sieve. The dried T. lobayense powder (100 g) was mixed with 3500 mL of distilled water, and extracted two times by HPH at 80 MPa. Then, the supernatant was evaporated in vacuo, concentrated, and precipitated with 4 equivalents of ethanol [12]. The resulted precipitates were collected, dissolved in distilled water, and subjected to the Sevag method to remove protein [13]. After the protein was completely removed, the supernatant was concentrated, dialyzed, and lyophilized. The yield of the T. lobayense polysaccharide extract by HPH was found to be 17.3 ± 0.2%. The total saccharide content was 99.2%. The UV spectrum of the extract showed no absorption at 260 and 280 nm for nucleic acid and protein, respectively. Conventional extraction was carried out according to a previously reported method [14]. The dried T. lobayense powder (100 g) was mixed with 3500 mL of water at 83 ◦ C and incubated for 2.5 h. After centrifugation the supernatant was concentrated by evaporation in vacuo, precipitated by ethanol, re-dissolved, subjected to protein removal, dialyzed, and freeze-dried to obtain T. lobayense polysaccharides. The yield of T. lobayense polysaccharides by hot water extraction was found to be 12.3 ± 0.2%. The total saccharide content of the extract was 98.5%. The UV spectrum of the extract showed no absorption at 260 and 280 nm, suggesting no nucleic acid or protein content. 2.3. Isolation and purification of TLH-3 by QASP-UF and column chromatography The dried polysaccharide extract (100 g) was dissolved in 5000 mL distilled water, to which 5000 mL 10% hexadecyl trimethyl ammonium bromide (CTAB) solution was then added. After incubating the solution overnight, the precipitate was collected and re-dissolved with 10% NaCl. Then, 3 to 4 equivalents of ethanol were added to precipitate polysaccharides from the solution. The residue was lyophilized in a freeze-dryer to yield T. lobayense polysaccharides (TLH-S ). Finally, the dried TLH-S polysaccharides were dissolved and diluted to 2 mg/mL, which was then used for further purification of TLH-3 by ultrafiltration membranes with a MWCO of 10 kDa. The resulting solution was concentrated, dialyzed, and lyophilized to obtain TLH-3 powder.
High Performance Liquid Chromatography (HPLC) was used for determining the molecular weight of TLH-3 and TLH-3 [16]. Standard dextrans 2000, T-10, T-40, T-70 and T-100 were detected by ELSD-HPLC, and retention times were plotted against the logarithms of their respective molecular weights. TLH-3 and TLH-3 polysaccharides (4 mg each) were separately dissolved in distilled water (2 mL) and centrifuged. Then, each supernatant was subjected to ELSD–HPLC and eluted at a fixed flow rate (1 mL/min) by distilled water. The retention times of the polysaccharides were then plotted on the same graph as the standards, and their molecular weights could be determined. 2.5. Infrared spectroscopic analysis The IR spectra of the polysaccharides were determined using Fourier Transform IR Spectrophotometry (FT-IR). The purified polysaccharides were ground with KBr powder and then pressed into pellets for FT-IR measurement in the frequency range of 4000–400 cm−1 . 2.6. Analysis of monosaccharide composition The monosaccharide compositions of TLH-3 and TLH-3 were analyzed by HPLC. Each polysaccharide (10 mg) was dissolved in 5 mL 2 mol/L trifluoroacetic acid (TFA) at 110 ◦ C in a sealed-tube for 8 h. The resultant solution was concentrated using a rotary vacuum evaporator at 50 ◦ C and excess acid was removed until the obtained hydrolysate was neutral. After that the hydrolysate was added 1 mL distilled water, at which point it was considered ready for the following experiments. PMP derivatization of monosaccharides was carried out according to the method of Lv et al. [17] with proper modifications. Briefly, standard monosaccharides and the hydrolyzed samples were mixed with 50 L of a methanol solution of 1-phenyl-3methyl-5-pyrazolone (PMP, 0.5 mol/L) and 50 L NaOH(0.3 mol/L), and incubated at 70 ◦ C for 30 min. Then, the reaction mixture was neutralized with 50 L of 0.3 mol/L HCl. The resulting products were analyzed using HPLC coupled with UV detection. The operation conditions for the HPLC were as follows: the analytical column was an Agilent ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm, particle size 5 m). The flow rate, column temperature, and wavelength for UV detection were 1.0 mL/min, 25 ◦ C, and 245 nm, respectively. The mobile phase A was a mixture of phosphate buffer (50 mmol/L) – acetonitrile (85:15, v/v) and the mobile phase B was a mixture of phosphate buffer (50 mmol/L) – acetonitrile (60:40, v/v). The analysis was using a gradient elution of 15–23–15% phase B by a linear from 0–20–35 min. 2.7. Antioxidant activity 2.7.1. Assay of DPPH radical scavenging DPPH scavenging activity was measured according to the method of Yao et al. [18] with some modifications. TLH-3 and TLH-3
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solutions (2 mL) with different concentrations of the polysaccharides (50 g/mL–500 g/mL) were added to 2.5 mL of DPPH solution (1.3 × 10−4 mol/L). The reaction mixture was shaken vigorously and incubated for 30 min at room temperature. The absorbance was measured at 517 nm using a spectrophotometer (METASH, UV 5100B, Shanghai). VC was used as a positive control. The DPPH scavenging effect was calculated as follows: Scavenging effect =
1−
A1 A0
× 100%
where A0 is the absorbance value of the DPPH solution without sample and A1 is the absorbance value of a tested sample. 2.7.2. Assay of superoxide radical (O2 − ) scavenging Superoxide radical scavenging was measured according to the method of Wang et al. [6] with minor modifications. Briefly, 4.0 mL of Tris–HCl buffer (50 mM, PH 8.2), 4.2 mL of H2 O and 1 mL of sample at different concentrations (50 g/mL–500 g/mL) were incubated at 25 ◦ C for 20 min. Then, 0.3 mL of pyrogallol was added to the mixture, and the reaction was incubated at 25 ◦ C for 4 min. Finally, the absorbance of each mixture were measured at 320 nm using a spectrophotometer (METASH, UV 5100B, Shanghai). The scavenging effect on superoxide radical was calculated as follows:
Scavenging effect =
1−
A1 A0
× 100%
where A0 is the absorbance value of control without sample and A1 is the absorbance value of a tested sample. 2.8. Statistical analysis All experiments were performed at least three times with similar results obtained. Statistical analysis was conducted using the Statistical Product and Service Solutions (SPSS) software. Differences at P < 0.05 were considered significant.
3. Results and discussion 3.1. Extraction of polysaccharides Conventional extraction techniques such as hot water extraction have been widely used to extract polysaccharides. However, such techniques have some drawbacks, such as the long extraction times required, low extraction rates and high operating temperatures. In this study, HPH instead of hot water extraction (Fig. 1) was adopted for the extraction of polysaccharides. As shown in Table 1, the operating temperature for the hot water extraction was 83 ◦ C, whereas the temperature for HPH extraction was only 4 ◦ C. Therefore, energy consumption could be reduced with HPH. Moreover, the extraction time was greatly shortened from 2.5 h to 0.5 h using HPH. Clearly, the HPH technique significantly reduces extraction time and is therefore suitable for follow-up studies and large-scale production. In terms of extraction yield, cell walls could be broken easily and completely with the aid of HPH, and bioactive components could fully be released from the cells [19]. As a result, an increase in the extraction yield of the polysaccharides could be expected. As shown in Table 1, the extraction yields by HPH and the conventional method were 17.3% and 12.3%, respectively; a 41% increase in extraction yield was achieved by utilizing HPH. Thus, HPH shows a great potential for efficiently extracting bioactive polysaccharides. 3.2. Separation and purification of TLH-3 Our previous study found that T. lobayense polysaccharides consisted of three main components, the neutral polysaccharide TLH-1 and the acidic polysaccharides TLH-S (TLH-2, TLH-3). The molecular weights of these polysaccharides were 8.75 × 105 Da, 5.85 × 105 Da and 4.22 × 103 Da, respectively [6]. According to the acid–base properties of polysaccharides and the fact that quaternary ammonium salts can form insoluble precipitates with acidic polysaccharides, QASP instead of DEAE ion-exchange was utilized for separating TLH-1 from the crude polysaccharides; the resulting precipitates were TLH-S’ polysaccharides (Fig. 1b). Furthermore,
Fig. 1. Traditional and novel flow charts for the purification of TLH-3 (a) and TLH-3 (b) from T. lobayense.
L. Liu et al. / Process Biochemistry 50 (2015) 1146–1151 Table 1 Comparison of parameters for extraction of polysaccharides by the conventional method and high-pressure homogenization (HPH).
Extraction yield (%) Extraction time (h) Extraction temperature (◦ C)
Conventional method
HPH
12.3 2.5 83.0
17.3 0.5 4.0
Table 2 Comparison of effectiveness of purification of polysaccharides by column chromatography and quaternary ammonium salt precipitation with ultrafiltration (QASP-UF).
Cost ($) Amount of starting sample (g) Time (d) TLH-3 yield (%)
Column chromatography
QASP-UF
2000 0.01 8 3
350 100 2 5
taking into account the fact that the molecular weights of TLH-2 and TLH-3 are considerably different, UF instead of Superdex-75 chromatography could be used to separate and purify TLH-3 easily (Fig. 1b). A comparison of the effectiveness of purification of polysaccharides by QASP-UF and column chromatography is shown in Table 2. First, the price of the gel used in column chromatography was high. In addition, the time for required for separation of TLH-3 was up to 8 days, and the limit to the amount of starting sample that could be separated by column chromatography at a time was only 0.01 g. By utilizing QASP-UF, more than 100 g of a sample could be handled at a time, and the purification time could be reduced to 2
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days. Finally, the extraction yield of TLH-3 by QASP-UF was 66.7% higher than that by column chromatography. 3.3. Measurement of molecular weight of TLH-3 and TLH-3 The purities and molecular weights of TLH-3 and TLH-3 were determined by HPLC. As shown in Fig. 2, TLH-3 and TLH-3 both showed a single and symmetrical peak, indicating their homogeneity. The results of the ELSD-HPLC analysis showed that the average molecular weights of TLH-3 and TLH-3 were estimated to be 4.24 × 103 Da (Fig. 2a) and 4.23 × 103 Da (Fig. 2b), respectively. 3.4. Infrared spectroscopic analysis FT-IR has been widely used for the characterization of polysaccharides. The FT-IR spectra of TLH-3 and TLH-3 are shown in Fig. 3. Typical signals were clearly presented at 3394, 2931, 1643,1413 and 1037 cm−1 for TLH-3 and TLH-3 . A strong and broad absorption peak at 3394 cm−1 could be assigned for the stretching vibration of O H, and another weak absorption peak at 2931 cm−1 could attributed to the stretching vibration of C H. The band at 1643 cm−1 was attributed to the bending vibration of O H. Absorption at 1413 cm−1 was attributed to C H deformation vibration. The absorptions at 1037 cm−1 indicate a pyranose form of sugars. 3.5. Analysis of monosaccharide composition The monosaccharide compositions of TLH-3 and TLH3 are shown in Fig. 4. The polysaccharides were mainly composed of seven monosaccharides, and the molar ratio of monosaccharide compositions was basically the same between the two. The monosaccharide proportions in TLH-3
Fig. 2. Profiles of TLH-3 (a) and TLH-3 (b) in HPLC with Evaporative Light Scattering Detector (ELSD). Experimental conditions: the analytical column, flow rate, column temperature, and flow-rate were TSK gel G4000 PWXL (300 × 7.8 mm), 1.0 mL/min, 35 ◦ C, and distilled water, respectively.
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monosaccharide composition analysis, it can be concluded that TLH-3 and TLH-3 are the same polysaccharide.
3.6. Antioxidant activity
Fig. 3. IR spectra of the purified TLH-3 and TLH-3 .
and TLH-3 were rhamnose:mannose:glucuronic acid:galacturonic acid:glucose:galactose:arabinose = 0.05:0.16:0.03:0.02:1.58:1: 0.13 (Fig. 4a) and 0.07:0.23:0.02:0.02:1.57:1:0.11 (Fig. 4b), respectively. From the characterization of molecular weights, FT-IR, and
Different concentrations of TLH-3 and TLH-3 were tested for their DPPH radical scavenging activities, and the results are presented in Fig. 5a. The DPPH radical scavenging activities of TLH-3 and TLH-3 were both concentration dependent within the range of concentrations from 50 to 500 g/mL. The IC50 values of VC, TLH3 and TLH-3 were measured to be 113.3 g/mL, 123.5 g/mL and 111.7 g/mL, respectively. The scavenging activities of TLH-3 and TLH-3 were very close to that of VC. Superoxide radical scavenging activities of TLH-3 and TLH-3 were also tested, and the results are shown in Fig. 5b. The superoxide radical scavenging activities of TLH-3 and TLH-3 also varied in a concentration-dependent manner within the range of concentrations from 50 to 500 g/mL. The IC50 values of VC, TLH-3 and TLH-3 were measured to be 141.9 g/mL, 159.4 g/mL and 160.0 g/mL, respectively. The results demonstrate that TLH-3 and TLH-3 have appreciable scavenging activities on superoxide radicals. The antioxidant activity study shows that TLH-3 prepared by a traditional method and TLH-3 prepared by HPH and QASP-UF maintain their activities well throughout the purification process. HPH followed by QASP-UF treatment has the unique advantages of
Fig. 4. Monosaccharide compositions of TLH-3 (a) and TLH-3 (b). Experimental conditions: the analytical column, flow rate, column temperature, and wavelength for UV detection were Agilent ZORBAX Eclipse XDB-C18 column (4.6 × 150 mm, particle size 5 m), 1.0 mL/min, 25 ◦ C, and 245 nm, respectively. (The asterisk indicates solvent peak, 1—rhamnose, 2—mannose, 3—glucuronic acid, 4—galacturonic acid, 5—glucose, 6—galactose, 7—arabinose).
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Fig. 5. Scavenging effect of TLH-3, TLH-3 and VC at different concentrations on DPPH radicals (a) and superoxide radicals (b); values represent the mean ± SD, n = 5. VC abbreviation stands for Vitamin C as a positive control for antioxidant activity.
being able to efficiently extract polysaccharides with high bioactivities.
[6]
4. Conclusions [7]
In this paper, HPH technology was adopted to extract T. lobayense polysaccharides, and QASP-UF was used to separate and purify TLH-3 from the T. lobayense polysaccharides. The employed extraction protocol was of easily operated and highly efficient, which makes it more suitable for industrial application. Further study on the antioxidant activity of the polysaccharide (in terms of DPPH radical scavenging and superoxide radical (O2 − ) scavenging) shows that TLH-3 has activity for oxidation resistance comparable with that of VC. This work paves a new way forward for the large-scale and efficient extraction of bioactive polysaccharides. Acknowledgments We gratefully acknowledge the financial support for this study from the National Natural Science Foundation of China (31271817) and the Key Project of Science and Technology of Anhui (1501031099). References [1] Zhang M, Cui SW, Cheung PCK, Wang Q. Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci Technol 2007;18:4–19. [2] Wang HX, Ng TB, Ooi VEC. A protein with inhibitory activity on cell-free translation from cultured mycelia of the edible mushroom Tricholoma lobayense. Comp Biochem Physiol, B: Biochem Mol Biol 2000;125:247–53. [3] Kim BH, Choi D, Piao YL, Park S-S, Lee MK, Cha W-S, et al. Comparative study on the antioxidant and nitrite scavenging activity of fruiting body and mycelium extract from Pleurotus ferulae. Korean J Chem Eng 2012;29:1393–402. [4] Chen H, Wang Z, Qu Z, Fu L, Dong P, Zhang X. Physicochemical characterization and antioxidant activity of a polysaccharide isolated from oolong tea. Eur Food Res Technol 2009;229:629–35. [5] Liu F, Ooi VEC, Liu WK, Chang ST. Immunomodulation and antitumor activity of polysaccharide-protein complex from the culture filtrates of a local
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