Aquaculture 310 (2010) 13–19

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Effects of anthraquinones extracted from Rheum officinale Bail on the growth,

non-specific immune response of Macrobrachium rosenbergii
Bo Liu a,b,1,2, Xianping Ge a,b,1,2, Yanhui He b,1, Jun Xie a,b,⁎,1, Pao Xu a,b,⁎,1, Yijin He b,1, Qunlan Zhou b,1,
Liangkun Pan b,1, Ruli Chen b,1
a
Wuxi Fishery College, Nanjing Agriculture University, Wuxi 214081, China
b
Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center,
Chinese Academy of Fishery Sciences, Wuxi 214081, China

a r t i c l e i n f o a b s t r a c t

Article history: The effects of anthraquinone extracted from Rheum officinale Bail on growth, some non-specific immunite
Received 19 February 2010 parameters, and disease resistance of Macrobrachium rosenbergii were studied in this experiment. M. rosenbergii
Received in revised form 12 September 2010 were randomly divided into five groups: a control group was fed with basal diet, and four treated groups fed with
Accepted 15 September 2010
basal diet supplemented with 0.05%, 0.1%, 0.2%, and 0.4% anthraquinone extract for 8 weeks, respectively. The
growth, the variations of haemolymph lysozyme, alkaline phosphatase, nitrogen monoxide, total anti-oxidative
Keywords:
Macrobrachium rosenbergii
capacity, glutathione peroxidase, and malondialdehyde content and the relative level of hepatic HSP70 mRNA
Anthraquinone extract from Rheum officinale were investigated. The growth gain rate, length gain rate, haemolymph alkaline phosphatase activity and the
Bail relative level of hepatic HSP70 mRNA of M. rosenbergii fed with 0.1% anthraquinone extract for 6 or 8 weeks were
Immunity higher than those of the control. Weight gain rate and haemolymph alkaline phosphatase activity of M. rosenbergii
HSP70 fed with 0.2% anthraquinone extract for 8 weeks were higher than those of the control. However feed conversion
Growth ratios of M. rosenbergii fed with 0.1% and 0.2% anthraquinone extracts for 8 weeks were lower compared to those
of the control (P b 0.05). The haemolymph lysozyme activity and total anti-oxidative capacity in the group fed
with 0.05% anthraquinone extract for 8 weeks were significantly higher than those of the control. However, there
were no significant differences in haemolymph nitrogen monoxide concentrations and glutathione peroxidase
activities between all treatment groups and the control. The present study suggested that the ingestion of 0.1%–
0.2% anthraquinone extracts supplemented with a basal diet had the potential to stimulate immunity, increase
the gene expression of HSP70, and promote the growth of prawns.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction causing significant economic losses (Sung et al., 2000; Sarathi et al.,
2008).
Macrobrachium rosenbergii, also known as the giant river prawn, In order to control the disease, prophylactic chemo-therapeutants
the giant freshwater prawn or the Malaysian prawn, is the most and antibiotics have commonly been used in intensive aquaculture.
important cultivated freshwater shrimp species in the world. This However, the abuse of broad-spectrum chemo-therapeutants and
species (as well as other Macrobrachium) is commercially important antibiotics has resulted in an increased number of antibiotic-resistant
for its value as a food source. In China, the production of M. rosenbergii bacteria, impacting on the food safety for human (Cabello, 2006).
has been increasing in recent years. However, cultured M. rosenbergii Therefore, it is necessary to replace chemotherapeutic methods with
in China as well as in other parts of the world have suffered from serious non-chemotherapeutic ones, like vaccines, probiotics, immunostimu-
disease problem caused by viral infection and bacterial pathogens, lants, and natural therapeutics from plants (Verschuere et al., 2000;
Sakai, 1999; Yeh et al., 2009; Yin et al., 2009).
China has a rich background in traditional Chinese medicines and
⁎ Corresponding authors. Wuxi Fishery College, Nanjing Agriculture University, Wuxi most of them have been used as immunostimulants to treat human and
214081, China. Tel.: + 86 510 85557959; fax: +86 510 85553304. animal diseases for thousands of years (Tan and Vanitha, 2004). This is
E-mail addresses: [email protected] (B. Liu), [email protected] (X. Ge), [email protected] (J. Xie), because Chinese herbal medicines are easy and cheap to prepare, and
[email protected] (P. Xu).
1
The address of all authors: No.9 Shanshui East Road, Wuxi PC 214081, FFRC CAFS,
are effective with fewer side effects during treatment of diseases (Jian
China. and Wu, 2003, 2004). They contain many types of active components,
2
The first author: Bo Liu and Xian ping Ge. like organic acids, alkaloids, polysaccharides, anthraquinones, volatile

0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.09.020
14 B. Liu et al. / Aquaculture 310 (2010) 13–19

oils, flavonoids, glycosides, tannic acid substance, trace elements respectively. Anthraquinone extract (1.4 mg of emodin per ml extract)
and some unknown immune active factors, which can strengthen the from R. officinale was supplied by Zhixin Pharmaceutical Co., Ltd.,
metabolism of aquatic animals, improve the composition of protein Nanjing, China. Diets were produced in Wuxi Tongwei Feed Co., Ltd.,
and enzymes, and enhance the growth of animals (Cheng et al., 2002). China.
The immunostimulating activity of herbal components has been
most widely studied in fish (Jian and Wu, 2004; Sivaram et al., 2004; 2.2. Rearing management
Yin et al., 2006, 2009; Xie et al., 2008; Zhang et al., 2009), shrimps
(Immanuel et al., 2004; Yeh et al., 2009), rats, hens or human cell lines Freshwater prawns were acclimatized in concrete tanks for
(Shan et al., 1999; Chen et al., 2006; Ma and Shan, 2007), and in the 15 days and then fed by hand on the trial diet with the feeding
case of some herbs even the molecular mechanisms of their effect are amount about 5.0%–10.0% BW, three times a day: 6:00–6:30, 12:00–
known (Shao et al., 2004; Liu et al., 2004b; Chen et al., 2006; Lei and 12:30, and 18:00–18:30. An underground water source was used.
Zeng, 2008; Ma and Shan, 2007; Yuan et al., 2008). Anthraquinone Water was oxygenated during day and night using the aerator. Silt
extract from Rheum officinale Bail containing emodin, chrysophenol, was siphoned daily and one-third of water was exchanged in each
rhein, etc. has been used as an immunostimulant. In mammals the tank once a week; water temperature was measured every day, and
studies indicated that emodin could inhibit inflammation (Chang et water quality was checked every week. During the test period the
al., 1996), resist oxidization and scavenge oxygen free radicals (Huang water quality parameters on average were the following: water
et al., 1995), protect the liver (Lin et al., 1996) and increase immunity temperature 24.5 ±4.32 °C, DO N 5 mg l − 1 , NH 3 b 0.05 mg l − 1 ,
of animals (Wang et al., 1995). In aquaculture, especially in our lab H2S b 0.1 mg l− 1, pH 8.0–8.2. The feeding amount increased every
recent studies showed that anthraquinone extract from R. officinale other week and was based on body weight measurement every two
significantly enhanced immunity, stress resistance and growth weeks. After the completion of the test period of 56 days, haemolymph
performance of common carp (Cyprinus carpio) (Xie et al., 2008). and liver samples were collected, and weight gain was measured.
However, few reports have been found about the effects of Chinese
herbal medicines on haemolymph metabolites, anti-oxidization
2.3. Haemolymph and hepatopancreas sample collection
enzymes, and the expression of hepatic heat shock protein 70 of
aquatic animals (Lei and Zeng, 2008). The objectives of the present
Nine haemolymph samples were taken from each group at the end
study were to evaluate the effect of anthraquinone extract from
of 1st, 2nd, 3rd and 4th weeks after the initiation of feeding.
R. officinale on the growth and non-specific immune parameters of
Haemolymph was sampled from abdominal cavity or around the
M. rosenbergii, then to go further and study the molecular mechanisms
heart ventricle using 1 ml syringes kept at 4 °C. Alsever's solution was
of anthraquinone extract, and to provide a theoretical basis for
used as an anticoagulant; the proportion of haemolymph to antico-
prophylaxis of shrimp diseases by using anthraquinone extract.
agulant was 1:1 (Rodriguez et al., 1995). Hepatopancreas samples
were collected and frozen in liquid nitrogen and stored at −80 °C until
2. Materials and methods further analysis.

2.1. Freshwater prawn, Chinese medicine and diets

2.4. Measurement of haemolymph immune and anti-oxidization parameters
Freshwater prawns (M. rosenbergii) were supplied by fish farm
The haemolymph nitrogen monoxide concentration was deter-
of Freshwater Fisheries Research Center, Chinese Academy of
mined by the method of Zhu et al. (2006b). Lysozyme activity was
Fishery Sciences in China. Freshwater prawns were reared in our
determined by the method of Hua et al. (2005) in a Beckman Cx-4
experimental fish farm. They were allocated to 15 concrete tanks
type Auto Bio-chemical Analyzer. Test kits of nitrogen monoxide
(800 × 200 × 100 cm) and acclimatized for 15 days. Thereafter, fresh-
lysozyme activity were bought from Nanjing Jiancheng Biological
water prawns were randomly divided into five groups: one control and
Engineering Research Institute. Haemolymph alkaline phosphatase
four treatment groups, in triplicate (three tanks, 1000 individuals per
activity was determined by the colorimetric method of Pinoni and
tank, with an average body weight of 1.07 ± 0.11 g). After acclimati-
López Mañanes (2004) (test kit from Shanghai Fudan Zhangjiang Bio
zation, the control group and the four experimental groups were fed
Medical Co., Ltd., China) in a Beckman Cx-4 type Auto Bio-chemical
with the basic diet (see Table 1) and the basic diet supplied with 0.05%,
Analyzer (Beckman Coulter, USA).
0.1%, 0.2% and 0.4% of anthraquinone extract from R. officinale,
The haemolymph total anti-oxidative capacity, glutathione perox-
idase activity and malondialdehyde content were determined by the
Table 1 methods described by Rice-Evans and Miller (1994), Wang et al.
The basal diet and nutrition levels of M. rosenbergii. (1999) and Xiang and Wang (1990), respectively. Test kits of total
anti-oxidative capacity, glutathione peroxidase activity and malon-
Ingredients (%) Nutrition levels
dialdehyde content were bought from Nanjing Jiancheng Biological
Fish meal 24 Dry matter (%) 89.13 engineering Research Institute.
Soybean meal 20 Gross energy (kJ/g) 17.73
Peanut meal 16 Crude protein (%) 39.74
Rapeseed meal 8 Total phosphorus (%) 1.62 2.5. Measurement of HSP70
Extruded soybean 8 Calcium (%) 1.48
Wheat midding 17 Methionine + cystine (%) 1.10
HSP70 cDNAs were amplified by PCR using specific primers chosen
Fish oil 2 Lysine (%) 2.09
Lecithin 1 Threonine (%) 1.34 in their cDNA sequences in M. rosenbergii from GenBank (accession
Cholesterol 0.2 no. AY466445.1). Primers of β-actin were based on M. rosenbergii
Chlorinated choline 0.3 cDNA sequences in GenBank (accession no. AY626840.1). Primers were
Vitamin additive 0.5
as follows: (1) 5′-TGACAAGGGTCGCCTCAGTA-3′ and 5′-CAT-
Mineral additive 1
Calcium dihydrogen phosphate 2 TATCTTGTTGCGATCCTC-3′ for the HSP70 cDNA; (2) 5′-TCCGTAAG-
Total 100 GACCT GTATGCC-3′ and 5′-TCGGGAGGTGCGATGATTTT-3′ for the β-
Note: Gross energy (GE) kJ g− 1: protein 23.64 kJ g− 1, fat 39.54 kJ g− 1, carbohydrate
actin cDNA. All primers were synthesized by Shanghai Biocolor
17.15 kJ g− 1; and the others are measured in the nutrition levels. Vitamin and mineral BioScience & Technolgy Company, China. The PCR products were all
additives were provided by Nanjing Huamu Animal Research Institute. 100–150 bp long.
 B. Liu et al. / Aquaculture 310 (2010) 13–19 15

Total RNA was extracted from M. rosenbergii hepatopancreas with computes an expression ratio, based on real-time PCR efficiency and
Trizol reagent (Invitrogen Co.). RNA samples were treated with RQ1 the crossing point deviation of the sample versus a control. PCR
RNase-Free DNase (Dalian Takara Co. Limited, China), to avoid efficiency was determined with a standard curve, using serial dilution of
genomic DNA amplification. cDNA was generated from 500 ng cDNA; ΔΔCT = (CT,Target − CT,Bactin)time x − (CT,Target − CT,Bactin)time o.
DNase-treated RNA using ExScriptTM RT-PCR Kit (Dalian Takara Co.
Limited, China) and the reverse transcription PCR reaction solution 2.6. Data statistics and analysis
consisted of 500 ng RNA, 2 μl 5× Buffer, 0.5 μl dNTP Mixture (10 mM
each), 0.25 μl RNase Inhibitor (40 U μl− 1), 0.5 μl dT-AP Primer Duncan's multiple range tests (SPSS v11.5) were used to
(50 mM), 0.25 μl ExScript™ RTase (200 U μl− 1), and DEPC H2O, up determine the differences between groups. Significant differences
to 10 μl. The reaction conditions were as follows: 42 °C for 40 min, were expressed at a significance level of P b 0.05. All the results were
90 °C for 2 min, and 4 °C thereafter. expressed as means ± standard error ( X ± SE).
Real-time PCR was used to determine mRNA levels based on SYBR
GreenIfluorescence kit (Livak and Schmittgen, 2001). Real-time PCR 3. Results
was performed in a Mini Opticon Real-Time Detector (BIO-RAD, USA).
The fluorescent quantitative PCR reaction solution consisted of 12.5 μl 3.1. The effect of anthraquinone extract on the growth of freshwater
SYBR® premix Ex Taq™ (2×), 0.5 μl PCR Forward Primer (10 μM), 0.5 μl prawn
PCR Reverse Primer (10 μM), 2.0 μl RT reaction mix (cDNA solution),
and 9.5 μl dH2O. The reaction conditions were as follows: 95 °C for Weight gain rates of M. rosenbergii fed with 0.1% and 0.4%
3 min, followed by 45 cycles consisting of 95 °C for 10 s and 60 °C for anthraquinone extracts for 6 weeks and fed with 0.1% and 0.2%
20 s. The florescent flux was then recorded and the reaction continued anthraquinone extracts for 8 weeks were significantly higher than
at 72 °C for 3 min. The dissolution rate was measured between 65 °C and those of control (P b 0.05, Fig. 1A). In addition, the length gain rate of
90 °C. Each increase of 0.2 °C was maintained for 1 s and the fluorescent M. rosenbergii fed with 0.1% anthraquinone extract for 4, 6 or 8 weeks
flux was recorded. Relative quantification of the target gene transcript was higher than that of control (P b 0.05, Fig. 1C) and the length gain
(HSP70) with a chosen reference gene transcript (β-actin) was rate of M. rosenbergii supplemented with 0.4% anthraquinone extract
calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001). for 6 weeks was higher than that of control (P b 0.05, Fig. 1C). How-
This mathematical algorithm, which needs no calibration curve ever the feed conversion ratio of M. rosenbergii fed with 0.05%

(A)
1200 Control
* *
Weight gain rate(%)

1000 0.05%
* 0.10%
800 *
0.20%
600
0.40%
400
200
0
2 4 6 8
Time (weeks)
(B)
3
Feed conversion ratio

2.5
*
2
*
1.5 * *
1
0.5
0
2 4 6 8
Time (weeks)

(C)
200
Length gain rate (%)

*
* *
150
*
100

50

0
2 4 6 8
Time (weeks)

Fig. 1. Effects of anthraquinone extract from R. officinale Bail on weight gains (A), feed conversion ratio (B), length gain rate (C) of M. rosenbergii. Note: Data are expressed as the
mean of triplication of every group ± SE (n = 3), and compared at different dose using Duncan's multiple range tests (SPSS, Ver.11.5). Significant differences (ρ b 0.05) from the
control are marked with asterisks. Weight gain rate (%) = 100 × (Average terminal body weight –Average initial body weight) / Average initial body weight; Feed conversion
ratio = Feed consumption / Weight gain; Length gain rate (%) = 100 × (Average terminal body length –Average initial body length) / Average initial body length.
16 B. Liu et al. / Aquaculture 310 (2010) 13–19

(A) Control
80 *
0.05%

Lysozyme activity
70
60 0.10%

(ug/mL)
50 0.20%
40 0.40%
30
20
10
0
2 4 6 8
Time (weeks)

(B)
Alkaline phosphatase activity

150

* *
100
(U/L)

50

0
2 4 6 8
Time (weeks)

(C)
Nitrogen oxide content

20

15
(umol/L)

10

0
2 4 6 8
Time (weeks)

Fig. 2. Effects of anthraquinone extract from R. officinale Bail on haemolymph lysozyme (A), alkaline phosphatase (B) and nitrogen monoxide (C) of M. rosenbergii. Note: Data are
expressed as the mean of triplication of every group ± SE (n = 9), and compared at different dose using Duncan multiple range tests (SPSS, Ver.11.5). Significant differences (ρ b 0.05)
from the control are marked with asterisks.

anthraquinone extract for 4 weeks, 0.2% anthraquinone extract for increasing in all treated groups compared to the control (P N 0.05,
6 weeks, 0.1% and 0.2% anthraquinone extract for 8 weeks was lower Fig. 2C).
compared to the control (P b 0.05, Fig. 1B). There were no significant
differences in weight gain rate, length gain rate and feed conversion
ratio of the other treated groups compared to the control (P b 0.05). 3.3. The effects of anthraquinone extract on haemolymph anti-
oxidization enzymes activity of freshwater prawn
3.2. The effect of anthraquinone extract on haemolymph lysozyme,
alkaline phosphatase and nitrogen monoxide of freshwater prawn The haemolymph total anti-oxidative capacity significantly in-
creased in the group treated with 0.05% anthraquinone extract for 6 or
On the first 6 weeks, there were no significant differences in the 8 weeks compared to the control (P b 0.05, Fig. 3A). There were no
haemolymph lysozyme activities between the groups fed with significant differences in this parameter in any of the treated groups
anthraquinone extracts and the control group (P N 0.05, Fig. 2A). after 2 or 4 weeks of feeding although there was a tendency of higher
However, lysozyme activity in the group supplemented with 0.05% oxidizing activity in all treatment groups, compared to the control
anthraquinone extract for 8 weeks after the food intake was (P N 0.05, Fig. 3A).
significantly higher than that of the control (P b 0.05, Fig. 2A). Haemolymph glutathione peroxidase activity did not significantly
Haemolymph alkaline phosphatase activities in all treated groups change in treated groups, compared to the control group, during the
were not significantly different from the control, after 2 or 4 weeks of whole experiment (P N 0.05, Fig. 3B). Compared with the control,
feeding (P N 0.05, Fig. 2B). After 6 weeks, this parameter was hepatic malondialdehyde content significantly decreased in the group
significantly higher in the group treated with 0.1% anthraquinone fed with 0.4% anthraquinone extract for 8 weeks (P b 0.05, Fig. 3C).
extract and after 8 weeks, in the group treated with 0.2% anthraqui- There were no significant differences of this parameter in the treated
none extract, compared to the control (P b 0.05, Fig. 2B). groups after 2, 4 or 6 weeks of feeding compared to the control group
Haemolymph nitrogen monoxide concentrations were not signif- (P N 0.05, Fig. 3C). However, the haemolymph malondialdehyde
icantly different in any of the treated groups compared to the control concentrations in all treated groups trended downward especially in
(P N 0.05, Fig. 2C). However, this parameter showed a tendency of week 8 (P N 0.05, Fig. 3C).
 B. Liu et al. / Aquaculture 310 (2010) 13–19 17

(A)

Total antioxidative capacity

30 Control
* 0.05%
25
* 0.10%
20

(U/mL)
0.20%
15 0.40%
10
5
0
2 4 6 8
Time (weeks)

(B)
Glutathione peroxidase activity

1400
1200
1000
(U/mL)

800
600
400
200
0
2 4 6 8
Time (weeks)

(C)
25
Malondialdehyde content

20
(nmol/mL)

15

10 *

0
2 4 6 8
Time (weeks)

Fig. 3. Effects of anthraquinone extract from R. officinale Bail on haemolymph total anti-oxidative capacity (A), glutathione peroxidase activity (B), and malondialdehyde content
(C) of M. rosenbergii. Note: Data are expressed as the mean of triplication of every group ± SE (n = 9), and compared at different dose using Duncan multiple range tests (SPSS,
Ver.11.5). Significant differences (ρ b 0.05) from the control are marked with asterisks.

3.4. Effects of anthraquinone extract on the relative level of hepatic and in the group fed with 0.2% anthraquinone extract for 8 weeks,
HSP70 mRNA of freshwater prawn compared to the control (P b 0.05, Fig. 4). However, the level of
hepatic HSP70 mRNA did not differ significantly in any of the
The relative level of hepatic HSP70 mRNA significantly increased treated groups compared to the control after 2 or 4 weeks of feeding
in the group fed with 0.1% anthraquinone extract for 6 or 8 weeks, (P N 0.05, Fig. 4).
Relative level of HSP70 mRNA

Control
3 0.05%
* *
2.5 0.10%
(arbitrary units)

0.20%
2 * 0.40%
1.5
1
0.5
0
2 4 6 8
Time (weeks)

Fig. 4. Effects of anthraquinone extract from R. officinale Bail on the relative level of hepatic HSP70 mRNA of M. rosenbergii. Note: Data are expressed as the mean of triplication of every
group ± SE (n = 9), and compared at different dose using Duncan multiple range tests (SPSS, Ver.11.5). Significant differences (ρ b 0.05) from the control are marked with asterisks.
18 B. Liu et al. / Aquaculture 310 (2010) 13–19

4. Discussion 1995; Wang et al., 1995; Lin et al., 1996). In fish, dietary intake of
Chinese herbal extracts can also significantly enhance the antioxidant
This study was conducted to evaluate how different anthraquinone ability (Rao et al., 2006; Christybapita et al., 2007; Xie et al., 2008).
extracts from R. officinale affect haemolymph metabolites and immune Consistent with these studies, in the present study the haemolymph
parameters, as well as the expression of hepatic HSP70 in M. rosenbergii. total anti-oxidative capacity was enhanced after feeding with 0.05%
In aquaculture, immunostimulants can stimulate the innate immune anthraquinone extract for 6 or 8 weeks, while hepatic malondialde-
system by activation or increasing the activity of granulocytes and hyde content significantly decreased in the group fed with 0.4%
macrophages, and increasing the number of phagocytes (Sakai, 1999; anthraquinone extract for 8 weeks, compared with the control. Thus
Yin et al., 2006, 2009). They can also increase protein synthesis to anthraquinone extract can reduce lipidic superoxide harm to some
produce more of the molecules involved in innate metabolism system extent.
such as lysozyme, alkaline phosphatase, and nitrogen monoxide (Xie Heat shock proteins (HSPs) are conserved proteins induced by heat
et al., 2008; Rao et al., 2006; Liu et al., 2008). and numerous noxious stimuli, including high temperature, heavy
Lysozyme is the important part of haemolymph protective enzyme metals, oxygen free radicals, virus, and pathologic stresses (Kiang and
system in the crustacean. It is responsible for breaking down the Tsokos, 1998; Vega et al., 2006). The 70 kDa heat shock protein
polysaccharide wall of bacteria and thus contributes to protection (HSP70) is one of the most extensively studied heat shock proteins,
from pathologic infection (Hauge et al., 2002; Rojtinakem et al., 2002). which are also known to facilitate the transport of damaged proteins to
Many authors have reported that ingestion of immunostimulants, lysosomes and have general protective functions in all living
such as β-glucan, chitin, chitosan or the root of Astragalus membra- organisms (Kiang and Tsokos, 1998). Forsyth et al. (1997) reported
naceus enhanced the lysozyme activity in Atlantic salmon (Salmo of an increase in HSP70 mRNA in the liver and head kidney after coho
salar), turbot (Scophthalmus maximus), tilapia (Oreochromis niloticus) salmon (Oncorhynchus kisutch) were challenged with Renibacterium
and common carp (C. carpio) (Santarem et al., 1997; Paulsen et al., salmoninarum. Ackerman and Iwama (2001) reported that an active
2001; Gopalakannan and Arul, 2006; Yin et al., 2006, 2009). In our acute infection with Listonella anguillarum resulted in a classic stress
previous study with common carp (C. carpio jian), enhanced lysozyme response and the increase in hepatic and head kidney HSP70 in
activity was measured when fish were fed with anthraquinone extract rainbow trout. The studies indicated that Chinese herbs could improve
(Xie et al., 2008). Results of the recent experiment showed that the expression of HSP70 in rat (Chen et al., 2006), hen (Ma and Shan,
haemolymph lysozyme activity of the group fed with 0.05% 2007) and white shrimp (Lei and Zeng, 2008). In this study, the
anthraquinone extract for 8 weeks was significantly higher than expression of HSP70 significantly increased in the group fed with 0.1%
that of the control. anthraquinone extract for 6 or 8 weeks, and 0.2% anthraquinone
ALP is an important regulative enzyme which has been associated extract for 8 weeks, compared with the control. However, the
with different essential functions in all living organisms (Sukhanova relationship between Chinese herbs and HSP70 remains to be further
et al., 1996; Pinoni and López Mañanes, 2004). In Labeo rohita, the ALP investigated.
activity drastically increased after feeding of 0.01–0.05% Achyranthes Robertson et al. (1990) showed an increased protection against
aspera, compared to the control fish (Rao et al., 2006). In our bacterial infection in Atlantic salmon, which correlated with an
experiment, the haemolymph alkaline phosphatase activities were increment in serum lysozyme levels, phagocytic activity and bacte-
higher in the group fed with 0.1% anthraquinone extract for 6 weeks ricidal activity of head kidney leucocytes. In this study, similarly all the
and 0.2% anthraquinone extract for 8 weeks, than those of the control. results can be correlated with the resistance against bacterial infection
Nitrogen monoxide is a multiple bioactive molecule. In immune to some degree, including the increase of haemolymph lysozyme
system nitrogen monoxide produced by nitric-oxide synthases has anti- activities, alkaline phosphatase activities, anti-oxidation abilities and
malignant tumor, antibacterial, antiviral and immune-regulatory the decrease of hepatic malondialdehyde content in the groups fed
activities (Tafalla et al., 1999; He, 2000). In allogynogenetic crucian with 0.1–0.2% of anthraquinone extracts. Some researchers also have
carp, nitrogen monoxide concentrations drastically increased in the reported that Chinese herbs could protect the fish or shrimp from
doses of 0.02–0.03% plant extracts compared to the control fish (Liu pathogenic infection, increase immune factors such as blood lyso-
et al., 2008). In our experiment, there were no significant differences in zyme, the phagocytic activity, nitrogen monoxide content, phagocytic
haemolymph nitrogen monoxide concentrations between all treated activity, and so on (Francis et al., 2002; Xie et al., 2008; Zhang et al.,
groups and the control. However, this parameter showed a tendency of 2009) and indirectly increase growth of aquatic animal. Dietary
increasing in all treated groups compared to the control. All these results supplementation of Quillaja saponin increased the growth rate in
indicated that the feeding of 0.1–0.2% anthraquinone extracts for 6 or common carp (Francis et al., 2002). As oral administration of
8 weeks could improve the immunity of M. rosenbergii. However there anthraquinone extract was done in our previous study (Xie et al.,
were no significant differences in haemolymph lysozyme activities, 2008). In this study, the results showed that anthraquinone extracts in
alkaline phosphatase activities and nitrogen monoxide concentrations 0.1–0.2% could improve haemolymph lysozyme activities, alkaline
between all treatment groups and the control after 2 or 4 weeks of phosphatase activities and anti-oxidation abilities, and improve
feeding. So it is suggested that the long-term ingestion of anthraquinone weight and length gain. However, overdose of supplements of
extract could enhance the immune ability of M. rosenbergii. anthraquinone extracts would be unfavorable to shrimp growth.
The non-specific defense mechanisms of fish include neutrophil The overdose of Chinese herbs might cause imbalance of bacterial
activation, the production of peroxidase, oxidative free radicals and populations in the intestine (Liu et al., 2004a), result in immune
initiation of other inflammatory factors (Ainsworth et al., 1991). suppression and affect the palatability of feeds. However, answering
Aquatic animals maintain complex systems of multiple types of these questions needs further studies.
antioxidants, such as glutathione, catalase, superoxide dismutase and
various peroxidases. Activation of non-specific defense mechanisms in
fish is evident by increased total anti-oxidative capacity, glutathione 5. Conclusions
peroxidase activity, the reactive oxygen and nitrogen species (Lewis
et al., 1995; Itou et al., 1996), which can prevent the oxidation of other In short, all these results indicate that the dose of 0.1–0.2%
molecules, and protect the organism from oxidative damage, reduce anthraquinone extracts can increase the gene expression of HSP70,
lipid hydroperoxides. In mammals, Chinese herbal anthraquinone enhance haemolymph lysozyme activities, alkaline phosphatase activ-
extracts (such as emodin, chrysophenol, rhein, etc.) can clear oxygen ities, anti-oxidation abilities, and promote the growth of freshwater
free radicals and prevent the effect of oxide damage (Huang et al., prawns.
 B. Liu et al. / Aquaculture 310 (2010) 13–19 19

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