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Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome
Accepted Manuscript Title: Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome Author: Jie Hu Xuejing Jia Xiaobin Fang Peng Li Chengwei He Meiwan Chen PII: DOI: Reference:
S0141-8130(15)30235-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.12.046 BIOMAC 5642
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
14-9-2015 24-11-2015 14-12-2015
Please cite this article as: Jie Hu, Xuejing Jia, Xiaobin Fang, Peng Li, Chengwei He, Meiwan Chen, Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2015.12.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome Jie Hu#, Xuejing Jia#, Xiaobin Fang, Peng Li, Chengwei He* [email protected], Meiwan Chen* [email protected] State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China *Corresponding author at: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, N22-7038, Avenida da Universidade,Taipa, Macao, China. Tel: +853-88228516; Fax: +853-28841358 (Chengwei He, Ph.D.); State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, N22-6012, Avenida da Universidade, Taipa, Macao, China. Tel.: +853-88224675; Fax: +853-28841358 (Meiwan Chen, Ph.D.) #
These authors contributed equally to the work.
1
Highlights
Three novel polysaccharides were obtained from L. chuanxiong rhizome.
The extraction condition was optimized using orthogonal experimental design.
The polysaccharides showed potent in vitro antioxidant and anticancer activities.
2
Abstract Ultrasonic-assisted extraction technology was employed to prepare Ligusticum chuanxiong Hort polysaccharide. Single factor test and orthogonal experimental design were used to optimize the extraction conditions. The results showed that the optimal extraction conditions consisted of ultrasonic temperature of 80°C, ultrasonic time of 40 min and water to raw material ratio of 30 mL/g. Three novel polysaccharides fractions, LCX0, LCX1 and LCX2, were isolated and purified from the crude polysaccharides using DEAE-52 cellulose and Sephadex G-100 column chromatography. The molecular weight and monosaccharide composition of three LCX polysaccharides fractions were analyzed with gel permeation chromatography (GPC) and HPLC analysis, respectively. Furthermore, the antioxidant and in vitro anticancer activities of the polysaccharides were investigated. Compared with LCX0, LCX2 and LCX1 showed relative higher antioxidant activity and inhibitory activity to the growth of HepG2, SMMC7721, A549 and HCT-116 cells. It is suggested that the novel polysaccharides from rhizome of L. chuanxiong could be promising bioactive macromolecules for biomedical use.
Keywords:
Polysaccharides;
Ligusticum
chuanxiong;
Ultrasonic-assisted
extraction;
Antioxidant; Anticancer.
3
1 Introduction Ligusticum chuanxiong Hort (LCX), a well-known traditional Chinese herb medicine, has been widely used as an analgesic agent for centuries in the treatment of headache, traumatic, swelling pain, menstrual disorders and rheumatic diseases [1, 2]. More than 200 compounds, including essential oils, alkaloid, phenolic acids, actones, and other constituents, have been isolated and identified from LCX [3, 4]. A large number of pharmacological studies during the last decades had demonstrated the enormous medicinal potentials of LCX, including antioxidation [5], anti-inflammation [6] and neuroprotection [7], cardiovascular and cerebrovascular effects [8], anticancer and antineoplastic activities [9, 10]. All these studies strongly support the view that LCX has multiple beneficial therapeutic properties and is an effective therapeutic agent.
Researches on the polysaccharides from LCX had gained increasing attention over the past few years. Fan et al. reported the extraction and purification polysaccharides of LCX. Four homogeneous fractions were obtained and showed strong scavenging effect against O2•· and ·OH [11, 12]. Recently, studies on different extraction methods for polysaccharide from LCX had been developed, such as ultrasonic-assisted extraction [13, 14], microwave-assisted extraction [15], the traditional hot water extraction [16] and enzymatic-assisted extraction [17]. Yuan et al. obtained three purified polysaccharides (LCA, LCB and LCC) from LCX using boiling water extraction and ethanol precipitation [18]. In their work, the monosaccharide composition, molecular weight and the main skeleton of LCX polysaccharides were determined. Meanwhile, their antioxidant and antiproliferative activities were also investigated. All purified polysaccharides fractions exhibited potential antioxidant and anticancer activities. Among them, LCB showed the strongest
4
antioxidant and anticancer activity. Similar results were also reported that one purified polysaccharide fraction was isolated from the rhizome of LCX using high-pressure ultrasound-assisted extraction. Single-factor tests and response surface methodology were employed to optimize extraction conditions. Antioxidant activities of this polysaccharide fraction was further evaluated against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide radicals in vitro [19].
According to these publications, it was obvious that researches on polysaccharides of LCX in the last ten years was scattered, especially in monosaccharide composition, molecular weight, and bioactivities. Thus, in this work, we reported the optimal conditions for the ultrasonic-assisted extraction, isolation and purification of novel polysaccharides from the rhizome of LCX, determined their preliminary structural features, and antioxidant and anticancer activities in vitro.
2 Materials and methods 2.1 Materials, reagents and equipment
The rhizome powder of L. chuanxiong was purchased from the Chengdu traditional Chinese medicine market (Chengdu, Sichuan, China). DEAE-52 cellulose and Sephadex G-100 gel were all purchased from Solarbio Bioscience & Technology Co., Ltd (Shanghai, China). Monosaccharides standards, including mannose, rhamnose, galacturonic acid, glucose, galactose and
arabinose,
were
obtained
1-phenyl-3-methyl-5-pyrazolone
from (PMP),
Aladdin
Co.,
trifluoroacetic
Ltd acid
(Shanghai,
China).
(TFA),
3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl
5
(DPPH) and Vitamin C (Vc) were obtained from Sigma Co., Ltd (St. Louis, MO, USA). Ferric reducing antioxidant power (FRAP) and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay kit were purchased from Beyotime Institute of Biotechnology Co., Ltd (Shanghai, China). All reagents were of analytical grade, except for acetonitrile, which was of HPLC grade. Water was supplied by Milli-Q Plus system (Millipore, Bedford, USA). A Branson 8510 ultrasonic generator (Branson Ultrasonics Corporation, Danbury, CT) was used for extraction. A Flexstation Ⅲ Multi-Mode Microplate Reader (Molecular Devices) was used for absorbance determination. Infrared was recorded on spectrum 100 FT-IR spectrometer.
2.2 Extraction methods
One gram of dried rhizome powder of LCX was refluxed with 50 mL of 80% ethanol for 1 h to remove lipids. After that, the filtered residues were placed in a 50 mL tube and added with corresponding volume of water. Then the residues were ultrasonically extracted at different extraction conditions. After extraction, the supernatant was separated by filtration. Then it was condensed by rotatory evaporator and ethanol was slowly added to the condensed solution. After 12h at 4 °C, the resulting precipitate was collected and it was redissolve to remove free proteins by Sevag method. Last the crude polysaccharides were obtained after drying for 12h at 50 °C. The total polysaccharide yield was determined by the method of phenol-sulphuric acid method using glucose as the standard reference [20].
2.3 Orthogonal experiment design
Single-factor test was employed to determine the preliminary range of the extraction variables,
6
namely, ultrasonic temperature (X1), ultrasonic time (X2), and water to raw material ratios (X3). Then a three-factor, three-level orthogonal extracting test was applied to optimize the extraction condition. As shown in Table 1, different factors and levels for orthogonal test were designed based on the results of single-factor experiments. Table 2 represented the experimental variables and 9 experimental points.
2.4 Isolation and purification of polysaccharides of LCX
The crude polysaccharide of LCX (0.5 g) was reconstituted in 35 mL distilled water, and the supernatant was collected by centrifugation. Then the supernatant was applied to a DEAE-52 cellulose chromatography column (5.5 × 40 cm) and eluted with different concentrations of NaCl solution (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mol/L). Fractions eluted with 0, 0.1 and 0.2 mol/L NaCl were collected, dialyzed, and lyophilized. These three polysaccharides fractions were then denoted as LCX0, LCX1 and LCX2 and further purified by gel-filtration (2.0 × 100 cm) on Sephadex G-100 column using distilled water as the eluant at a flow rate of 0.4 mL/min.
2.5 Monosaccharide composition of purified polysaccharides fractions
2.5.1 Hydrolysis of polysaccharides
The polysaccharide samples (10 mg), LCX0, LCX1 and LCX2, were dissolved with 2 mol/L trifluoroacetic acid (10 mL) in a sealed flask, respectively. The flask was placed in an oven at 110 o
C for 3 h under nitrogen atmosphere. After being cooled to room temperature, the flask was
opened and methanol was added into it. Then the reaction mixture was evaporated to dryness under a reduced pressure. After that, the same amount of methanol was added and dried again by 7
the same method above, and the procedure was repeated thrice for trifluoroacetic acid to be removed. The dried hydrolyzed polysaccharide samples were dissolved in 2 mL of water for subsequent derivatization.
2.5.2 Derivatization of monosaccharides with PMP
The procedure employed for the PMP derivatization of monosaccharides was carried out as described by Honda et al. with slight modifications [21]. Briefly, 100 μL of hydrolysed polysaccharides (5 mg/mL) or monosaccharide standards were transferred into a tube, then 0.3 mol/L NaOH (200 μL) and 0.5 mol/L PMP-methanol solution (200 μL) were added and mixed. The mixture was allowed to react for 90 min at 70 °C, then cooled to room temperature and neutralized with 200 μL 0.3 mol/L HCl. After that, chloroform was added, and the mixture was shaken vigorously. The organic layer was carefully discarded, and the extraction process was repeated thrice. Finally, the aqueous layer was filtered through a 0.45 μm membrane for HPLC analysis.
2.5.3 HPLC analysis
The analysis of PMP-labeled monosaccharides were carried out on Waters e2695 HPLC system equipped with a reverse phase C18 column (250×4.6 mm) as previous method [22]. The wavelength for UV detection was 245 nm. The mobile phase consisted of 0.1 mol/L phosphate buffer (pH 6.7) with (A) 83% and (B) 17% acetonitrile, at a flow rate of 1 mL/min.
2.6 GPC analysis
8
The molecular weight distributions of the purified fractions were analyzed using gel permeation chromatography (GPC) on connecting GPC columns: TSK gel G4000PWXL (Tosoh CO., Tokyo, Japan). Milli-Q water was selected as mobile phase, and the flow rate was set at 0.5 mL/min. Purified polysaccharide fractions was dissolved in distilled water at the concentration of 10 mg/mL, respectively, and 100 μL of sample was injected each time. GPC peaks were detected with a refractive index (RI) detector. The number average molecular weight (Mn), the weight average molecular weight (Mw), and the polydispersity (Mw/Mn) were calculated using molecular weight calculation software connected to the HPLC integration system. Polyethylene oxide/glycol easivials were chosen as standard materials.
2.7 Fourier transform infrared (FT-IR) spectroscopy analysis
Purified polysaccharide fractions (2 mg) and potassium bromide were mixed, grounded, and pressed into a pellet. Spectra was then recorded in the absorbance mode from 4000 cm−1 to 400 cm−1 on a Perkin-Elmer FT-IR spectrometer.
2.8 Antioxidant activities
The reducing power of purified polysaccharide fractions was determined using the method of Jayaprakasha et al. [23]. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay was performed following Shimada et al. [24]. The ABTS radical scavenging activity assay was carried out using the method of Re et al. [25]. The ferric reducing antioxidant power (FRAP) assay was measured using the method of Benzie and Strain [26].
2.9 Anticancer activities 9
In this study, HepG2 and SMMC7721 liver carcinoma cell lines, MCF-7 and MDA-MB-231 breast cancer cell lines, A2780 ovarian cancer cell line, HCT-116 colon cancer cell line, A549 lung cancer cell line, SGC-7901 gastric cancer cell line and Hela cervical cancer cell line were employed to evaluate the anticancer effect of purified polysaccharide fractions by MTT assay. Cancer cells were plated at 5000 per well in 96-well plate and were allowed to adhere and spread for 24 h. Then the cells were treated with LCX0, LCX1 and LCX2 solution at different concentrations (25 to 1000 μg/mL). The wells were then incubated for 48 h. MTT solution (10 mg/mL in PBS) was added to each well. After incubating for another 4 h, the supernatants were aspirated. The formazan crystals in each well were dissolved in 100 μL of DMSO. The amount of purple formazan was determined by measuring the absorbance at 540 nm.
2.10 Statistical analysis
All data are presented as means ± standard deviation (SD). All experiments were repeated three times.
3 Results and discussion 3.1 Effect of ultrasonic-assisted extraction conditions on the yield of LCX polysaccharides
3.1.1 Ultrasonic temperature
The effect of different ultrasonic temperature on extraction yield of polysaccharides from the rhizome of LCX was investigated. Ultrasonic temperature was set at 40, 50, 60, 70, and 80 oC while other extraction conditions were given as followings: ultrasonic time of 20 min, water to
10
raw material ratio of 20 mL/g. As shown in Fig. 1a, extraction yield of polysaccharides from the rhizome of L. chuanxiong increased with the increasing ultrasonic temperature and reached the maximum value (10.4%) when extraction temperature was 70 oC. The extraction yield started to decrease when extraction temperature was over 80 oC. Therefore, 70 oC was chosen as the optimum temperature for extraction.
3.1.2 Ultrasonic time
To assess the influence of ultrasonic time on the extraction yield of LCX polysaccharides, the extraction process was carried out using different ultrasonic time of 5, 10, 20, 30, 40, and 50 min. The other extraction parameters were set at ultrasonic temperature 70 oC and water to raw material ratio of 30 mL/g. Fig. 1b showed that the extraction yield obviously increased when the ultrasonic time increased from 5 to 30 min, and then the yield slightly decreased from 30 to 50 min. Thus, the preferred ultrasonic time was chosen as 30 min.
3.1.3 Water to raw material ratio
Different ratios of water to raw material (5, 10, 20, 30, 40, and 50 mL/g) on the extraction yield of LCX polysaccharides was shown in Fig. 1c. The ultrasonic temperature was set at 70 oC and ultrasonic time was fixed as 30 min. When the ratio of water to raw material increased, LCX polysaccharides yield rose and reached a peak value at ratio of 40. Thus, 40 mL/g was selected as the optimum point for the water to raw material ratio in the orthogonal experiments.
3.2 Optimization for extraction of LCX polysaccharides
11
An orthogonal L9 (3)4 test was designed based on the results of single factor experiments in order to optimize the extraction of LCX polysaccharides. Three factors with three variation levels, X1 (ultrasonic temperature, 60, 70, 80 oC), X2 (ultrasonic time, 20, 30, 40 min) and X3 (water to raw material ratio, 30, 40 and 50 mL/g), were chosen for optimization (Table 1). The results and analysis of orthogonal test are presented in Table 2. From the results, the maximum extraction yield of LCX polysaccharides was 19.49%. However, it could not be considered as the best extraction conditions. Because of R2 > R1 > R3, the influence of extraction parameters decreased in the order: X2 > X1 > X3. The ultrasonic time was found to be the most important parameter in the LCX polysaccharides extraction process. The maximum yield of LCX polysaccharides was obtained when ultrasonic temperature, ultrasonic time and water to raw material ratio were 80 °C, 40 min and 30 mL/g, respectively. Through confirmatory test, the highest yield of LCX polysaccharides was 20.75 ± 1.18%.
3.3 Purification and characterization
3.3.1 Isolation and purification of LCX polysaccharides
The crude polysaccharide extracts were subsequently isolated by DEAE-52 cellulose anion-exchange column. After eluting with 0, 0.1, and 0.2 mol/L NaCl, fractions, LCX0, LCX1 and LCX2, were obtained (Fig. 2a). Then LCX0, LCX1 and LCX2 were further purified by Sephadex G-100 column (Fig. 2b, 2c and 2d). All fractions were in a single peak, suggesting that they were homogeneous polysaccharide. In addition, no absorption at 260 nm and 280 nm was detected (data not shown), indicating these fractions were absence of nucleic acid and protein.
12
3.3.2 GPC analysis
The molecular weight distributions of three purified fractions were determined by GPC. The data showed that LCX0, LCX1 and LCX2 had different molecular weights of 2620.3, 1614.7 and 3525.5 kDa (Fig. 3 and Table 3). This result further confirmed that all purified fractions were homogeneous polysaccharide. Their huge molecular weight gained our interests, for it was different from other findings. Fan et al. isolated four homogeneous polysaccharides, with the molecular weights of 31, 52, 90, and 36 kDa, respectively [11, 12]. Yuan et al. obtained three purified polysaccharides, LCA, LCB and LCC, with molecular weight of 28.3, 12.3, and 63.1 kDa, respectively [18]. Another study conducted by Sun et al., LCXP-1, LCXP-2 and LCXP-3 were isolated from rhizome of L. chuanxiong, with molecular weight of 11.9, 20.8 and 701.9 kDa [27]. From these studies, we can see that molecular weight distributions of LCX polysaccharides are different in each study. Many factors can cause this difference, including extraction and purification methods and conditions, the sources of the rhizome of L. chuanxiong, etc.
3.3.3 Monosaccharide composition analysis of LCX polysaccharides by HPLC
In the present study, different fractions of LCX polysaccharides were hydrolyzed with trifluoroacetic acid, and then their monosaccharide components were determined by PMP derivatization assay and were analyzed with HPLC analysis. The HPLC results of seven PMP-labeled monosaccharide standards were shown in Fig. 4a, and the monosaccharide compositions of LCX0, LCX1 and LCX2 were identified and presented in Fig. 4b, 4c and 4d, respectively. As shown in Fig. 4, LCX0, LCX1 and LCX2 were composed of mannose (Man), rhamnose (Rha), galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara). 13
They had the same monosaccharide composition but different ratio. LCX0 primarily consisted of Man, Rha, GalA, Glc, Gal and Ara in molar ratio of 0.013:0.034:0.07:1:0.25:0.28. LCX1 and LCX2 contained monosaccharide components of Man, Rha, GalA, Glc, Gal and Ara in molar ratio of 0.001:0.013:1:0.038:0.046:0.045 and 0.018:0.152:1:0.723:0.758:0.478, respectively. Obviously, glucose was the main monosaccharide in LCX0 while galacturonic acid was the significant predominant component in both LCX1 and LCX2. It is noteworthy that the monosaccharide composition of LCX polysaccharides is different from previous studies [18, 27]. In this study, polysaccharide fractions LCX1 and LCX2, especially LCX1, were mainly comprised of galacturonic acid, while it was not detected by Yuan et al. [18]. However, the result of Sun et al. was somewhat in agreement with our results, especially LCP-3, which contained small amount of galacturonic acid [27]. The differences between these results and our findings may be attributed to the different extraction and purification methods and conditions, and different sources of the rhizome of L. chuanxiong.
3.3.4 FT-IR analysis
The structure of LCX0, LCX1 and LCX2 were further analyzed by FT-IR (Fig. 5). From the FT-IR spectra, LCX0, LCX1 and LCX2 had similar structure. The broad and strong absorption at 3400 cm-1 was assigned to the stretching vibration of -OH groups. The peak at 2932 cm−1 was corresponding to the stretching vibration of -CH2 groups, while 1417 cm−1 was the symmetrical deformation vibration of -CH2. The absorption bands between 1000 cm−1 and 1145 cm−1 belonged to the characteristic absorption of C-O-C groups. In addition, the peaks at 1644 cm−1 and 1332 cm−1 were observed, which strongly indicating the presence of COO− groups on the LCX purified
14
polysaccharides chains. This observation further confirmed that LCX purified polysaccharides fractions contained COO− groups, which was in accordance to the monosaccharide composition analysis. The difference between LCX0, LCX1 and LCX2 was that LCX0 had an addition peak at 1726 cm−1, which might be belonged to the stretching vibration of carbonyl group.
3.4 Antioxidant activities
3.4.1 Reducing power
The reducing power serves as an important indicator to evaluate polysaccharides’ potential antioxidant activities [28]. In the present study, the K3Fe(CN)6 reduction method was employed to evaluate the antioxidant capacity of LCX purified polysaccharides fractions. The reducing power of LCX0, LCX1 and LCX2 was shown in Fig. 6a. It was clear that the reducing power of LCX1 and LCX2 increased with increasing sample concentration, while the reducing power of LCX0 was slightly affected. Their reducing power decreased in the order of LCX2, LCX1 and LCX0. The present results revealed that LCX purified polysaccharides fractions, especially LCX2, showed potently reducing power in vitro. Generally, reducing power was associated with reductones. This result suggested that LCX2 might contain more reductone-associated and hydroxide groups than LCX0 and LCX1 [29].
3.4.2 DPPH radical scavenging activity
The scavenging effect of DPPH radicals is a common antioxidant assay. Antioxidants can protect against excessive free radicals that have been implicated in the etiology of many diseases [28]. In this work, DPPH radical scavenging effects of LCX purified polysaccharides fractions were 15
measured. As shown in Fig. 6b, LCX1 and LCX2 showed dose-dependent scavenging effect, but they were weaker than that of Vitamin C in each concentration. The IC50 of LCX2 and LCX1 were estimated at 2.95 mg/mL and above 8 mg/mL, respectively. However, LCX0 showed no DPPH radical scavenging activity even at 8 mg/mL. The difference of their DPPH scavenging ability may be contributed to their monosaccharide composition, content of hydroxy and carboxy groups, and hydrogen donation ability [30].
3.4.3 ABTS radical scavenging activity
ABTS radical scavenging activity is also commonly used to determine antioxidant activity of a compound. In this study, the scavenging abilities of different LCX purified polysaccharides fractions on ABTS radicals were measured. The results were shown in Fig. 6c. The ABTS radical scavenging activities of LCX0, LCX1 and LCX2 increased with their concentrations, from 0.0625 to 8.0 mg/mL. Similar to the results of reducing power and DPPH radical scavenging activity, LCX2 exhibited the strongest scavenging ability. The IC50 of LCX2 was 0.57 mg/mL, which was significantly higher than that of LCX1 and LCX0 (IC50 = 2.57 mg/mL and above 8.0mg/mL, respectively). However, even the scavenging effect of LCX2 increased obviously with increasing concentration, it remained lower than that of Vitamin C at each dosage.
3.4.4 Ferric reducing antioxidant power (FRAP)
FRAP assay is also generally used to evaluate total antioxidant capacity of polysaccharides. The antioxidant activities of different LCX purified polysaccharides fractions are evaluated via the ability of reducing the Fe3+-TPTZ complex to the Fe2+-TPTZ complex [31]. The results of LCX2,
16
LCX1 and LCX0 were shown in Fig. 6d. The FRAP values of LCX2 was higher than those of LCX1 and LCX0 at each concentration. At the concentration of 8.0 mg/mL, the FRAP values of LCX2, LCX1 and LCX0 were 0.71, 0.28 and 0.11 mmol/L, respectively. This result suggested that three LCX purified polysaccharides fractions possessed potently antioxidant abilities, especially LCX2. However, compared with Vitamin C, their antioxidant capacities were weak.
3.5 Anticancer activities
It had been reported that polysaccharides played a certain role in anticancer activity [32]. In this study, MTT assay was employed to evaluate the anticancer effect of three fractions on different cancer cell lines. The cells were treated with various concentrations of LCX0, LCX1 and LCX2 for 48 h. As shown in Fig. 7, all three fractions exhibited concentration-dependent inhibition activities to HepG2, SMMC7721, A549 and HCT-116 cells. Compared with those of A549 and HCT-116 cells, there was a significant increase of inhibitory rate for HepG2 and SMMC7721 cells. This result was similar to the previous study [18]. In addition, LCX2 and LCX1 possessed relative higher anticancer activity in vitro than LCX0 in these four test cells. However, three purified fractions did not inhibit the growth of MDA-MB-231, MCF-7, Hela, A2780 and SGC-7901 cells, even at a high concentration of 1000 μg/mL, the inhibitory rate in suppressing the growth of these cells was lower than 10% (data not shown), suggesting that the purified polysaccharide fractions of L. chuanxiong rhizome exhibited selective anticancer activity in vitro.
It was reported that the anticancer activity of polysaccharides could be influenced by monosaccharide composition, molecular weight, form, degree of branching, and solubility [33]. In addition, studies showed that excessive levels of reactive oxygen species were correlated well 17
with the generation and malign transformation of cancer cells [34]. If polysaccharides could enhance the level of antioxidants and decrease the level of reactive oxygen species in cancer cells, they might inhibit the cancer cell growth [35, 36]. In the present study, LCX2 and LCX1 showed stronger reducing power, as evidenced by the relative higher scavenging activity on DPPH and ABTS radicals, and higher FRAP value than that of LCX0, indicating the close relationship between the anticancer activities of LCX2 and LCX1 and their antioxidant activities. Taken together, our results implied that the novel LCX polysaccharides could be developed as potential natural products for cancer prevention and treatment with low toxicity. However, the anticancer mechanisms of these polysaccharides fractions remain to be investigated in the future.
4 Conclusion In summary, orthogonal experimental design was successfully applied to isolate LCX polysaccharides using ultrasonic-assisted extraction. Results showed that ultrasonic temperature, ultrasonic time and water to raw material ratio significantly affected the LCX polysaccharides yield. The optimal conditions for LCX polysaccharides extraction were as followed: ultrasonic temperature 80 °C, ultrasonic time 40 min and water to raw material ratio 30 mL/g.
Three novel purified polysaccharides, LCX0, LCX1 and LCX2, were obtained from LCX. Their estimated weight was around 2620.3, 1614.7 and 3525.5 kDa, respectively. LCX0, LCX1 and LCX2 comprised of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose. They had the same monosaccharide composition but different ratio. Compared with LCX0, LCX1 and LCX2 exhibited stronger antioxidant activities and inhibitory activity to the growth of HepG2, SMMC7721, A549 and HCT-116 cells. The differences might be contributed to the chemical 18
components and molecular weights of polysaccharides.
Overall, LCX2 and LCX1 possess strong antioxidant and anticancer activities, and have the potential to be developed as preventive and therapeutic agents for cancers and other diseases. In addition, we assume that the novel polysaccharides of high molecular weight are important bioactive constituents accounting for the medicinal properties of L. chuanxiong rhizome.
Conflict of interest The authors declare no conflict of interest.
Acknowledgement This study was supported by the Macao Science and Technology Development Fund (102/2012/A3), the Research Fund of the University of Macau (MYRG2014-00033-ICMS-QRCM, MYRG2014-00051-ICMS-QRCM,
MYRG107(Y1-L3)-ICMS13-HCW,
MYRG2015-00081-ICMS-QRCM), and the National Natural Science Foundation of China (81403120).
19
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[21] S. Honda, E. Akao, S. Suzuki, M. Okuda, K. Kakehi, J. Nakamura, Analytical Biochemistry, 180 (1989) 351-357. [22] J. Dai, Y. Wu, S.W. Chen, S. Zhu, H.P. Yin, M. Wang, J. Tang, Carbohydrate Polymers, 82 (2010) 629-635. [23] G.K. Jayaprakasha, R.P. Singh, K.K. Sakariah, Food Chemistry, 73 (2001) 285-290. [24] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura, Journal of Agricultural and Food Chemistry, 40 (1992) 945-948. [25] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radical Biology and Medicine, 26 (1999) 1231-1237. [26] I.F.F. Benzie, J.J. Strain, Analytical Biochemistry, 239 (1996) 70-76. [27] X.C. Sun, J. Yan, G. He, L.L. Zhang, Y. Yi, X.J. Gou, Journal of Sichuan Agricultural University, 29 (2011) 56-60. [28] T.P.A. Devasagayam, J.C. Tilak, K.K. Boloor, K.S. Sane, S.S. Ghaskadbi, R.D. Lele, The Journal of the Association of Physicians of India, 52 (2004) 794-804. [29] M.H. Gordon, The mechanism of antioxidant action in vitro, in: Food Antioxidants, Springer, 1990, pp. 1-18. [30] X.J. Jia, L.H. Dong, Y. Yang, S. Yuan, Z.W. Zhang, M. Yuan, Carbohydrate Polymers, 95 (2013) 195-199. [31] X.J. Jia, C.B. Ding, S. Yuan, Z.W. Zhang, L. Du, M. Yuan, Carbohydrate Polymers, 99 (2014) 319-324. [32] A.Z. Zong, H.Z. Cao, F.S. Wang, Carbohydrate Polymers, 90 (2012) 1395-1410. [33] S. Wasser, Applied Microbiology and Biotechnology, 60 (2002) 258-274. [34] H. Wiseman, B. Halliwell, Biochemical Journal, 313 (1996) 17-29. [35] R.J. Xu, H. Ye, Y. Sun, Y.Y. Tu, X.X. Zeng, Food and Chemical Toxicology, 50 (2012) 2473-2480. [36] L.Y. Tong, C.C. Chuang, S.Y. Wu, L. Zuo, Cancer Letters, 367 (2015) 18-25.
21
Figure Captions
Figure 1 Effects of different extraction parameters: (a) ultrasonic temperature, (b) ultrasonic time, and (c) water to raw material ratio on the yield of LCX polysaccharides (%).
22
Figure 2 (a) Elution curve of crude polysaccharide extracted from the rhizome of L. chuanxiong by a DEAE-52 cellulose anion-exchange column. Purification of polysaccharide fractions of LCX0 (b), LCX1 (c) and LCX2 (d) by a Sephadex G-100 column.
23
Figure 3 GPC analysis of LCX0, LCX1 and LCX2.
24
Figure 4 HPLC analysis of monosaccharide composition in LCX purified polysaccharides (Man, mannose; Rha, Rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glucose; Gal, galactose; Ara, Arabinose). (a) Standards of monosaccharide; (b) LCX0; (c) LCX1; and (d) LCX2.
25
Figure 5 FT-IR spectra of LCX0, LCX1, and LCX2.
26
Figure 6 (a) Reducing power of LCX0, LCX1 and LCX2. (b) DPPH radical scavenging activities of LCX0, LCX1 and LCX2. (c) ABTS radical scavenging activities of LCX0, LCX1 and LCX2. (d) Ferric reducing antioxidant power (FRAP) of LCX0, LCX1 and LCX2. Data are means ± SD of values obtained in three separate experiments.
27
Figure 7 In vitro anticancer activity of LCX0, LCX1 and LCX2 against HepG2, SMMC7721, HCT-116 and A549 cancer cells. Cell viability was determined by MTT colorimetric assay.
28
Tables Table 1 Factors and levels for orthogonal test Levels Variable 1
2
3
X1
Ultrasound temperature (oC)
60
70
80
X2
Ultrasonic time (min)
20
30
40
X3
Water to raw material ratio (mL/g)
30
40
50
29
Table 2 Analysis of L9 (3)4 test results (X1)
ultrasonic (X2) ultrasonic (X3) Water to raw The yield of LCX
No. temperature (oC) time (min)
material ratio (mL/g) polysaccharides (%)
1
1
1
1
15.57
2
1
2
2
13.04
3
1
3
3
16.98
4
2
1
2
18.02
5
2
2
3
13.36
6
2
3
1
19.44
7
3
1
3
19.49
8
3
2
1
17.05
9
3
3
2
18.08
K1
15.20
17.69
17.35
K2
16.94
14.48
16.38
K3
18.21
18.67
16.61
Ra
3.01
3.68
0.97
a
R refers to the result of extreme analysis.
30
Table 3 GPC analysis: Molecular weight distribution of LCX purified polysaccharides fractions. Mn
Mw
Mw/Mn
LCX0
1404516
2620257
1.87
LCX1
1033541
1614687
1.56
LCX2
1539409
3525507
2.29
31
S0141-8130(15)30235-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.12.046 BIOMAC 5642
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
14-9-2015 24-11-2015 14-12-2015
Please cite this article as: Jie Hu, Xuejing Jia, Xiaobin Fang, Peng Li, Chengwei He, Meiwan Chen, Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2015.12.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome Jie Hu#, Xuejing Jia#, Xiaobin Fang, Peng Li, Chengwei He* [email protected], Meiwan Chen* [email protected] State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China *Corresponding author at: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, N22-7038, Avenida da Universidade,Taipa, Macao, China. Tel: +853-88228516; Fax: +853-28841358 (Chengwei He, Ph.D.); State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, N22-6012, Avenida da Universidade, Taipa, Macao, China. Tel.: +853-88224675; Fax: +853-28841358 (Meiwan Chen, Ph.D.) #
These authors contributed equally to the work.
1
Highlights
Three novel polysaccharides were obtained from L. chuanxiong rhizome.
The extraction condition was optimized using orthogonal experimental design.
The polysaccharides showed potent in vitro antioxidant and anticancer activities.
2
Abstract Ultrasonic-assisted extraction technology was employed to prepare Ligusticum chuanxiong Hort polysaccharide. Single factor test and orthogonal experimental design were used to optimize the extraction conditions. The results showed that the optimal extraction conditions consisted of ultrasonic temperature of 80°C, ultrasonic time of 40 min and water to raw material ratio of 30 mL/g. Three novel polysaccharides fractions, LCX0, LCX1 and LCX2, were isolated and purified from the crude polysaccharides using DEAE-52 cellulose and Sephadex G-100 column chromatography. The molecular weight and monosaccharide composition of three LCX polysaccharides fractions were analyzed with gel permeation chromatography (GPC) and HPLC analysis, respectively. Furthermore, the antioxidant and in vitro anticancer activities of the polysaccharides were investigated. Compared with LCX0, LCX2 and LCX1 showed relative higher antioxidant activity and inhibitory activity to the growth of HepG2, SMMC7721, A549 and HCT-116 cells. It is suggested that the novel polysaccharides from rhizome of L. chuanxiong could be promising bioactive macromolecules for biomedical use.
Keywords:
Polysaccharides;
Ligusticum
chuanxiong;
Ultrasonic-assisted
extraction;
Antioxidant; Anticancer.
3
1 Introduction Ligusticum chuanxiong Hort (LCX), a well-known traditional Chinese herb medicine, has been widely used as an analgesic agent for centuries in the treatment of headache, traumatic, swelling pain, menstrual disorders and rheumatic diseases [1, 2]. More than 200 compounds, including essential oils, alkaloid, phenolic acids, actones, and other constituents, have been isolated and identified from LCX [3, 4]. A large number of pharmacological studies during the last decades had demonstrated the enormous medicinal potentials of LCX, including antioxidation [5], anti-inflammation [6] and neuroprotection [7], cardiovascular and cerebrovascular effects [8], anticancer and antineoplastic activities [9, 10]. All these studies strongly support the view that LCX has multiple beneficial therapeutic properties and is an effective therapeutic agent.
Researches on the polysaccharides from LCX had gained increasing attention over the past few years. Fan et al. reported the extraction and purification polysaccharides of LCX. Four homogeneous fractions were obtained and showed strong scavenging effect against O2•· and ·OH [11, 12]. Recently, studies on different extraction methods for polysaccharide from LCX had been developed, such as ultrasonic-assisted extraction [13, 14], microwave-assisted extraction [15], the traditional hot water extraction [16] and enzymatic-assisted extraction [17]. Yuan et al. obtained three purified polysaccharides (LCA, LCB and LCC) from LCX using boiling water extraction and ethanol precipitation [18]. In their work, the monosaccharide composition, molecular weight and the main skeleton of LCX polysaccharides were determined. Meanwhile, their antioxidant and antiproliferative activities were also investigated. All purified polysaccharides fractions exhibited potential antioxidant and anticancer activities. Among them, LCB showed the strongest
4
antioxidant and anticancer activity. Similar results were also reported that one purified polysaccharide fraction was isolated from the rhizome of LCX using high-pressure ultrasound-assisted extraction. Single-factor tests and response surface methodology were employed to optimize extraction conditions. Antioxidant activities of this polysaccharide fraction was further evaluated against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide radicals in vitro [19].
According to these publications, it was obvious that researches on polysaccharides of LCX in the last ten years was scattered, especially in monosaccharide composition, molecular weight, and bioactivities. Thus, in this work, we reported the optimal conditions for the ultrasonic-assisted extraction, isolation and purification of novel polysaccharides from the rhizome of LCX, determined their preliminary structural features, and antioxidant and anticancer activities in vitro.
2 Materials and methods 2.1 Materials, reagents and equipment
The rhizome powder of L. chuanxiong was purchased from the Chengdu traditional Chinese medicine market (Chengdu, Sichuan, China). DEAE-52 cellulose and Sephadex G-100 gel were all purchased from Solarbio Bioscience & Technology Co., Ltd (Shanghai, China). Monosaccharides standards, including mannose, rhamnose, galacturonic acid, glucose, galactose and
arabinose,
were
obtained
1-phenyl-3-methyl-5-pyrazolone
from (PMP),
Aladdin
Co.,
trifluoroacetic
Ltd acid
(Shanghai,
China).
(TFA),
3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl
5
(DPPH) and Vitamin C (Vc) were obtained from Sigma Co., Ltd (St. Louis, MO, USA). Ferric reducing antioxidant power (FRAP) and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay kit were purchased from Beyotime Institute of Biotechnology Co., Ltd (Shanghai, China). All reagents were of analytical grade, except for acetonitrile, which was of HPLC grade. Water was supplied by Milli-Q Plus system (Millipore, Bedford, USA). A Branson 8510 ultrasonic generator (Branson Ultrasonics Corporation, Danbury, CT) was used for extraction. A Flexstation Ⅲ Multi-Mode Microplate Reader (Molecular Devices) was used for absorbance determination. Infrared was recorded on spectrum 100 FT-IR spectrometer.
2.2 Extraction methods
One gram of dried rhizome powder of LCX was refluxed with 50 mL of 80% ethanol for 1 h to remove lipids. After that, the filtered residues were placed in a 50 mL tube and added with corresponding volume of water. Then the residues were ultrasonically extracted at different extraction conditions. After extraction, the supernatant was separated by filtration. Then it was condensed by rotatory evaporator and ethanol was slowly added to the condensed solution. After 12h at 4 °C, the resulting precipitate was collected and it was redissolve to remove free proteins by Sevag method. Last the crude polysaccharides were obtained after drying for 12h at 50 °C. The total polysaccharide yield was determined by the method of phenol-sulphuric acid method using glucose as the standard reference [20].
2.3 Orthogonal experiment design
Single-factor test was employed to determine the preliminary range of the extraction variables,
6
namely, ultrasonic temperature (X1), ultrasonic time (X2), and water to raw material ratios (X3). Then a three-factor, three-level orthogonal extracting test was applied to optimize the extraction condition. As shown in Table 1, different factors and levels for orthogonal test were designed based on the results of single-factor experiments. Table 2 represented the experimental variables and 9 experimental points.
2.4 Isolation and purification of polysaccharides of LCX
The crude polysaccharide of LCX (0.5 g) was reconstituted in 35 mL distilled water, and the supernatant was collected by centrifugation. Then the supernatant was applied to a DEAE-52 cellulose chromatography column (5.5 × 40 cm) and eluted with different concentrations of NaCl solution (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mol/L). Fractions eluted with 0, 0.1 and 0.2 mol/L NaCl were collected, dialyzed, and lyophilized. These three polysaccharides fractions were then denoted as LCX0, LCX1 and LCX2 and further purified by gel-filtration (2.0 × 100 cm) on Sephadex G-100 column using distilled water as the eluant at a flow rate of 0.4 mL/min.
2.5 Monosaccharide composition of purified polysaccharides fractions
2.5.1 Hydrolysis of polysaccharides
The polysaccharide samples (10 mg), LCX0, LCX1 and LCX2, were dissolved with 2 mol/L trifluoroacetic acid (10 mL) in a sealed flask, respectively. The flask was placed in an oven at 110 o
C for 3 h under nitrogen atmosphere. After being cooled to room temperature, the flask was
opened and methanol was added into it. Then the reaction mixture was evaporated to dryness under a reduced pressure. After that, the same amount of methanol was added and dried again by 7
the same method above, and the procedure was repeated thrice for trifluoroacetic acid to be removed. The dried hydrolyzed polysaccharide samples were dissolved in 2 mL of water for subsequent derivatization.
2.5.2 Derivatization of monosaccharides with PMP
The procedure employed for the PMP derivatization of monosaccharides was carried out as described by Honda et al. with slight modifications [21]. Briefly, 100 μL of hydrolysed polysaccharides (5 mg/mL) or monosaccharide standards were transferred into a tube, then 0.3 mol/L NaOH (200 μL) and 0.5 mol/L PMP-methanol solution (200 μL) were added and mixed. The mixture was allowed to react for 90 min at 70 °C, then cooled to room temperature and neutralized with 200 μL 0.3 mol/L HCl. After that, chloroform was added, and the mixture was shaken vigorously. The organic layer was carefully discarded, and the extraction process was repeated thrice. Finally, the aqueous layer was filtered through a 0.45 μm membrane for HPLC analysis.
2.5.3 HPLC analysis
The analysis of PMP-labeled monosaccharides were carried out on Waters e2695 HPLC system equipped with a reverse phase C18 column (250×4.6 mm) as previous method [22]. The wavelength for UV detection was 245 nm. The mobile phase consisted of 0.1 mol/L phosphate buffer (pH 6.7) with (A) 83% and (B) 17% acetonitrile, at a flow rate of 1 mL/min.
2.6 GPC analysis
8
The molecular weight distributions of the purified fractions were analyzed using gel permeation chromatography (GPC) on connecting GPC columns: TSK gel G4000PWXL (Tosoh CO., Tokyo, Japan). Milli-Q water was selected as mobile phase, and the flow rate was set at 0.5 mL/min. Purified polysaccharide fractions was dissolved in distilled water at the concentration of 10 mg/mL, respectively, and 100 μL of sample was injected each time. GPC peaks were detected with a refractive index (RI) detector. The number average molecular weight (Mn), the weight average molecular weight (Mw), and the polydispersity (Mw/Mn) were calculated using molecular weight calculation software connected to the HPLC integration system. Polyethylene oxide/glycol easivials were chosen as standard materials.
2.7 Fourier transform infrared (FT-IR) spectroscopy analysis
Purified polysaccharide fractions (2 mg) and potassium bromide were mixed, grounded, and pressed into a pellet. Spectra was then recorded in the absorbance mode from 4000 cm−1 to 400 cm−1 on a Perkin-Elmer FT-IR spectrometer.
2.8 Antioxidant activities
The reducing power of purified polysaccharide fractions was determined using the method of Jayaprakasha et al. [23]. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay was performed following Shimada et al. [24]. The ABTS radical scavenging activity assay was carried out using the method of Re et al. [25]. The ferric reducing antioxidant power (FRAP) assay was measured using the method of Benzie and Strain [26].
2.9 Anticancer activities 9
In this study, HepG2 and SMMC7721 liver carcinoma cell lines, MCF-7 and MDA-MB-231 breast cancer cell lines, A2780 ovarian cancer cell line, HCT-116 colon cancer cell line, A549 lung cancer cell line, SGC-7901 gastric cancer cell line and Hela cervical cancer cell line were employed to evaluate the anticancer effect of purified polysaccharide fractions by MTT assay. Cancer cells were plated at 5000 per well in 96-well plate and were allowed to adhere and spread for 24 h. Then the cells were treated with LCX0, LCX1 and LCX2 solution at different concentrations (25 to 1000 μg/mL). The wells were then incubated for 48 h. MTT solution (10 mg/mL in PBS) was added to each well. After incubating for another 4 h, the supernatants were aspirated. The formazan crystals in each well were dissolved in 100 μL of DMSO. The amount of purple formazan was determined by measuring the absorbance at 540 nm.
2.10 Statistical analysis
All data are presented as means ± standard deviation (SD). All experiments were repeated three times.
3 Results and discussion 3.1 Effect of ultrasonic-assisted extraction conditions on the yield of LCX polysaccharides
3.1.1 Ultrasonic temperature
The effect of different ultrasonic temperature on extraction yield of polysaccharides from the rhizome of LCX was investigated. Ultrasonic temperature was set at 40, 50, 60, 70, and 80 oC while other extraction conditions were given as followings: ultrasonic time of 20 min, water to
10
raw material ratio of 20 mL/g. As shown in Fig. 1a, extraction yield of polysaccharides from the rhizome of L. chuanxiong increased with the increasing ultrasonic temperature and reached the maximum value (10.4%) when extraction temperature was 70 oC. The extraction yield started to decrease when extraction temperature was over 80 oC. Therefore, 70 oC was chosen as the optimum temperature for extraction.
3.1.2 Ultrasonic time
To assess the influence of ultrasonic time on the extraction yield of LCX polysaccharides, the extraction process was carried out using different ultrasonic time of 5, 10, 20, 30, 40, and 50 min. The other extraction parameters were set at ultrasonic temperature 70 oC and water to raw material ratio of 30 mL/g. Fig. 1b showed that the extraction yield obviously increased when the ultrasonic time increased from 5 to 30 min, and then the yield slightly decreased from 30 to 50 min. Thus, the preferred ultrasonic time was chosen as 30 min.
3.1.3 Water to raw material ratio
Different ratios of water to raw material (5, 10, 20, 30, 40, and 50 mL/g) on the extraction yield of LCX polysaccharides was shown in Fig. 1c. The ultrasonic temperature was set at 70 oC and ultrasonic time was fixed as 30 min. When the ratio of water to raw material increased, LCX polysaccharides yield rose and reached a peak value at ratio of 40. Thus, 40 mL/g was selected as the optimum point for the water to raw material ratio in the orthogonal experiments.
3.2 Optimization for extraction of LCX polysaccharides
11
An orthogonal L9 (3)4 test was designed based on the results of single factor experiments in order to optimize the extraction of LCX polysaccharides. Three factors with three variation levels, X1 (ultrasonic temperature, 60, 70, 80 oC), X2 (ultrasonic time, 20, 30, 40 min) and X3 (water to raw material ratio, 30, 40 and 50 mL/g), were chosen for optimization (Table 1). The results and analysis of orthogonal test are presented in Table 2. From the results, the maximum extraction yield of LCX polysaccharides was 19.49%. However, it could not be considered as the best extraction conditions. Because of R2 > R1 > R3, the influence of extraction parameters decreased in the order: X2 > X1 > X3. The ultrasonic time was found to be the most important parameter in the LCX polysaccharides extraction process. The maximum yield of LCX polysaccharides was obtained when ultrasonic temperature, ultrasonic time and water to raw material ratio were 80 °C, 40 min and 30 mL/g, respectively. Through confirmatory test, the highest yield of LCX polysaccharides was 20.75 ± 1.18%.
3.3 Purification and characterization
3.3.1 Isolation and purification of LCX polysaccharides
The crude polysaccharide extracts were subsequently isolated by DEAE-52 cellulose anion-exchange column. After eluting with 0, 0.1, and 0.2 mol/L NaCl, fractions, LCX0, LCX1 and LCX2, were obtained (Fig. 2a). Then LCX0, LCX1 and LCX2 were further purified by Sephadex G-100 column (Fig. 2b, 2c and 2d). All fractions were in a single peak, suggesting that they were homogeneous polysaccharide. In addition, no absorption at 260 nm and 280 nm was detected (data not shown), indicating these fractions were absence of nucleic acid and protein.
12
3.3.2 GPC analysis
The molecular weight distributions of three purified fractions were determined by GPC. The data showed that LCX0, LCX1 and LCX2 had different molecular weights of 2620.3, 1614.7 and 3525.5 kDa (Fig. 3 and Table 3). This result further confirmed that all purified fractions were homogeneous polysaccharide. Their huge molecular weight gained our interests, for it was different from other findings. Fan et al. isolated four homogeneous polysaccharides, with the molecular weights of 31, 52, 90, and 36 kDa, respectively [11, 12]. Yuan et al. obtained three purified polysaccharides, LCA, LCB and LCC, with molecular weight of 28.3, 12.3, and 63.1 kDa, respectively [18]. Another study conducted by Sun et al., LCXP-1, LCXP-2 and LCXP-3 were isolated from rhizome of L. chuanxiong, with molecular weight of 11.9, 20.8 and 701.9 kDa [27]. From these studies, we can see that molecular weight distributions of LCX polysaccharides are different in each study. Many factors can cause this difference, including extraction and purification methods and conditions, the sources of the rhizome of L. chuanxiong, etc.
3.3.3 Monosaccharide composition analysis of LCX polysaccharides by HPLC
In the present study, different fractions of LCX polysaccharides were hydrolyzed with trifluoroacetic acid, and then their monosaccharide components were determined by PMP derivatization assay and were analyzed with HPLC analysis. The HPLC results of seven PMP-labeled monosaccharide standards were shown in Fig. 4a, and the monosaccharide compositions of LCX0, LCX1 and LCX2 were identified and presented in Fig. 4b, 4c and 4d, respectively. As shown in Fig. 4, LCX0, LCX1 and LCX2 were composed of mannose (Man), rhamnose (Rha), galacturonic acid (GalA), glucose (Glc), galactose (Gal) and arabinose (Ara). 13
They had the same monosaccharide composition but different ratio. LCX0 primarily consisted of Man, Rha, GalA, Glc, Gal and Ara in molar ratio of 0.013:0.034:0.07:1:0.25:0.28. LCX1 and LCX2 contained monosaccharide components of Man, Rha, GalA, Glc, Gal and Ara in molar ratio of 0.001:0.013:1:0.038:0.046:0.045 and 0.018:0.152:1:0.723:0.758:0.478, respectively. Obviously, glucose was the main monosaccharide in LCX0 while galacturonic acid was the significant predominant component in both LCX1 and LCX2. It is noteworthy that the monosaccharide composition of LCX polysaccharides is different from previous studies [18, 27]. In this study, polysaccharide fractions LCX1 and LCX2, especially LCX1, were mainly comprised of galacturonic acid, while it was not detected by Yuan et al. [18]. However, the result of Sun et al. was somewhat in agreement with our results, especially LCP-3, which contained small amount of galacturonic acid [27]. The differences between these results and our findings may be attributed to the different extraction and purification methods and conditions, and different sources of the rhizome of L. chuanxiong.
3.3.4 FT-IR analysis
The structure of LCX0, LCX1 and LCX2 were further analyzed by FT-IR (Fig. 5). From the FT-IR spectra, LCX0, LCX1 and LCX2 had similar structure. The broad and strong absorption at 3400 cm-1 was assigned to the stretching vibration of -OH groups. The peak at 2932 cm−1 was corresponding to the stretching vibration of -CH2 groups, while 1417 cm−1 was the symmetrical deformation vibration of -CH2. The absorption bands between 1000 cm−1 and 1145 cm−1 belonged to the characteristic absorption of C-O-C groups. In addition, the peaks at 1644 cm−1 and 1332 cm−1 were observed, which strongly indicating the presence of COO− groups on the LCX purified
14
polysaccharides chains. This observation further confirmed that LCX purified polysaccharides fractions contained COO− groups, which was in accordance to the monosaccharide composition analysis. The difference between LCX0, LCX1 and LCX2 was that LCX0 had an addition peak at 1726 cm−1, which might be belonged to the stretching vibration of carbonyl group.
3.4 Antioxidant activities
3.4.1 Reducing power
The reducing power serves as an important indicator to evaluate polysaccharides’ potential antioxidant activities [28]. In the present study, the K3Fe(CN)6 reduction method was employed to evaluate the antioxidant capacity of LCX purified polysaccharides fractions. The reducing power of LCX0, LCX1 and LCX2 was shown in Fig. 6a. It was clear that the reducing power of LCX1 and LCX2 increased with increasing sample concentration, while the reducing power of LCX0 was slightly affected. Their reducing power decreased in the order of LCX2, LCX1 and LCX0. The present results revealed that LCX purified polysaccharides fractions, especially LCX2, showed potently reducing power in vitro. Generally, reducing power was associated with reductones. This result suggested that LCX2 might contain more reductone-associated and hydroxide groups than LCX0 and LCX1 [29].
3.4.2 DPPH radical scavenging activity
The scavenging effect of DPPH radicals is a common antioxidant assay. Antioxidants can protect against excessive free radicals that have been implicated in the etiology of many diseases [28]. In this work, DPPH radical scavenging effects of LCX purified polysaccharides fractions were 15
measured. As shown in Fig. 6b, LCX1 and LCX2 showed dose-dependent scavenging effect, but they were weaker than that of Vitamin C in each concentration. The IC50 of LCX2 and LCX1 were estimated at 2.95 mg/mL and above 8 mg/mL, respectively. However, LCX0 showed no DPPH radical scavenging activity even at 8 mg/mL. The difference of their DPPH scavenging ability may be contributed to their monosaccharide composition, content of hydroxy and carboxy groups, and hydrogen donation ability [30].
3.4.3 ABTS radical scavenging activity
ABTS radical scavenging activity is also commonly used to determine antioxidant activity of a compound. In this study, the scavenging abilities of different LCX purified polysaccharides fractions on ABTS radicals were measured. The results were shown in Fig. 6c. The ABTS radical scavenging activities of LCX0, LCX1 and LCX2 increased with their concentrations, from 0.0625 to 8.0 mg/mL. Similar to the results of reducing power and DPPH radical scavenging activity, LCX2 exhibited the strongest scavenging ability. The IC50 of LCX2 was 0.57 mg/mL, which was significantly higher than that of LCX1 and LCX0 (IC50 = 2.57 mg/mL and above 8.0mg/mL, respectively). However, even the scavenging effect of LCX2 increased obviously with increasing concentration, it remained lower than that of Vitamin C at each dosage.
3.4.4 Ferric reducing antioxidant power (FRAP)
FRAP assay is also generally used to evaluate total antioxidant capacity of polysaccharides. The antioxidant activities of different LCX purified polysaccharides fractions are evaluated via the ability of reducing the Fe3+-TPTZ complex to the Fe2+-TPTZ complex [31]. The results of LCX2,
16
LCX1 and LCX0 were shown in Fig. 6d. The FRAP values of LCX2 was higher than those of LCX1 and LCX0 at each concentration. At the concentration of 8.0 mg/mL, the FRAP values of LCX2, LCX1 and LCX0 were 0.71, 0.28 and 0.11 mmol/L, respectively. This result suggested that three LCX purified polysaccharides fractions possessed potently antioxidant abilities, especially LCX2. However, compared with Vitamin C, their antioxidant capacities were weak.
3.5 Anticancer activities
It had been reported that polysaccharides played a certain role in anticancer activity [32]. In this study, MTT assay was employed to evaluate the anticancer effect of three fractions on different cancer cell lines. The cells were treated with various concentrations of LCX0, LCX1 and LCX2 for 48 h. As shown in Fig. 7, all three fractions exhibited concentration-dependent inhibition activities to HepG2, SMMC7721, A549 and HCT-116 cells. Compared with those of A549 and HCT-116 cells, there was a significant increase of inhibitory rate for HepG2 and SMMC7721 cells. This result was similar to the previous study [18]. In addition, LCX2 and LCX1 possessed relative higher anticancer activity in vitro than LCX0 in these four test cells. However, three purified fractions did not inhibit the growth of MDA-MB-231, MCF-7, Hela, A2780 and SGC-7901 cells, even at a high concentration of 1000 μg/mL, the inhibitory rate in suppressing the growth of these cells was lower than 10% (data not shown), suggesting that the purified polysaccharide fractions of L. chuanxiong rhizome exhibited selective anticancer activity in vitro.
It was reported that the anticancer activity of polysaccharides could be influenced by monosaccharide composition, molecular weight, form, degree of branching, and solubility [33]. In addition, studies showed that excessive levels of reactive oxygen species were correlated well 17
with the generation and malign transformation of cancer cells [34]. If polysaccharides could enhance the level of antioxidants and decrease the level of reactive oxygen species in cancer cells, they might inhibit the cancer cell growth [35, 36]. In the present study, LCX2 and LCX1 showed stronger reducing power, as evidenced by the relative higher scavenging activity on DPPH and ABTS radicals, and higher FRAP value than that of LCX0, indicating the close relationship between the anticancer activities of LCX2 and LCX1 and their antioxidant activities. Taken together, our results implied that the novel LCX polysaccharides could be developed as potential natural products for cancer prevention and treatment with low toxicity. However, the anticancer mechanisms of these polysaccharides fractions remain to be investigated in the future.
4 Conclusion In summary, orthogonal experimental design was successfully applied to isolate LCX polysaccharides using ultrasonic-assisted extraction. Results showed that ultrasonic temperature, ultrasonic time and water to raw material ratio significantly affected the LCX polysaccharides yield. The optimal conditions for LCX polysaccharides extraction were as followed: ultrasonic temperature 80 °C, ultrasonic time 40 min and water to raw material ratio 30 mL/g.
Three novel purified polysaccharides, LCX0, LCX1 and LCX2, were obtained from LCX. Their estimated weight was around 2620.3, 1614.7 and 3525.5 kDa, respectively. LCX0, LCX1 and LCX2 comprised of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose. They had the same monosaccharide composition but different ratio. Compared with LCX0, LCX1 and LCX2 exhibited stronger antioxidant activities and inhibitory activity to the growth of HepG2, SMMC7721, A549 and HCT-116 cells. The differences might be contributed to the chemical 18
components and molecular weights of polysaccharides.
Overall, LCX2 and LCX1 possess strong antioxidant and anticancer activities, and have the potential to be developed as preventive and therapeutic agents for cancers and other diseases. In addition, we assume that the novel polysaccharides of high molecular weight are important bioactive constituents accounting for the medicinal properties of L. chuanxiong rhizome.
Conflict of interest The authors declare no conflict of interest.
Acknowledgement This study was supported by the Macao Science and Technology Development Fund (102/2012/A3), the Research Fund of the University of Macau (MYRG2014-00033-ICMS-QRCM, MYRG2014-00051-ICMS-QRCM,
MYRG107(Y1-L3)-ICMS13-HCW,
MYRG2015-00081-ICMS-QRCM), and the National Natural Science Foundation of China (81403120).
19
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Figure Captions
Figure 1 Effects of different extraction parameters: (a) ultrasonic temperature, (b) ultrasonic time, and (c) water to raw material ratio on the yield of LCX polysaccharides (%).
22
Figure 2 (a) Elution curve of crude polysaccharide extracted from the rhizome of L. chuanxiong by a DEAE-52 cellulose anion-exchange column. Purification of polysaccharide fractions of LCX0 (b), LCX1 (c) and LCX2 (d) by a Sephadex G-100 column.
23
Figure 3 GPC analysis of LCX0, LCX1 and LCX2.
24
Figure 4 HPLC analysis of monosaccharide composition in LCX purified polysaccharides (Man, mannose; Rha, Rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glucose; Gal, galactose; Ara, Arabinose). (a) Standards of monosaccharide; (b) LCX0; (c) LCX1; and (d) LCX2.
25
Figure 5 FT-IR spectra of LCX0, LCX1, and LCX2.
26
Figure 6 (a) Reducing power of LCX0, LCX1 and LCX2. (b) DPPH radical scavenging activities of LCX0, LCX1 and LCX2. (c) ABTS radical scavenging activities of LCX0, LCX1 and LCX2. (d) Ferric reducing antioxidant power (FRAP) of LCX0, LCX1 and LCX2. Data are means ± SD of values obtained in three separate experiments.
27
Figure 7 In vitro anticancer activity of LCX0, LCX1 and LCX2 against HepG2, SMMC7721, HCT-116 and A549 cancer cells. Cell viability was determined by MTT colorimetric assay.
28
Tables Table 1 Factors and levels for orthogonal test Levels Variable 1
2
3
X1
Ultrasound temperature (oC)
60
70
80
X2
Ultrasonic time (min)
20
30
40
X3
Water to raw material ratio (mL/g)
30
40
50
29
Table 2 Analysis of L9 (3)4 test results (X1)
ultrasonic (X2) ultrasonic (X3) Water to raw The yield of LCX
No. temperature (oC) time (min)
material ratio (mL/g) polysaccharides (%)
1
1
1
1
15.57
2
1
2
2
13.04
3
1
3
3
16.98
4
2
1
2
18.02
5
2
2
3
13.36
6
2
3
1
19.44
7
3
1
3
19.49
8
3
2
1
17.05
9
3
3
2
18.08
K1
15.20
17.69
17.35
K2
16.94
14.48
16.38
K3
18.21
18.67
16.61
Ra
3.01
3.68
0.97
a
R refers to the result of extreme analysis.
30
Table 3 GPC analysis: Molecular weight distribution of LCX purified polysaccharides fractions. Mn
Mw
Mw/Mn
LCX0
1404516
2620257
1.87
LCX1
1033541
1614687
1.56
LCX2
1539409
3525507
2.29
31