Research Article Received: 4 February 2012;
Revised: 17 April 2012;
Accepted: 21 April 2012
Published online in Wiley Online Library: 12 June 2012
(wileyonlinelibrary.com) DOI 10.1002/pca.2376
Pulsed Electric Field Extraction Enhanced Anti-coagulant Effect of Fungal Polysaccharide from Jew’s Ear (Auricularia auricula) Changtian Li,a,b Xinxin Maoa and Baojun Xuc* Introduction – As a Chinese herbal medicine, Jew’s ear has been known for its anti-coagulant effects. Hence it is worthwhile developing an effective technique to extract active components. Objective – To find the optimal extraction condition and to identify the best strain to yield fungal polysaccharide with anti-coagulant activity. Methodology – Three strains of Jew’s ear from Jilin Province, named as 988, DY 18 and FS 02, and three extraction techniques, namely, high intensity pulsed electric fields (HIPEF), microwave-assisted extraction method (MAEM) and ultrasonic-assisted extraction method (UAEM), were applied to optimise the extraction conditions. The crude extracts and polysaccharides were further determined for anti-coagulant activities. Results – All extracts prolonged blood clotting time as compared to reagent control. The HIPEF exhibited the most remarkable effect among the three extraction techniques. The anti-coagulant activities of extracts were enhanced with increasing electric field strength when the field strength reached 24 kV/cm. Conclusion – Current results suggest that the HIPEF technique will be an effective method in the manufacture of bioactive natural polysaccharide. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: Pulsed electric field; extraction; anti-coagulant activity; Jew’s ear; polysaccharide
Introduction
36
Edible mushroom Jew’s ear (Auricularia auricula) is cultured widely in China. The fruit body of A. auricula from Jilin Province is famous worldwide for its high quality. Jew’s ear has long been widely used in Chinese cuisines, and it is acknowledged to be a healthy food due to the high carbohydrate, protein and mineral (Ca, P and Fe) contents in its fruit body (Hobbs, 1995). It has also been used for centuries as a medicinal mushroom by many herbalists. Modern researches into possible medical applications have variously concluded that Jew’s ear has anti-oxidant, antitumour, hypoglycaemic, anti-coagulant and cholesterol-lowering properties. Bor et al. (2006) reported that water extracts from Jew’s ear showed strong anti-oxidant activity and free-radical-scavenging ability in vitro. An animal research reported in vivo anti-oxidant activity of Jew’s ear, in which water soluble polysaccharide from Jew’s ear significantly decreased the level of malondialdehyde and increased superoxide dismutase and glutathione peroxidase activities in mice where ageing is induced by D-galactose (Zhang et al., 2011). Other experiments concluded that glucans isolated from Jew’s ear showed potent anti-tumour properties when used on mice artificially implanted with Sarcoma 180 tumours (Misaki et al., 1981; Ma et al., 2010). It was further verified that the polysaccharide of Jew’s ear exerted its anti-tumour effect via macrophage activation (Yu et al., 2009). Research on genetically diabetic mice showed that a polysaccharide from Jew’s ear had a hypoglycemic effect (Yuan et al., 1998). One in vitro study reported that Jew’s ear may be effective in stopping platelet binding, with possible uses regarding hypercholesterolemia. Research has shown that Jew’s ear could be used to lower
Phytochem. Anal. 2013, 24, 36–40
cholesterol levels (Francia et al., 1999). Several studies have reported the anti-coagulant effects of Jew’s ear (Yoon et al, 2003; Fan et al., 2007), in which Jew’s ear exhibited ability to extend blood clotting time in phlebitis (or deep-vein thrombosis). Prothrombin and thrombin play critical roles in thrombosis, since they convert fibrinogen to fibrin for clot formation and strongly induce platelet aggregation (Mann, 1997). Therefore, in order to maximally extract active components from active fungal strains, the anti-coagulant activities of crude fungal polysaccharides from three strains of Jew’s ear were compared. The anti-coagulant activities of polysaccharides extracted by three extraction techniques, namely, high intensity pulsed electric fields (HIPEF), microwave-assisted extraction method (MAEM) and ultrasonic-assisted extraction method (UAEM), were also compared. The anti-coagulant activities of the extracts were evaluated by comparing prothrombin time (PT) and activated partial thrombin time (APTT).
* Correspondence to: Baojun Xu, Food Science and Technology Program, Beijing Normal University – Hong Kong Baptist University United International College, 28, Jinfeng Road, Tangjiawan, Zhuhai, Guangdong 519085, China. Email:
[email protected] a
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun, Jilin 130118, China
b
Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Changchun 130118, China
c
Food Science and Technology Program, Beijing Normal University – Hong Kong Baptist University United International College, Zhuhai, Guangdong 519085, China
Copyright © 2012 John Wiley & Sons, Ltd.
Anti-Coagulant Effect of Jew’s Ear Polysaccharide
Experimental Mushroom materials Three strains (988, DY 18 and FS 02) of Jew’s ear (A. auricula) were collected from Hang-song-dian in Jiaohe County, Jilin Province, China. The fungal fruit bodies were identified by Professor Yu Li at the Engineering Research Center of Edible and Medicinal Fungi, Ministry of Education, Changchun, China. Samples were dried initially at 70 C in an oven, ground in a food mixer and sieved through 40-mesh. Chemicals and reagents The APTT and PT (ISI = 1.96) assay kits were obtained from Thermo Scientific (USA). Sodium heparin from porcine intestinal mucosa (177 U/mg) was obtained from Sigma Chemical Co. (Hudson, NH, USA). Normal plasma was obtained by centrifugation (3000 g, 15 min) of the mixture (10:1, v/v) of 3.8% sodium citrate with human whole blood from ten individual healthy donors, and frozen at 60 C until further use. All other chemicals were of analytical grade. Assays of anti-coagulant activity Activated partial thromboplastin time (APTT) and prothrombin time (PT) clotting assays were performed using normal human plasma as described by Anderson et al. (1976). The anti-coagulant activity was expressed as Clotting time (unit in second) with the heparin (177 U/mg) as control (Jaques, 1979). For APTT assay, citrated human plasma (90 mL) was mixed with sample extract (10 mL), and then APTT reagent (100 mL) was added to the mixture and incubated for 3 min at 37 C. Thereafter clotting time was recorded in a coagulometer (model Thrombotimer 4, Behnk Elektronik, Norderstedt, Germany). In PT assay, plasma (90 mL) was mixed with sample extract (10 mL), and incubated for 3 min, and then PT reagent (200 mL, preincubated for over 15 min at 37 C) was added to the mixture and clotting time was recorded by the coagulometer.
Figure 1. Flow chart of treatment in high-intensity pulsed electric fields (HIPEF).
(model G80F20CN2L-B8, Galanz Group, Foshan, China) for 1 min, the sample vessels were allowed to cool to room temperature after microwave thermal extraction. The extracts were centrifuged at 3500 g for 15 min. The supernatants were filtered through Whatman filters. The extracts were stored at 4 C in a cold room for further analysis within 48 h. Ultrasonic-assisted extraction method Samples were extracted in triplicate using the method described by Yin et al. (2008). Briefly, 1 g samples were extracted with 30 mL of distilled water by vortexing for 30 s followed by sonication in a 125 W, 60 Hz sonication room (Xinyi-IID, Xinyi Company, Shanghai, China) for 5 min. After extraction, the extracts were subjected to centrifugation (3500 g, 15 min), the supernatants filtered through Whatman filter paper. The extracts were stored at 4 C in a cold room for further analysis within 48 h.
High intensity pulsed electric fields
Extraction of crude polysaccharides
Each strain of Jew’s ear (20 g) was suspended in 600 mL water, and extracted in the HIPEF facility at Jilin University (Changchun, China). The HIPEF produced mono-polar wave pulses, the treatment flow rate was 5 L/min and it was controlled by a pump (model D100A, Minsks, Xi’an, China). The treatment chamber consisted of two separate stainless-steel electrodes. The treatment temperature was kept below 40 C using a cooling coil (Fig. 1). The electric pulses delivered the following specific characteristics, such as shape, polarity, width and difference of potential. The electric current generated across the electrodes and the pulse frequency were monitored using a digital oscilloscope (Hewlett Packard 54600B, Agilent, Santa Clara, CA, USA). Samples were treated under an electric field strength ranging from 16 to 36 kV/cm (Table 1) for 12 ms at frequencies between 25 and 250 Hz, pulse number at 6, at pH 8, liquid:solid ratio at 30:1 and the flow speed at 5 L/min. After HIPEF treatment, the extracts were centrifuged at 3500 g for 15 min. The supernatants were filtered through Whatman filters. The extracts were stored at 4 C in a cold room for further analysis within 48 h.
The crude aqueous extracts from mushroom were concentrated in a rotary evaporator and precipitated with four volumes of 100% ethanol. The resulting precipitate was collected by centrifugation, washed with ethanol, and lyophilised. The lyophilised material was generically identified as crude polysaccharide through a colour reaction test in test tubes and stored at ambient temperature until further use. Statistical analyses Data are presented as means SEM of three measurements. Statistical analyses of data were performed by one-way ANOVA followed by Fisher’s protected least significant difference posthoc multiple procedure using software SPSS 12.0. Differences were considered to be significant at p < 0.05.
Results and Discussion Comparison on anti-coagulant effects of different fungal strains from Jew’s ear
Dried powders of mushroom sample (1 g) were weighed into a Teflon vessel and 30 mL of water were added. The microwave vessels were sealed. Samples were extracted in a 500 W microwave
The aqueous extracts from three strains of mushroom Jew’s ear were prepared and evaluated for their inhibitory activities against blood coagulation. All these extracts prolonged blood
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Microwave-assisted extraction method
C. Li et al. clotting time as assessed by the APTT and PT tests when compared with the control group (22.9 s in the APTT test and 15 s in the PT test; Fig. 2). The blood clotting time of control groups
in both the APTT and PT tests in the current investigation were very close to those recently reported (Lau et al., 2009). The longest clotting times (62.5 s in the APTT test and 29.5 s in the PT
Table 1. Electric field strength of HIPEF Variables
Sample
Electric field strength (kV/cm) Pulse number
70 60
PEF 2
PEF 3
PEF 4
PEF 5
PEF 6
16 6
20 6
24 6
28 6
32 6
36 6
70
a
AA
65 60 APTT PT
55
Clotting time (s)
50
c
40 35
a
b
30
D D B
APTT PT
50
b
45
55
Clotting time (s)
65
PEF 1
b
25
d
20
45
a
40
b
35
a
30
b b
b
c
25
c
20
c
15
15
10
10
5
5 0
0 988-HIPEF
DY18-HIPEF
FS02-HIPEF
988-HIPEF
Control
988-UAEM
988-MAEM
Samples
Control
Samples
70 60
70 65
B
55
55
a
50
Clotting time (s)
60
APTT PT
50
45 40
b
35
a
a
30
c b
25
d c
20
Clotting time (s)
65
E E B
a APTT PT
40
a
35
b
30
10
10
5
5
c
0
0 988-UAEM
DY18-UAEM
FS02-UAEM
DY18-HIPEF
Control
DY18-UAEM
Samples
65 55
a Clotting time (s)
Clotting time (s)
60
APTT PT
50 45
30
Control
70
C C
55
35
DY18-MAEM
Samples
70
40
c
20 15
60
b
25
15
65
b
b
45
b a
a
c b
25
d c
20
50 45
APTT PT
a
40 35 30
a
25
b
b b
c b c
20
15
15
10
10
5
F E B
5
0
0 988-MAEM
DY18-MAEM
FS02-MAEM
Control
FS02-HIPEF
Samples
FS02-UAEM
FS02-MAEM
Control
Samples
38
Figure 2. Anti-coagulant activity of aqueous extracts from Jew’s ear using different extraction methods: (A–C) three strains extracted using high-intensity pulsed electric fields (HIPEF), the ultrasonic-assisted extraction method (UAEM) and the microwave-assisted extraction method MAEM, respectively; (D–F) extractions from the three stains 988, DY18 and FS02, respectively. Data are expressed as mean SEM from triplicate tests. Values marked by the same small case letter (either activated partial thromboplastin time (APTT) and prothrombin time (PT) tests) are not significantly different (p < 0.05).
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Anti-Coagulant Effect of Jew’s Ear Polysaccharide test) were found in the extract from strain DY 18 extracted by the HIPEF technique. The aqueous extracts from the DY 18 strain showed a significantly longer clotting time (p < 0.05) as compared with those for extracts from the other strains 988 and FS 02 (Fig. 2A, B and C). Overall, the extract from the DY 18 strain showed the highest anti-coagulant activity; the extracts from FS 02 and 988 strains did show APTT and PT activity, but they were significantly lower than the DY 18 strain.
As shown in Fig. 2D, E and F, no matter which strain was used, the blood clotting time values of aqueous extracts from HIPEF extraction were significantly (p < 0.05) higher than those from UAEM and MAEM in both APTT and PT tests. All aqueous extracts from the three strains (988, DY 18 and FS 02) exhibited significantly (p < 0.05) higher blood clotting time as compared with the control group (Fig. 2D, E and F). The HIPEF extract of Jew’s ear had a higher anti-coagulant activity than the extracts from UAEM and MAEM. There were no significant (p > 0.05) differences in the blood clotting time values between MAEM and UAEM (Fig. 2D, E and F). The experiments showed that the HIPEF technique was an effective way to extract anti-coagulant active components from the Jew’s ear. The HIPEF technique extracts compounds under low temperature in a short time, and such a technique is safe and time saving. The HIPEF treatment can extract perfectly and quickly, because HIPEF can cause electroporation and damage of membranes (Zakhem et al., 2006). Our results suggest that HIPEF is more effective by using water as the extraction solvent as it accelerates the release of polysaccharide. Polysaccharide extraction with the HIPEF method will be a very useful technique in the manufacture of natural extracts. Optimisation of HIPEF extraction The HIPEF treatment time for another mushroom (Inonotus obliquus) has been studied in a previous report (Yin et al., 2008), which indicated that HIPEF treatment time could be longer than a threshold value at the applied electric field strength. In the current study, the anti-coagulant activities of the aqueous extracts from the mushroom Jew’s ear extracted by HIPEF were further studied by measuring their effects on the APTT and PT at various levels of electric field strength. The anti-coagulant activity increased with the increasing electric field strength (Table 1) between 0 and 24 kV/cm. However, there were no significant (p > 0.05) diffderences in terms of APTT time and PT time (in the treatments PEF 3, PEF4, PEF5 and PEF6; Fig. 3), which indicated that blood clotting time did not increase when the HIPEF electric field strength exceeded 24 kV/cm. The optimal extraction conditions were found to be as follows: HIPEF strength at 24 kV/cm, pulse number at 6, at pH 8, liquid:solid ratio at 30:1, at which the clotting time was extended to 67.56 s in the APTT test and 30.56 s in the PT test with the DY 18 stain.
b
b
b
b
a
c d c
c
b
b
b
b
e d
Figure 3. Anti-coagulant activity from HIPEF extract from Jew’s ear (strain DY 18). Data are expressed as mean SEM from triplicate tests. Values marked by the same small case letter among different treatments (either activated partial thromboplastin time (APTT) and prothrombin time (PT) tests) are not significantly different (p < 0.05). CK: control.
exhibited a higher anti-coagulant activity than the crude aqueous extract when tested for the APTT and PT in vitro. The APTT values increased dose-dependently as the polysaccharide concentration increased, resulting in almost threefold increases in APTT values at 0.8 mg/mL of polysaccharide (Fig. 4). The PT increased with increments of the polysaccharide concentration, the increases were not as significant (p > 0.05) as for APTT in the low concentration range (0–0.6 mg/mL; Fig. 4): the APTT prolongation was found under low concentration while the PT prolongation was not significant. The polysaccharide did not affect the blood clotting time in the PT test up to 0.4 mg/mL, but increased about four times when the concentration increased to 0.8 mg/mL. This suggests that polysaccharides from Jew’s ear might mainly inhibit several targets involved in the intrinsic or common pathway, with less inhibition in the extrinsic pathway of the blood clotting process, this mechanism has been proposed in a previous report (Yoon et al., 2003). To confirm the anti-coagulant activity of crude polysaccharide, one portion of the aqueous extract from Jew’s ear was further extracted with 85% ethanol to yield the components of the low molecular part and the insoluble (in 85% ethanol) part. The low molecular fraction (85% ethanol extract) and the 85% ethanol insoluble part (polysaccharide) were further tested for APTT and PT activities. No clotting time prolongation was observed on the 85% ethanol extract in the APTT and PT tests. It suggested
250 APTT PT
200
Coltting time (s)
Effects of extraction techniques on anti-coagulant activities of aqueous extracts from Jew’s ear
a
150
100
50
Anti-coagulation activities of fungal polysaccharide extracted by HIPEF
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0
0.2
0.4
0.6
0.8
1
1.2
The concentration of crude polysaccharide (mg/mL) Figure 4. Concentration-dependent anti-coagulant activity of the crude polysaccharide precipitated from extract of Jew’s ear using high-intensity pulsed electric fields (HIPEF). Data are expressed as mean SEM from triplicate tests.
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The HIPEF aqueous extract was further fractionated using precipitation with absolute ethanol in order to isolate and characterise the anti-coagulant active substances. The predominant components in the precipitate were fungal polysaccharides. The results shown in Fig. 4 were performed with crude polysaccharide. The crude polysaccharide
0
C. Li et al. that the components with low molecular weight were not active components of Jew’s ear. Therefore, the crude polysaccharide from Jew’s ear was verified to be the active component in inhibiting platelet aggregation, and delaying blood clotting in the current study. Current findings suggest that crude polysaccharide from Jew’s ear extracted by the HIPEF technique could be developed as a functional food component with potential anti-coagulant and anti-platelet activity.
Conclusions The aqueous extract from the edible mushroom Jew’s ear extracted by HIPEF can significantly extend the blood clotting time in terms of APTT and PT; the polysaccharide component from Jew’s ear was a main source of the anti-coagulant compounds. Evaluation of the mushroom polysaccharide as an agent in thrombosis therapy requires further in vivo studies. The HIPEF method will be an effective method in the manufacture of natural extracts. Acknowledgements This project was supported by the Jilin Committee of Science and Technology (Grant No. 2010ZX09401-305).
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