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Molluscicidal activity and physiological toxicity of Macleaya cordata alkaloids components on snail Oncomelania hupensis
Pesticide Biochemistry and Physiology xxx (xxxx) xxx–xxx
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Molluscicidal activity and physiological toxicity of Macleaya cordata alkaloids components on snail Oncomelania hupensis Wenshan Kea,⁎, Xiong Lina, Zhengshen Yua, Qiqiang Sunb, Qian Zhangb a b
School of Life Science, Hubei University, Wuhan 430062, PR China Research Institute of Forestry Chinese Academy of Forestry, Beijing 100086, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Macleaya cordata (Willd) R. Br, Oncomelania hupensis Molluscicidal activity Enzyme activity Isozyme
In order to search new local plant molluscicides for the control of the vectors of schistosomiasis, leaves of Macleaya cordata (Willd) R. Br. were used to extract and separate alkaloid components by thinner acid method and column chromatography, and the molluscicidal effect of alkaloid components against snail Oncomelania hupensis was determined by bioassay. The results showed that 7 alkaloid components (AN1-7) were obtained after extracting and separating alkaloids from the leaves of M. cordata, where AN2 was found being the most toxic against snail O. hupensis with 48 h LC50 and LC90 values of AN2 of 6.35 mg/L and 121.23 mg/L, respectively. Responses of some critical enzymes to AN2, including activities of Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate transaminase (AST), Malic dehydrogenase (MDH) and Succinate dehydrogenase (SDH) in both cephalopodium and liver, were also detected through experiments, which also explored esterase isozyme (EST) exposed to AN2 in liver tissue. The results showed that AN2 significantly inhibited the activities of SDH, MDH and esterase isozyme, as AN2 significantly stimulated the activities of ALP, ALT and AST to increase at a low concentration (e.g. 25 mg/L), while significantly inhibited the activities of these enzymes at a high concentration (100 mg/L). These results indicated that AN2 not only inhibited protein synthesis, and respiratory chain oxidative phosphorylation, but also caused hepatocellular injury and reduced the detoxification ability of liver.
1. Introduction Schistosomiasis is the second most prevalent endemic disease after malaria in tropical and subtropical regions [1] and remains a serious disease in China (Schistosoma japonicum) by devastating human health [2]. Recent monitoring data revealed that schistosomiasis was re-emerging in China, especially along the Yangtze River and in the lakes region of Jiangxi and Hunan provinces [3]. As the snail Oncomelania hupensis is the only intermediate host of Schistosoma japonicum that causes schistosomiasis in China, the extermination of snails is efficient for control of schistosomiasis. Niclosamide, the only commercially available molluscicide recommended by WHO for large scale use in schistosomiasis control [4], however, is limited by its high cost of synthetic compounds, along with increasing concern of possible snail resistance to these compounds and their toxicity in non-target organisms, which have raised the study of plant molluscicides [5]. The use of plants with molluscicidal properties is a technology that is simple, inexpensive, biodegradable and appropriate for local control of the snail vector, especially in rural areas of developing countries where schistosomiasis is endemic [6].
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As a perennial plant, Macleaya cordata (Willd) R. Br. is a member of the Macleaya genus in the family of Papaveraceae, which is mainly distributed in North America, Europe, South and Northwest China. M. cordata has been widely used as a folk herbal medicine in China to cure cervical cancer and thyroid cancer, and to relieve insect bites and ringworm infection [7,8]. Currently, M. cordata is utilized as a traditional Chinese medicine for the treatment of inflamed wounds, arthritis, rheumatism arthralgia, and trichomonas vagi-nalis [9]. In our initial study, it was found that plant M. cordata had strong molluscicidal activity against snails O. hupensis, with the molluscicidal effect stronger than that of Nerium indicum Mill and Arisaema erubescens Schott [10,11]. The objectives of this study were: (1) to furtherly examine the molluscicidal activities of M. cordata extracts and try to find out the strongest molluscicidal component in the plant; and (2) to reveal the possible physiological toxicity mechanism of the molluscicidal component from activity changes in Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate transaminase (AST), Malic dehydrogenase (MDH), Succinate dehydrogenase (SDH) and esterase isoenzyme (EST).
Corresponding author. E-mail address: [email protected] (W. Ke).
http://dx.doi.org/10.1016/j.pestbp.2017.08.016 Received 21 March 2017; Received in revised form 17 August 2017; Accepted 27 August 2017 0048-3575/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Ke, W., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.08.016
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2. Materials and methods
2.5. Enzyme assay
2.1. Oncomelania hupensis
According to the testing result of molluscicidal activity above, the strongest molluscicidal active component of alkaloid (AN2) was used to examine the snail enzyme assay. Two hundred snails were randomly divided into four groups and exposed to each concentration level of AN2 (0, 25 mg/L, 50 mg/L, and 100 mg/L) for 48 h, with dechlorinated water as the control. Afterwards, the snails were washed with dechlorinated water, and their cephalopodium and liver tissues were excised for biochemical analysis - a process recommended by Li [15]. Phosphatic buffer solutions (pH 6.8) were added (1 drop/snail) to the cephalopodium and liver which were then homogenated in ice incubation condition, where the homogenates were centrifuged at 4000 rpm for 10 min at 0 °C. Then, one part of supernatant was stored at − 20 °C for the measurement of enzyme activities and electrophoresis. The processes of preparation of snail cephalopodium and liver were replicated for 3 times in the same conditions.
Adult O. hupensis snails (9–11 mm in length) were collected from farmfield of Taihu countryside of Jingzhou, Hubei Province, China, amongst which the healthy ones were selected by cercariae escaping method to get rid of the infected [12]. Then the snails were kept in a laboratory at 20 °C for a week before experiment. 2.2. Plants Leaves of the plant M. cordata at vegetative growth phase before flowering were collected from Lushan Mountain in central China, with their fresh and dry weights recorded. 2.3. Extracts of alkaloids The total alkaloids of M. cordata were extracted according to the modified ethanol extract method [13,14]. Weigh dryly pulverized M. cordata powder for 500 g with 95% ethanol at M:V = 1:3 added, and immerse it for 3 days. Filter the ethanol for the distillation concentration in a cyclotron evaporimeter. With the immersion repeated for 3 times, mix and dry the concentrate to obtain the extractum. Then, solve the total alkaloid extractum of M. cordata by 1% hydrochloric acid solution, extract it via chloroform at a volume ratio of 1:1, remove the lipids and pigments in acid water, and keep the acid aqueous phase. Adjust the pH of acid water to around 6.8 through ammonia, and remove the tannin fraction by filtration. Finally, continue to adjust the pH of acid water to 8.0–9.0, and again, extract the solution via chloroform at a volume ratio of 1:1, retain the chloroform phase, and remove the chloroform layer via the cyclotron distillation, so as to obtain the total alkaloids of M. cordata. Components of the total alkaloids were separated with column chromatography. D-101 Macroporous resin, selected as the stationary phase, with ethanol as the moving phase, was columned by the wet packing column method. Firstly fill the chromatographic column with distilled water, and weigh the macroporous resin for 10 g to gradually pour the resion into the chromatographic column with a glass rod for drainage. Then fill the samples as the columning was finished. For the last step, elution was formed by water, gradient aqueous ethanol (10%, 20%, …, 90%), and absolute ethanol in turn with each moving phase of 100 ml. The moving speed was controlled, as every 5 ml was collected in a tube until the eluent was detected as no precipitation via Dragendorff's reagent. The collected liquors were further analyzed by TLC(thin-layer chromatography) method. Development solvent (chloroform-methanol (19:1)) was used to separate the alkaloids and iodine vapor was used to test the separation point in TLC. Rf values were measured after finishing of the separation, whereas the liquids from different tubes with same Rf values were merged into one tube as one component. Liquids of seven alkaloid components (NO. 1–7) (AN1-AN7) were gained in the end and then dried (freeze drying) for next experiment.
2.5.1. Assay of enzyme activities The enzyme activities were measured according to Guilbault et al.'s [16] methods of enzyme kinetic assay. Enzyme activities were expressed as the amount of substrate hydrolyzed or production liberated in 1 mol/min/g protein in the supernatant. Total protein level of supernatants was estimated according to Bradford's method [17]. Enzyme activities were expressed as the amount of substrate hydrolyzed or production liberated in 1 mol/min/g protein in the supernatant. ALP kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the ALP activities were determined at 405 nm with 4nitrophenyl phosphate as the substrate. ALT, AST, LDH, SDH and MDH kits were purchased from Shanghai AILEX Technology Co., Ltd., and the enzyme activities were determined at 340 nm.
2.5.2. Electrophoresis assay of EST Similar to the snail treatments as above, two treatment levels (0 and LC50) were designed. Having exposure for 24 h and 48 h, the snails were carefully crushed so that the liver tissues could be extracted. Phosphatic buffer solutions (pH 6.8) were added (1 drop/snail) to the liver tissues, which were then homogenated on ice. Then the homogenates were centrifuged at 8000 g for 10 min at 0 °C. The supernatant was stored at − 20 °C for enzyme electrophoresis. The supernatant (10 μL each sample, 2 replicates) of snail liver tissues was electrophoresed with a vertical electrophoresis system ECP3000. Polyacrylamide gel electrophoresis (PAGE) was carried out with 20 mA constant current until the tracing dye reached the bottom of the gel. The chemicals were purchased from Sigma, with the separating gel consisted of 7.5% (acrylamide + bisacrylamide) and 0.04 mol/L Tris-HCl, with pH of 8.8; the stacking gel consisted of 3.75% (acrylamide + bisacrylamide) and 0.01 mol/L Tris-HCl, with pH of 6.8; and the buffer was Tris-glycine buffer, with pH of 8.3. After electrophoresis, gels were stained, washed, fixed and then photographed. Acetic acid (7.5%) was the stationary phase.
2.4. Testing for molluscicidal activity Seven different alkaloids components separated from leaves of M. cordata were respectively dissolved with distilled water and three treatments (0, 25 mg/L, 50 mg/L and 100 mg/L) were made to test the molluscicidal activity. The snails were collected in a nylon mesh bag (30 snails per bag) and immersed into 500 mL solution of a known concentration in the extracts. For each treatment, five bags (30 snails per bag) of snails were exposed for 24, 48, 72, 96 and 120 h, respectively, with dechlorinated tap water exposure as the control. For checking the mortality, no response to a needle probe under dissecting microscope was the evidence of death. The experiments were conducted at 21 °C, each of which was replicated three times.
2.6. Data analysis The mortalities of snails were expressed as the mean of three replicates. The effect of alkaloid components on O. hupensis was expressed by LC50 and LC90 and their 95% confidence limit (95% cl). The results of enzyme activities were expressed as means ± SE of the three replicates. One way ANOVA and SSR (Duncan's repeat comparison) were used to detect significant differences (P < 0.05 or 0.01).
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Table 1 Mortality of snails Oncomelania hupensis exposed to 7 alkaloid components extracted from Macleaya cordata leaves under laboratory conditions. Component
Concentration (mg/L)
Mortality of snails (%) 24 h
AN 1
AN 2
AN 3
AN 4
AN 5
AN 6
AN 7
NIC DW
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 1
Table 3 Effect of AN2 on enzyme activity in cephalopodium (u/g prot) of snail Oncomelania hupensis after 48 h exposure.
20.00 13.33 10.00 56.67 46.67 36.67 56.67 36.67 33.33 46.67 40.00 13.33 26.67 26.67 23.33 20.00 6.67 10.00 3.33 3.33 0 83.33 0
48 h 50.00 36.67 26.67 83.33 76.67 73.33 80.00 73.33 40.00 73.33 50.00 36.67 66.67 56.67 40.00 40.00 20.00 16.67 23.33 10.00 6.67 100 6.67
72 h
96 h
56.67 43.33 40.00 90.00 83.33 83.33 80.00 73.33 43.33 73.33 56.67 50.00 70.00 60.00 50.00 46.67 26.67 16.67 26.67 16.67 10.00 100 10.00
70.00 60.00 56.67 90.00 90.00 83.33 80.00 73.33 50.00 80.00 66.67 66.67 70.00 63.33 56.67 46.67 26.67 16.67 26.67 16.67 10.00 100 10.00
120 h 76.67 63.33 56.67 96.67 90.00 83.33 90.00 73.33 56.67 86.67 70.00 66.67 76.67 66.67 60.00 56.67 36.67 23.33 33.33 23.33 16.67 100 10.00
Group (mg/L)
MDH
DW AN2
2450.23 2083.94 1971.62 1598.97
⁎⁎
25 50 100
SDH ± ± ± ±
23.49 10.92⁎⁎ 74.43⁎⁎ 82.57⁎⁎
21.85 18.75 17.53 16.01
ALP ± ± ± ±
0.21 0.65⁎⁎ 0.37⁎⁎ 3.49⁎⁎
1382.51 1536.12 1494.91 1273.86
± ± ± ±
28.76 37.28⁎⁎ 114.62⁎⁎ 125.61⁎⁎
Compared to DW control, P < 0.01.
Table 4 Effect of AN2 on enzyme activity in liver (u/g prot) of snail Oncomelania hupensis after 48 h exposure. Group (mg/L)
ALP
DW AN2
198.46 211.09 190.56 185.59
⁎ ⁎⁎
25 50 100
MDH ± ± ± ±
3.58 20.67⁎ 19.75⁎ 33.34⁎
1823.86 1678.09 1532.46 1465.82
SDH ± ± ± ±
39.48 48.91⁎⁎ 78.32⁎⁎ 142.7⁎⁎
10.42 ± 0.32 8.80 ± 0.47⁎⁎ 7.79 ± 0.23⁎⁎ 6.46 ± 0.88⁎⁎
Compared to DW (dechlorinated water) control, P < 0.05. Compared to DW control, P < 0.01.
at a high concentration (100 mg/L) as compared to the DW control (Tables 3 and 4). ALT and AST activities were also measured in both cephalopodium and liver tissues after 48 h exposure to different concentrations of AN2. The results showed that the AN2 at a low concentration (e.g. 25 mg/L) caused significantly increase of ALT and AST activities in both cephalopodium (Fig. 1) and in liver (Fig. 2) tissues as compared to the DW control (P < 0.05 or 0.01). Then, the activities of ALT and AST began to reduce with the increasing of AN2 concentration and were significantly lower at a high concentration than those of the control (P < 0.05 or 0.01, Figs. 1 and 2).
Notes: AN 1-7, No.1–7 alkaloid components; NIC, niclosamide; DW, dechlorinated water. The data in the table above is the mean of 3 repetitions.
3. Results 3.1. Molluscicidal activity Molluscicidal activities of seven alkaloid components (AN1-7) of the leaves of M. cordata against snail O. hupensis were determined by the snail bioassay. It was found that AN2 was the most toxic and AN7 was the least toxic against the snails amongst all 7 alkaloid components. The mortality of snails was higher than 70% (73.33%) after 25 mg/L AN2 exposure for 48 h (Table 1). The 48 h LC50 and LC90 values of AN2 were 6.35 mg/L and 121.23 mg/L (Table 2), respectively. The order of toxicity of all seven alkaloid components was AN2 > AN3 > AN5 > AN4 > AN1 > AN6 > AN7.
3.3. Electrophoresis assay of EST The response of EST to drug was more intensive in liver than in cephalopodium [18]. Therefore, the toxicity of AN2 against the liver tissue was examined by applying LC50 AN2 to the snails. Five EST bands were showed in the experiment and the activities of EST1 and EST2 were strongest in the five bands (Fig. 3). For the EST bands, especially EST1 and EST2, the width narrowed down and the color became shallow as the treatment time went on when compared to the control. The activity of EST began to decrease after 24 h exposure to LC50 AN2 (lane B), and obviously decreased after 48 h exposure (lane C) as compared to the control.
3.2. Enzyme activities In vivo, 48 h exposure to different concentrations of AN2 altered the activities of different enzymes in different body tissues of snails. AN2 significantly inhibited the activities of MDH and SDH in both cephalopodium (Table 3) and liver tissues (Table 4) as compared to the DW (dechlorinated water) control (P < 0.01). For ALP, however, AN2 could cause a significant increase of ALP activity at a low concentration (e.g. 25 mg/L) in cephalopodium and liver while a significant reduction Table 2 LC50 and LC90 of 7 alkaloid components extracted from Macleaya cordata leaves on snails Oncomelania hupensis after 48 h exposure. Component
LC50 (mg/L)
Confidence interval (95%)
LC90 (mg/L)
Confidence interval (95%)
AN1 AN2 AN3 AN4 AN5 AN6 AN7
93.49 6.35 30.27 43.83 39.56 213.82 369.63
55.64–111.27 0.92–20.87 12.72–49.77 22.67–68.70 9.40–79.45 157.69–290.35 263.21–532.67
799.05 121.23 146.71 279.34 528.46 3890.45 17,119.34
243.04–860.91 101.69–159.84 87.67–176.31 129.18–425.37 456.64–595.52 – –
Fig. 1. Activities of ALT and AST in snail's cephalopodium after 48 h exposure to AN2.
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The molluscicidal activity of AN2 was stronger than that of the extracts of Nerium indicum Mill (LC50 13.2 mg/L), which was found in our lab before [18]. Although the molluscicidal activity of the plant extracts is lower than that of the standard chemical molluscicide niclosamide (LC50 is around 0.1 mg/L), the plant molluscicides are much safer to human beings and environment [18]. And AN2 could inhibit escaping of snails, which prevents the molluscicidal effect of some pestcides such as plant Nerium indicum extracts and chemical molluscicide niclosamide from reducing due to snail escaping. It was reported that more than twenty active alkaloid components were found in M. cordata plant [23]. The main alkaloids are sanguinarine (SG), chelerythrine (CHE), dihydrosanguinarine (DHSG), dihydrochelerythrine (DHCHE), protopine (PR), and allocryptopine (AL). Higher physiological activity was also found in M. cordata plant [24]. In the experiments of this study, AN2 had the strongest molluscicidal activity against the snails in 7 components (Table 1). During TLC analysis, no stratification was found in AN2 (RF value, 0.36) (Table 5), indicating that AN2 might be a monomer. AN2 showed orange color under visible light and positive reaction with Dragendorff's reagent. With HPLC-MS analysis, the mass spectrogram of AN2 (Fig. 4) was identified with that of standard Sanguinarine, indicating that AN2 should be the Sanguinarine, while other alkaloid components need further investigations. Some toxic compounds might affect the activities of some vital enzymes in different body tissues and lead to the death of snails [25]. ALT level and AST level, especially the former one, which can sensitively supervise the hepatocellular injury, are commonly measured clinically as biomarkers for liver health [26,27]. When treating with AN2 at a lower concentration (29 mg/L), the activities of ALT and AST in cephalopodium (Fig. 1) and liver (Fig. 2) were significantly increased, indicating an acute response of the snail toxicosis to AN2. When treating at a high concentration (100 mg/L), however, the activities of ALT and AST were significantly inhibited (Figs. 1 and 2). The result indicated that hepatic failure might have occurred in the snail liver after treated at a high concentration, manifesting that the degree of intoxication are concentration- and time-dependent [25]. In the experiments of this study, the activity of ALP in both cephalopodium (Table 3) and liver (Table 4) of snails significantly increased when treating with AN2 at a low concentration (e.g. 25 mg/L) while significantly decreased when treating at a high concentration (100 mg/L). The same result was also reported for the n-butanol extract of Arisaema tuber [25] and the benzinum extract of Ginkgo biloba sarcotesta against snail O. hupensis[28]. It had been reported that ALP had critical roles on protein synthesis [29], shell formation [30], and other secretory activities in gastropods [31]. Therefore, with the increase of the concentration of drug AN2, protein synthesis and some secretory activities in the snails were disturbed. AN2 also significantly inhibited the activities of SDH and MDH in both cephalopodium and liver. The inhibition showed concentrationdependent effects (Tables 3 and 4). Chen [28] also reported that the extract of G. biloba sarcotesta by benzinum (EGSB) and arecoline (ARE) also inhibited the activities of SDH and MDH. SDH is the only enzyme that participates in both the citric acid cycle and the electron transport chain in the inner mitochondrial membrane of eukaryotes [32]. MDH resided in NADH respiratory chain, which was a vital enzyme in Tricarboxylic acid cycle and played critical roles on the oxidative phosphorylation [33]. Therefore, another cause of AN2’s molluscicidal action may be the inhibition of oxidative phosphorylation and the reduction of ATP energy production, which cause snails of O. hupensis to die. These results indicated that AN2 interfered not only the protein synthesis in the liver and cephalopodium, but also the respiratory chain. EST is an important detoxification enzyme in vivo and also one of the enzymes with the strongest reaction to environmental stimulation, which can be regarded as a pathological index for intoxication of snails [20,25]. Exposure to sublethal dose of AN2 significantly altered the expression of EST in liver tissues of snails. In the experiments of this
Fig. 2. Activities of ALT and AST in snail's liver after 48 h exposure to AN2.
ESTˍ
EST 2 EST 3 EST 4 EST 5 A
B
C
Fig. 3. EST profiles. Lane A represents samples of liver tissues exposed to dechlorinated water control. Lane B and C represent samples of liver tissues exposed to LC50 AN2 for 24 h and 48 h, respectively. About 7 μg protein is loaded in each lane.
4. Discussion Some reports showed that the alkaloids extracted from M. cordata were often used for various bioactivities, such as antitumor [19,20], treating type II diabetes [9], algaecidal [21] and insecticidal activity [22], with few reports to show M. cordata used as a molluscicide against snail O. hupensis[10,11]. In the experiment of this study, seven components (AN1-7) were separated from the total alkaloids extracted from M. cordata leaves, whose Rf values and extract ratios were as listed in Table 5. The experiments on the molluscicidal activity revealed that, the 7 alkaloid components all had molluscicidal activity against snail O. hupensis, amongst of which the AN2 is the most toxic. The LC50 and LC90 values of AN2 were 6.35 mg/L and 121.23 mg/L (Table 2), respectively.
Table 5 Extraction ratio of 7 alkaloids from Macleaya cordata leaves. Sample
Content (g)
Rf value
Percentage (%) in total alkaoids
Extraction ratio (%)
No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 Total
0.8658 0.7754 0.7984 0.7426 0.9660 2.5342 1.5072 8.1896
0.24 0.36 0.48/0.90 0.29/0.70 0.25 0.35/0.56 0.71 –
10.57 9.47 9.75 9.07 11.80 30.94 18.40 100
0.17 0.16 0.16 0.15 0.19 0.51 0.30 1.64
Note: extraction ratio = weight of component/total alkaloids weight.
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A: Standard sample
B: AN2
Fig. 4. The mass spectrogram of standard sample (A) and AN2 (B) with HPLC-MS analysis. [10] W.S. Ke, S.J. Chen, J.L. Yang, Molluscicidal activity of 5 drug plant species on Oncomelania hupensis, Mod. Prev. Med. 34 (2007) 5–8 (in Chinese). [11] W.S. Ke, Z.S. Yu, B. Zeng, W.X. Wang, M.Y. Wu, Mollucicddal activity of Macleaya cordata mixed with fertilizers against Oncomelania hupensis and its effect on rice germination and growth, Res. Environ. Yangze Basin 21 (2012) 472–476 (in Chinese). [12] S.B. Mao, Schistosome Biology and Schistosomiasis Control (M), People's Med. Pub. House, Beijing, 1991, pp. 72–121 (in Chinese). [13] J.H. Xu, J.J. Zhang, W. Wang, L.L. Pan, Study on alkaloid content change of Macleaya cordata in different growing environments, Mod. Chin. Med. 10 (2008) 27–29 (in Chinese). [14] J.S. Yu, J.P. Yu, Study on separation and purification of total alkaloids from Macleaya cordata, J. Chin. Med. Mat. 30 (2007) 1008–1012 (in Chinese). [15] Y.D. Li, J.M. Yang, The study on effect of microbe and microbial pesticides killing Oncomelania hupensis, Acta Hydrobiol. Sin. 29 (2005) 203–205 (in Chinese). [16] G.G. Guilbault, H.N. Miao, S.G. Chen, Handbook of Enzymatic Methods Analysis, Shanghai Science and Technology Press, Shanghai, 1983. [17] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 249–254. [18] H. Wang, W.M. Cai, W.X. Wang, J.M. Yang, Molluscicidal activity of Nerium indicum mill, Pterocarya stenoptera DC, and Rumex japonicum Houtt against Oncomelania hupensis, Biomed. Environ. Sci. 19 (2006) 245–248 (in Chinese). [19] H.L. Qin, P. Wang, Z.H. Li, The establishment of the control substance and 1H nuclear magnetic resonance fingerprint of Macleaya microcarpa (Maxim.) Fedde, Chin, J. Anal. Chem. 32 (2004) 1165–1170 (in Chinese). [20] M. Liu, Y.L. Lin, X.R. Chen, C.C. Liao, W.K. Poo, In vitro assessment of Macleaya cordata crude extract bioactivity and anticancer properties in normal and cancerous human lung cells, Exp. Toxicol. Pathol. 65 (2013) 775–787. [21] S. Jana, T. Eva, B. Hana, P. Hana, M. Pavel, HPLC quantification of seven quaternary benzo[c]phenanthridine alkaloids in six species of the family Papaveraceae, J. Pharm. Biomed. 44 (2007) 283–287. [22] G. Feng, J. Zhang, X.W. Li, J.T. Feng, X. Zhang, Insecticidal activity of alkaloids from Macleaya microcarpa against several species of insect pests, J. Zhejiang Univ. (Agric. Life Sci.) 34 (2008) 187–192 (in Chinese). [23] M.L. Wu, Z.D. Zhang, Q.J. Xu, R.R. Xie, Q.Q. Li, Study advance on plant Macleaya cordata, Asia-Pacific Trad. Med. 5 (2009) 144–145 (in Chinese). [24] P. Kosina, J. Gregorova, J. Gruz, J. Vacek, M. Kolar, M. Vogel, W. Roos, K. Naumann, V. Simanek, J. Ulrichova, Phytochemical and antimicrobial characterization of Macleaya cordata herb, Fitoterapia 81 (2010) 1006–1012. [25] W.S. Ke, J.L. Yang, Z. Meng, A.N. Ma, Evaluation of molluscicidal activities of Arisaema tubers extracts on the snail Oncomelania hupensis, Pestic. Biochem. Physiol. 92 (2008) 129–132. [26] C.S. Wang, T.T. Chang, W.J. Yao, S.T. Wang, P. Chou, Impact of increasing alanine aminotransferase levels within normal range on incident diabetes, J. Formos. Med. Assoc. Taiwan Yi Zhi 111 (2012) 201–208 (in Chinese). [27] N. Ghouri, D. Preiss, N. Sattar, Liver enzymes, nonalcoholic fatty liver disease, and incident cardiovascular disease: a narrative review and clinical perspective of prospective data, Hepatology 52 (2010) 1156–1161. [28] S.X. Chen, L. Wu, X.M. Yang, X.G. Jiang, L.G. Li, R.X. Zhang, L. Xia, S.H. Shao, Comparative molluscicidal action of extract of Ginko biloba sarcotesta, arecoline and niclosamide on snail hosts of Schistosoma japonicum, Pestic. Biochem. Physiol. 89 (2007) 237–241. [29] B. Pilo, M.B. Asnani, R.V. Shah, Studies on wound healing and repair in pigeon. III. Histochemical studies on acid and alkaline phosphatase activity during the process, J. Anim. Morphol. Physiol. 19 (1972) 205–212. [30] L.P.M. Timmermans, Studies on shell formation in mollusks, Neth, J. Zool. 19 (1969) 17–36. [31] A.M. Ibrahim, M.G. Migazi, E.S. Dexian, Histochemical localization of alkaline phosphatase activity in the alimentary tract of the snail Marisa coruarietlis (l.), Bull. Zool. Soc. Egypt 26 (1974) 94–105. [32] K.S. Oyedotun, B.D. Lemire, The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies, J. Biol. Chem. 279 (2004) 9424–9431. [33] C.R. Goward, D.J. Nicholls, Malate dehydrogenase: a model for structure, evolution, and catalysis, Prot. Sci. 3 (1994) 1883–1888.
study, EST bands narrowed down and the color became shallow as the treating time went on (Fig. 3), with some bands (e.g. EST 3 and 4) even vanished (lane C in Fig. 3), which indicated that activities of EST were obviously inhibited. In subsequent stages (48 h treatment), because of surpassing the physiological limit of normal functions in vivo, substance metabolism and energy metabolism became disrupted, and the synthesis of EST was impeded [20]. In the dying organisms, the synthesis of EST almost ceased, which might be another cause for the molluscicidal activity [26]. In conclusion, AN2 amongst seven alkaloid components extracted from M. cordata leaves had a stronger molluscicidal activity against the snail O. hupensis. With comparison of standard samples, AN2 should be the Sanguinarine. The physiological toxic mechanisms of AN2 against snails might disturb normal physiological metabolism of snails such as inhibiting protein synthesis and oxidative phosphorylation in the respiratory chain, and weakening the detoxification ability of liver, which caused snails to die in the end. Plant M. cordata is widely distributed in China and often used as Chinese traditional herb drug. This study showed that the plant M. cordata had a stronger mollusicicial activity against snails, which could be a potential plant molluscicide against snails. Acknowledgements This work was supported by the National Science and Technology Support Program (No. 2015BAD07B07). References [1] M. Njoroge, N.M. Njuguna, P. Mutai, D.S.B. Ongarora, P.W. Smith, K. Chibale, Recent approaches to chemical discovery and development against malaria and the neglected tropical diseases human African trypanosomiasis and schistosomiasis, Chem. Rev. 114 (2014) 11138–11163. [2] M.G. Chen, Z. Feng, Schistosomiasis control in China, Parasitol. Int. 48 (1999) 11–19. [3] D. Li, X. Fu, Design of hydraulic structures to prevent the spread of intermediate snails hosts of schistosomiasis, Irrig. Drain. Syst. 20 (2006) 69–82 (in Chinese). [4] WHO (World Health Organization), The Control of Schistosomiasis: Second Report of the WHO Expert Committee, WHO Tech. Rep. Series, No. 830, WHO, Geneva, 1993. [5] B. Karademir, C. Sibel Ozden, B. Alpertunga, Effects of trichlorfon on malondialdehyde and antioxidant system in human erythrocytes, Toxicol. in Vitro 21 (2007) 1538–1544. [6] T.D. Brackenbury, C.C. Appleton, Plant Molluscicides in South Africa: a registration dilemma, Parasitol. Today 14 (1998) 83–84. [7] G.P. Pi, P. Ren, J.M. Yu, R.F. Shi, Z. Yuan, C.H. Wang, Separation of sanguinarine and chelerythrine in Macleaya cordata (Willd) R. Br. based on methyl acrylate-codivinylbenzene macroporous adsorbents, J. Chromatogr. A 1192 (2008) 17–24. [8] Z.X. Qing, P. Cheng, X.B. Liu, Y.S. Liu, J.G. Zeng, W. Wang, Structural speculation and identification of alkaloids in Macleaya cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening procedure, Rapid Commun. Mass Spectrom. 28 (2014) 1033–1044. [9] C.M. Sai, N.B. Qin, C.C. Jia, D.H. Li, K.B. Wang, Y.H. Pei, J. Bai, Z.L. Li, H.M. Hua, Macleayine, a new alkaloid from Macleaya cordata, Chin. Chem. Lett. 27 (2016) 1717–1720 (in Chinese).
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Molluscicidal activity and physiological toxicity of Macleaya cordata alkaloids components on snail Oncomelania hupensis Wenshan Kea,⁎, Xiong Lina, Zhengshen Yua, Qiqiang Sunb, Qian Zhangb a b
School of Life Science, Hubei University, Wuhan 430062, PR China Research Institute of Forestry Chinese Academy of Forestry, Beijing 100086, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Macleaya cordata (Willd) R. Br, Oncomelania hupensis Molluscicidal activity Enzyme activity Isozyme
In order to search new local plant molluscicides for the control of the vectors of schistosomiasis, leaves of Macleaya cordata (Willd) R. Br. were used to extract and separate alkaloid components by thinner acid method and column chromatography, and the molluscicidal effect of alkaloid components against snail Oncomelania hupensis was determined by bioassay. The results showed that 7 alkaloid components (AN1-7) were obtained after extracting and separating alkaloids from the leaves of M. cordata, where AN2 was found being the most toxic against snail O. hupensis with 48 h LC50 and LC90 values of AN2 of 6.35 mg/L and 121.23 mg/L, respectively. Responses of some critical enzymes to AN2, including activities of Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate transaminase (AST), Malic dehydrogenase (MDH) and Succinate dehydrogenase (SDH) in both cephalopodium and liver, were also detected through experiments, which also explored esterase isozyme (EST) exposed to AN2 in liver tissue. The results showed that AN2 significantly inhibited the activities of SDH, MDH and esterase isozyme, as AN2 significantly stimulated the activities of ALP, ALT and AST to increase at a low concentration (e.g. 25 mg/L), while significantly inhibited the activities of these enzymes at a high concentration (100 mg/L). These results indicated that AN2 not only inhibited protein synthesis, and respiratory chain oxidative phosphorylation, but also caused hepatocellular injury and reduced the detoxification ability of liver.
1. Introduction Schistosomiasis is the second most prevalent endemic disease after malaria in tropical and subtropical regions [1] and remains a serious disease in China (Schistosoma japonicum) by devastating human health [2]. Recent monitoring data revealed that schistosomiasis was re-emerging in China, especially along the Yangtze River and in the lakes region of Jiangxi and Hunan provinces [3]. As the snail Oncomelania hupensis is the only intermediate host of Schistosoma japonicum that causes schistosomiasis in China, the extermination of snails is efficient for control of schistosomiasis. Niclosamide, the only commercially available molluscicide recommended by WHO for large scale use in schistosomiasis control [4], however, is limited by its high cost of synthetic compounds, along with increasing concern of possible snail resistance to these compounds and their toxicity in non-target organisms, which have raised the study of plant molluscicides [5]. The use of plants with molluscicidal properties is a technology that is simple, inexpensive, biodegradable and appropriate for local control of the snail vector, especially in rural areas of developing countries where schistosomiasis is endemic [6].
⁎
As a perennial plant, Macleaya cordata (Willd) R. Br. is a member of the Macleaya genus in the family of Papaveraceae, which is mainly distributed in North America, Europe, South and Northwest China. M. cordata has been widely used as a folk herbal medicine in China to cure cervical cancer and thyroid cancer, and to relieve insect bites and ringworm infection [7,8]. Currently, M. cordata is utilized as a traditional Chinese medicine for the treatment of inflamed wounds, arthritis, rheumatism arthralgia, and trichomonas vagi-nalis [9]. In our initial study, it was found that plant M. cordata had strong molluscicidal activity against snails O. hupensis, with the molluscicidal effect stronger than that of Nerium indicum Mill and Arisaema erubescens Schott [10,11]. The objectives of this study were: (1) to furtherly examine the molluscicidal activities of M. cordata extracts and try to find out the strongest molluscicidal component in the plant; and (2) to reveal the possible physiological toxicity mechanism of the molluscicidal component from activity changes in Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate transaminase (AST), Malic dehydrogenase (MDH), Succinate dehydrogenase (SDH) and esterase isoenzyme (EST).
Corresponding author. E-mail address: [email protected] (W. Ke).
http://dx.doi.org/10.1016/j.pestbp.2017.08.016 Received 21 March 2017; Received in revised form 17 August 2017; Accepted 27 August 2017 0048-3575/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Ke, W., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.08.016
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2. Materials and methods
2.5. Enzyme assay
2.1. Oncomelania hupensis
According to the testing result of molluscicidal activity above, the strongest molluscicidal active component of alkaloid (AN2) was used to examine the snail enzyme assay. Two hundred snails were randomly divided into four groups and exposed to each concentration level of AN2 (0, 25 mg/L, 50 mg/L, and 100 mg/L) for 48 h, with dechlorinated water as the control. Afterwards, the snails were washed with dechlorinated water, and their cephalopodium and liver tissues were excised for biochemical analysis - a process recommended by Li [15]. Phosphatic buffer solutions (pH 6.8) were added (1 drop/snail) to the cephalopodium and liver which were then homogenated in ice incubation condition, where the homogenates were centrifuged at 4000 rpm for 10 min at 0 °C. Then, one part of supernatant was stored at − 20 °C for the measurement of enzyme activities and electrophoresis. The processes of preparation of snail cephalopodium and liver were replicated for 3 times in the same conditions.
Adult O. hupensis snails (9–11 mm in length) were collected from farmfield of Taihu countryside of Jingzhou, Hubei Province, China, amongst which the healthy ones were selected by cercariae escaping method to get rid of the infected [12]. Then the snails were kept in a laboratory at 20 °C for a week before experiment. 2.2. Plants Leaves of the plant M. cordata at vegetative growth phase before flowering were collected from Lushan Mountain in central China, with their fresh and dry weights recorded. 2.3. Extracts of alkaloids The total alkaloids of M. cordata were extracted according to the modified ethanol extract method [13,14]. Weigh dryly pulverized M. cordata powder for 500 g with 95% ethanol at M:V = 1:3 added, and immerse it for 3 days. Filter the ethanol for the distillation concentration in a cyclotron evaporimeter. With the immersion repeated for 3 times, mix and dry the concentrate to obtain the extractum. Then, solve the total alkaloid extractum of M. cordata by 1% hydrochloric acid solution, extract it via chloroform at a volume ratio of 1:1, remove the lipids and pigments in acid water, and keep the acid aqueous phase. Adjust the pH of acid water to around 6.8 through ammonia, and remove the tannin fraction by filtration. Finally, continue to adjust the pH of acid water to 8.0–9.0, and again, extract the solution via chloroform at a volume ratio of 1:1, retain the chloroform phase, and remove the chloroform layer via the cyclotron distillation, so as to obtain the total alkaloids of M. cordata. Components of the total alkaloids were separated with column chromatography. D-101 Macroporous resin, selected as the stationary phase, with ethanol as the moving phase, was columned by the wet packing column method. Firstly fill the chromatographic column with distilled water, and weigh the macroporous resin for 10 g to gradually pour the resion into the chromatographic column with a glass rod for drainage. Then fill the samples as the columning was finished. For the last step, elution was formed by water, gradient aqueous ethanol (10%, 20%, …, 90%), and absolute ethanol in turn with each moving phase of 100 ml. The moving speed was controlled, as every 5 ml was collected in a tube until the eluent was detected as no precipitation via Dragendorff's reagent. The collected liquors were further analyzed by TLC(thin-layer chromatography) method. Development solvent (chloroform-methanol (19:1)) was used to separate the alkaloids and iodine vapor was used to test the separation point in TLC. Rf values were measured after finishing of the separation, whereas the liquids from different tubes with same Rf values were merged into one tube as one component. Liquids of seven alkaloid components (NO. 1–7) (AN1-AN7) were gained in the end and then dried (freeze drying) for next experiment.
2.5.1. Assay of enzyme activities The enzyme activities were measured according to Guilbault et al.'s [16] methods of enzyme kinetic assay. Enzyme activities were expressed as the amount of substrate hydrolyzed or production liberated in 1 mol/min/g protein in the supernatant. Total protein level of supernatants was estimated according to Bradford's method [17]. Enzyme activities were expressed as the amount of substrate hydrolyzed or production liberated in 1 mol/min/g protein in the supernatant. ALP kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the ALP activities were determined at 405 nm with 4nitrophenyl phosphate as the substrate. ALT, AST, LDH, SDH and MDH kits were purchased from Shanghai AILEX Technology Co., Ltd., and the enzyme activities were determined at 340 nm.
2.5.2. Electrophoresis assay of EST Similar to the snail treatments as above, two treatment levels (0 and LC50) were designed. Having exposure for 24 h and 48 h, the snails were carefully crushed so that the liver tissues could be extracted. Phosphatic buffer solutions (pH 6.8) were added (1 drop/snail) to the liver tissues, which were then homogenated on ice. Then the homogenates were centrifuged at 8000 g for 10 min at 0 °C. The supernatant was stored at − 20 °C for enzyme electrophoresis. The supernatant (10 μL each sample, 2 replicates) of snail liver tissues was electrophoresed with a vertical electrophoresis system ECP3000. Polyacrylamide gel electrophoresis (PAGE) was carried out with 20 mA constant current until the tracing dye reached the bottom of the gel. The chemicals were purchased from Sigma, with the separating gel consisted of 7.5% (acrylamide + bisacrylamide) and 0.04 mol/L Tris-HCl, with pH of 8.8; the stacking gel consisted of 3.75% (acrylamide + bisacrylamide) and 0.01 mol/L Tris-HCl, with pH of 6.8; and the buffer was Tris-glycine buffer, with pH of 8.3. After electrophoresis, gels were stained, washed, fixed and then photographed. Acetic acid (7.5%) was the stationary phase.
2.4. Testing for molluscicidal activity Seven different alkaloids components separated from leaves of M. cordata were respectively dissolved with distilled water and three treatments (0, 25 mg/L, 50 mg/L and 100 mg/L) were made to test the molluscicidal activity. The snails were collected in a nylon mesh bag (30 snails per bag) and immersed into 500 mL solution of a known concentration in the extracts. For each treatment, five bags (30 snails per bag) of snails were exposed for 24, 48, 72, 96 and 120 h, respectively, with dechlorinated tap water exposure as the control. For checking the mortality, no response to a needle probe under dissecting microscope was the evidence of death. The experiments were conducted at 21 °C, each of which was replicated three times.
2.6. Data analysis The mortalities of snails were expressed as the mean of three replicates. The effect of alkaloid components on O. hupensis was expressed by LC50 and LC90 and their 95% confidence limit (95% cl). The results of enzyme activities were expressed as means ± SE of the three replicates. One way ANOVA and SSR (Duncan's repeat comparison) were used to detect significant differences (P < 0.05 or 0.01).
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Table 1 Mortality of snails Oncomelania hupensis exposed to 7 alkaloid components extracted from Macleaya cordata leaves under laboratory conditions. Component
Concentration (mg/L)
Mortality of snails (%) 24 h
AN 1
AN 2
AN 3
AN 4
AN 5
AN 6
AN 7
NIC DW
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 1
Table 3 Effect of AN2 on enzyme activity in cephalopodium (u/g prot) of snail Oncomelania hupensis after 48 h exposure.
20.00 13.33 10.00 56.67 46.67 36.67 56.67 36.67 33.33 46.67 40.00 13.33 26.67 26.67 23.33 20.00 6.67 10.00 3.33 3.33 0 83.33 0
48 h 50.00 36.67 26.67 83.33 76.67 73.33 80.00 73.33 40.00 73.33 50.00 36.67 66.67 56.67 40.00 40.00 20.00 16.67 23.33 10.00 6.67 100 6.67
72 h
96 h
56.67 43.33 40.00 90.00 83.33 83.33 80.00 73.33 43.33 73.33 56.67 50.00 70.00 60.00 50.00 46.67 26.67 16.67 26.67 16.67 10.00 100 10.00
70.00 60.00 56.67 90.00 90.00 83.33 80.00 73.33 50.00 80.00 66.67 66.67 70.00 63.33 56.67 46.67 26.67 16.67 26.67 16.67 10.00 100 10.00
120 h 76.67 63.33 56.67 96.67 90.00 83.33 90.00 73.33 56.67 86.67 70.00 66.67 76.67 66.67 60.00 56.67 36.67 23.33 33.33 23.33 16.67 100 10.00
Group (mg/L)
MDH
DW AN2
2450.23 2083.94 1971.62 1598.97
⁎⁎
25 50 100
SDH ± ± ± ±
23.49 10.92⁎⁎ 74.43⁎⁎ 82.57⁎⁎
21.85 18.75 17.53 16.01
ALP ± ± ± ±
0.21 0.65⁎⁎ 0.37⁎⁎ 3.49⁎⁎
1382.51 1536.12 1494.91 1273.86
± ± ± ±
28.76 37.28⁎⁎ 114.62⁎⁎ 125.61⁎⁎
Compared to DW control, P < 0.01.
Table 4 Effect of AN2 on enzyme activity in liver (u/g prot) of snail Oncomelania hupensis after 48 h exposure. Group (mg/L)
ALP
DW AN2
198.46 211.09 190.56 185.59
⁎ ⁎⁎
25 50 100
MDH ± ± ± ±
3.58 20.67⁎ 19.75⁎ 33.34⁎
1823.86 1678.09 1532.46 1465.82
SDH ± ± ± ±
39.48 48.91⁎⁎ 78.32⁎⁎ 142.7⁎⁎
10.42 ± 0.32 8.80 ± 0.47⁎⁎ 7.79 ± 0.23⁎⁎ 6.46 ± 0.88⁎⁎
Compared to DW (dechlorinated water) control, P < 0.05. Compared to DW control, P < 0.01.
at a high concentration (100 mg/L) as compared to the DW control (Tables 3 and 4). ALT and AST activities were also measured in both cephalopodium and liver tissues after 48 h exposure to different concentrations of AN2. The results showed that the AN2 at a low concentration (e.g. 25 mg/L) caused significantly increase of ALT and AST activities in both cephalopodium (Fig. 1) and in liver (Fig. 2) tissues as compared to the DW control (P < 0.05 or 0.01). Then, the activities of ALT and AST began to reduce with the increasing of AN2 concentration and were significantly lower at a high concentration than those of the control (P < 0.05 or 0.01, Figs. 1 and 2).
Notes: AN 1-7, No.1–7 alkaloid components; NIC, niclosamide; DW, dechlorinated water. The data in the table above is the mean of 3 repetitions.
3. Results 3.1. Molluscicidal activity Molluscicidal activities of seven alkaloid components (AN1-7) of the leaves of M. cordata against snail O. hupensis were determined by the snail bioassay. It was found that AN2 was the most toxic and AN7 was the least toxic against the snails amongst all 7 alkaloid components. The mortality of snails was higher than 70% (73.33%) after 25 mg/L AN2 exposure for 48 h (Table 1). The 48 h LC50 and LC90 values of AN2 were 6.35 mg/L and 121.23 mg/L (Table 2), respectively. The order of toxicity of all seven alkaloid components was AN2 > AN3 > AN5 > AN4 > AN1 > AN6 > AN7.
3.3. Electrophoresis assay of EST The response of EST to drug was more intensive in liver than in cephalopodium [18]. Therefore, the toxicity of AN2 against the liver tissue was examined by applying LC50 AN2 to the snails. Five EST bands were showed in the experiment and the activities of EST1 and EST2 were strongest in the five bands (Fig. 3). For the EST bands, especially EST1 and EST2, the width narrowed down and the color became shallow as the treatment time went on when compared to the control. The activity of EST began to decrease after 24 h exposure to LC50 AN2 (lane B), and obviously decreased after 48 h exposure (lane C) as compared to the control.
3.2. Enzyme activities In vivo, 48 h exposure to different concentrations of AN2 altered the activities of different enzymes in different body tissues of snails. AN2 significantly inhibited the activities of MDH and SDH in both cephalopodium (Table 3) and liver tissues (Table 4) as compared to the DW (dechlorinated water) control (P < 0.01). For ALP, however, AN2 could cause a significant increase of ALP activity at a low concentration (e.g. 25 mg/L) in cephalopodium and liver while a significant reduction Table 2 LC50 and LC90 of 7 alkaloid components extracted from Macleaya cordata leaves on snails Oncomelania hupensis after 48 h exposure. Component
LC50 (mg/L)
Confidence interval (95%)
LC90 (mg/L)
Confidence interval (95%)
AN1 AN2 AN3 AN4 AN5 AN6 AN7
93.49 6.35 30.27 43.83 39.56 213.82 369.63
55.64–111.27 0.92–20.87 12.72–49.77 22.67–68.70 9.40–79.45 157.69–290.35 263.21–532.67
799.05 121.23 146.71 279.34 528.46 3890.45 17,119.34
243.04–860.91 101.69–159.84 87.67–176.31 129.18–425.37 456.64–595.52 – –
Fig. 1. Activities of ALT and AST in snail's cephalopodium after 48 h exposure to AN2.
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The molluscicidal activity of AN2 was stronger than that of the extracts of Nerium indicum Mill (LC50 13.2 mg/L), which was found in our lab before [18]. Although the molluscicidal activity of the plant extracts is lower than that of the standard chemical molluscicide niclosamide (LC50 is around 0.1 mg/L), the plant molluscicides are much safer to human beings and environment [18]. And AN2 could inhibit escaping of snails, which prevents the molluscicidal effect of some pestcides such as plant Nerium indicum extracts and chemical molluscicide niclosamide from reducing due to snail escaping. It was reported that more than twenty active alkaloid components were found in M. cordata plant [23]. The main alkaloids are sanguinarine (SG), chelerythrine (CHE), dihydrosanguinarine (DHSG), dihydrochelerythrine (DHCHE), protopine (PR), and allocryptopine (AL). Higher physiological activity was also found in M. cordata plant [24]. In the experiments of this study, AN2 had the strongest molluscicidal activity against the snails in 7 components (Table 1). During TLC analysis, no stratification was found in AN2 (RF value, 0.36) (Table 5), indicating that AN2 might be a monomer. AN2 showed orange color under visible light and positive reaction with Dragendorff's reagent. With HPLC-MS analysis, the mass spectrogram of AN2 (Fig. 4) was identified with that of standard Sanguinarine, indicating that AN2 should be the Sanguinarine, while other alkaloid components need further investigations. Some toxic compounds might affect the activities of some vital enzymes in different body tissues and lead to the death of snails [25]. ALT level and AST level, especially the former one, which can sensitively supervise the hepatocellular injury, are commonly measured clinically as biomarkers for liver health [26,27]. When treating with AN2 at a lower concentration (29 mg/L), the activities of ALT and AST in cephalopodium (Fig. 1) and liver (Fig. 2) were significantly increased, indicating an acute response of the snail toxicosis to AN2. When treating at a high concentration (100 mg/L), however, the activities of ALT and AST were significantly inhibited (Figs. 1 and 2). The result indicated that hepatic failure might have occurred in the snail liver after treated at a high concentration, manifesting that the degree of intoxication are concentration- and time-dependent [25]. In the experiments of this study, the activity of ALP in both cephalopodium (Table 3) and liver (Table 4) of snails significantly increased when treating with AN2 at a low concentration (e.g. 25 mg/L) while significantly decreased when treating at a high concentration (100 mg/L). The same result was also reported for the n-butanol extract of Arisaema tuber [25] and the benzinum extract of Ginkgo biloba sarcotesta against snail O. hupensis[28]. It had been reported that ALP had critical roles on protein synthesis [29], shell formation [30], and other secretory activities in gastropods [31]. Therefore, with the increase of the concentration of drug AN2, protein synthesis and some secretory activities in the snails were disturbed. AN2 also significantly inhibited the activities of SDH and MDH in both cephalopodium and liver. The inhibition showed concentrationdependent effects (Tables 3 and 4). Chen [28] also reported that the extract of G. biloba sarcotesta by benzinum (EGSB) and arecoline (ARE) also inhibited the activities of SDH and MDH. SDH is the only enzyme that participates in both the citric acid cycle and the electron transport chain in the inner mitochondrial membrane of eukaryotes [32]. MDH resided in NADH respiratory chain, which was a vital enzyme in Tricarboxylic acid cycle and played critical roles on the oxidative phosphorylation [33]. Therefore, another cause of AN2’s molluscicidal action may be the inhibition of oxidative phosphorylation and the reduction of ATP energy production, which cause snails of O. hupensis to die. These results indicated that AN2 interfered not only the protein synthesis in the liver and cephalopodium, but also the respiratory chain. EST is an important detoxification enzyme in vivo and also one of the enzymes with the strongest reaction to environmental stimulation, which can be regarded as a pathological index for intoxication of snails [20,25]. Exposure to sublethal dose of AN2 significantly altered the expression of EST in liver tissues of snails. In the experiments of this
Fig. 2. Activities of ALT and AST in snail's liver after 48 h exposure to AN2.
ESTˍ
EST 2 EST 3 EST 4 EST 5 A
B
C
Fig. 3. EST profiles. Lane A represents samples of liver tissues exposed to dechlorinated water control. Lane B and C represent samples of liver tissues exposed to LC50 AN2 for 24 h and 48 h, respectively. About 7 μg protein is loaded in each lane.
4. Discussion Some reports showed that the alkaloids extracted from M. cordata were often used for various bioactivities, such as antitumor [19,20], treating type II diabetes [9], algaecidal [21] and insecticidal activity [22], with few reports to show M. cordata used as a molluscicide against snail O. hupensis[10,11]. In the experiment of this study, seven components (AN1-7) were separated from the total alkaloids extracted from M. cordata leaves, whose Rf values and extract ratios were as listed in Table 5. The experiments on the molluscicidal activity revealed that, the 7 alkaloid components all had molluscicidal activity against snail O. hupensis, amongst of which the AN2 is the most toxic. The LC50 and LC90 values of AN2 were 6.35 mg/L and 121.23 mg/L (Table 2), respectively.
Table 5 Extraction ratio of 7 alkaloids from Macleaya cordata leaves. Sample
Content (g)
Rf value
Percentage (%) in total alkaoids
Extraction ratio (%)
No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 Total
0.8658 0.7754 0.7984 0.7426 0.9660 2.5342 1.5072 8.1896
0.24 0.36 0.48/0.90 0.29/0.70 0.25 0.35/0.56 0.71 –
10.57 9.47 9.75 9.07 11.80 30.94 18.40 100
0.17 0.16 0.16 0.15 0.19 0.51 0.30 1.64
Note: extraction ratio = weight of component/total alkaloids weight.
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A: Standard sample
B: AN2
Fig. 4. The mass spectrogram of standard sample (A) and AN2 (B) with HPLC-MS analysis. [10] W.S. Ke, S.J. Chen, J.L. Yang, Molluscicidal activity of 5 drug plant species on Oncomelania hupensis, Mod. Prev. Med. 34 (2007) 5–8 (in Chinese). [11] W.S. Ke, Z.S. Yu, B. Zeng, W.X. Wang, M.Y. Wu, Mollucicddal activity of Macleaya cordata mixed with fertilizers against Oncomelania hupensis and its effect on rice germination and growth, Res. Environ. Yangze Basin 21 (2012) 472–476 (in Chinese). [12] S.B. Mao, Schistosome Biology and Schistosomiasis Control (M), People's Med. Pub. House, Beijing, 1991, pp. 72–121 (in Chinese). [13] J.H. Xu, J.J. Zhang, W. Wang, L.L. Pan, Study on alkaloid content change of Macleaya cordata in different growing environments, Mod. Chin. Med. 10 (2008) 27–29 (in Chinese). [14] J.S. Yu, J.P. Yu, Study on separation and purification of total alkaloids from Macleaya cordata, J. Chin. Med. Mat. 30 (2007) 1008–1012 (in Chinese). [15] Y.D. Li, J.M. Yang, The study on effect of microbe and microbial pesticides killing Oncomelania hupensis, Acta Hydrobiol. Sin. 29 (2005) 203–205 (in Chinese). [16] G.G. Guilbault, H.N. Miao, S.G. Chen, Handbook of Enzymatic Methods Analysis, Shanghai Science and Technology Press, Shanghai, 1983. [17] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 249–254. [18] H. Wang, W.M. Cai, W.X. Wang, J.M. Yang, Molluscicidal activity of Nerium indicum mill, Pterocarya stenoptera DC, and Rumex japonicum Houtt against Oncomelania hupensis, Biomed. Environ. Sci. 19 (2006) 245–248 (in Chinese). [19] H.L. Qin, P. Wang, Z.H. Li, The establishment of the control substance and 1H nuclear magnetic resonance fingerprint of Macleaya microcarpa (Maxim.) Fedde, Chin, J. Anal. Chem. 32 (2004) 1165–1170 (in Chinese). [20] M. Liu, Y.L. Lin, X.R. Chen, C.C. Liao, W.K. Poo, In vitro assessment of Macleaya cordata crude extract bioactivity and anticancer properties in normal and cancerous human lung cells, Exp. Toxicol. Pathol. 65 (2013) 775–787. [21] S. Jana, T. Eva, B. Hana, P. Hana, M. Pavel, HPLC quantification of seven quaternary benzo[c]phenanthridine alkaloids in six species of the family Papaveraceae, J. Pharm. Biomed. 44 (2007) 283–287. [22] G. Feng, J. Zhang, X.W. Li, J.T. Feng, X. Zhang, Insecticidal activity of alkaloids from Macleaya microcarpa against several species of insect pests, J. Zhejiang Univ. (Agric. Life Sci.) 34 (2008) 187–192 (in Chinese). [23] M.L. Wu, Z.D. Zhang, Q.J. Xu, R.R. Xie, Q.Q. Li, Study advance on plant Macleaya cordata, Asia-Pacific Trad. Med. 5 (2009) 144–145 (in Chinese). [24] P. Kosina, J. Gregorova, J. Gruz, J. Vacek, M. Kolar, M. Vogel, W. Roos, K. Naumann, V. Simanek, J. Ulrichova, Phytochemical and antimicrobial characterization of Macleaya cordata herb, Fitoterapia 81 (2010) 1006–1012. [25] W.S. Ke, J.L. Yang, Z. Meng, A.N. Ma, Evaluation of molluscicidal activities of Arisaema tubers extracts on the snail Oncomelania hupensis, Pestic. Biochem. Physiol. 92 (2008) 129–132. [26] C.S. Wang, T.T. Chang, W.J. Yao, S.T. Wang, P. Chou, Impact of increasing alanine aminotransferase levels within normal range on incident diabetes, J. Formos. Med. Assoc. Taiwan Yi Zhi 111 (2012) 201–208 (in Chinese). [27] N. Ghouri, D. Preiss, N. Sattar, Liver enzymes, nonalcoholic fatty liver disease, and incident cardiovascular disease: a narrative review and clinical perspective of prospective data, Hepatology 52 (2010) 1156–1161. [28] S.X. Chen, L. Wu, X.M. Yang, X.G. Jiang, L.G. Li, R.X. Zhang, L. Xia, S.H. Shao, Comparative molluscicidal action of extract of Ginko biloba sarcotesta, arecoline and niclosamide on snail hosts of Schistosoma japonicum, Pestic. Biochem. Physiol. 89 (2007) 237–241. [29] B. Pilo, M.B. Asnani, R.V. Shah, Studies on wound healing and repair in pigeon. III. Histochemical studies on acid and alkaline phosphatase activity during the process, J. Anim. Morphol. Physiol. 19 (1972) 205–212. [30] L.P.M. Timmermans, Studies on shell formation in mollusks, Neth, J. Zool. 19 (1969) 17–36. [31] A.M. Ibrahim, M.G. Migazi, E.S. Dexian, Histochemical localization of alkaline phosphatase activity in the alimentary tract of the snail Marisa coruarietlis (l.), Bull. Zool. Soc. Egypt 26 (1974) 94–105. [32] K.S. Oyedotun, B.D. Lemire, The quaternary structure of the Saccharomyces cerevisiae succinate dehydrogenase. Homology modeling, cofactor docking, and molecular dynamics simulation studies, J. Biol. Chem. 279 (2004) 9424–9431. [33] C.R. Goward, D.J. Nicholls, Malate dehydrogenase: a model for structure, evolution, and catalysis, Prot. Sci. 3 (1994) 1883–1888.
study, EST bands narrowed down and the color became shallow as the treating time went on (Fig. 3), with some bands (e.g. EST 3 and 4) even vanished (lane C in Fig. 3), which indicated that activities of EST were obviously inhibited. In subsequent stages (48 h treatment), because of surpassing the physiological limit of normal functions in vivo, substance metabolism and energy metabolism became disrupted, and the synthesis of EST was impeded [20]. In the dying organisms, the synthesis of EST almost ceased, which might be another cause for the molluscicidal activity [26]. In conclusion, AN2 amongst seven alkaloid components extracted from M. cordata leaves had a stronger molluscicidal activity against the snail O. hupensis. With comparison of standard samples, AN2 should be the Sanguinarine. The physiological toxic mechanisms of AN2 against snails might disturb normal physiological metabolism of snails such as inhibiting protein synthesis and oxidative phosphorylation in the respiratory chain, and weakening the detoxification ability of liver, which caused snails to die in the end. Plant M. cordata is widely distributed in China and often used as Chinese traditional herb drug. This study showed that the plant M. cordata had a stronger mollusicicial activity against snails, which could be a potential plant molluscicide against snails. Acknowledgements This work was supported by the National Science and Technology Support Program (No. 2015BAD07B07). References [1] M. Njoroge, N.M. Njuguna, P. Mutai, D.S.B. Ongarora, P.W. Smith, K. Chibale, Recent approaches to chemical discovery and development against malaria and the neglected tropical diseases human African trypanosomiasis and schistosomiasis, Chem. Rev. 114 (2014) 11138–11163. [2] M.G. Chen, Z. Feng, Schistosomiasis control in China, Parasitol. Int. 48 (1999) 11–19. [3] D. Li, X. Fu, Design of hydraulic structures to prevent the spread of intermediate snails hosts of schistosomiasis, Irrig. Drain. Syst. 20 (2006) 69–82 (in Chinese). [4] WHO (World Health Organization), The Control of Schistosomiasis: Second Report of the WHO Expert Committee, WHO Tech. Rep. Series, No. 830, WHO, Geneva, 1993. [5] B. Karademir, C. Sibel Ozden, B. Alpertunga, Effects of trichlorfon on malondialdehyde and antioxidant system in human erythrocytes, Toxicol. in Vitro 21 (2007) 1538–1544. [6] T.D. Brackenbury, C.C. Appleton, Plant Molluscicides in South Africa: a registration dilemma, Parasitol. Today 14 (1998) 83–84. [7] G.P. Pi, P. Ren, J.M. Yu, R.F. Shi, Z. Yuan, C.H. Wang, Separation of sanguinarine and chelerythrine in Macleaya cordata (Willd) R. Br. based on methyl acrylate-codivinylbenzene macroporous adsorbents, J. Chromatogr. A 1192 (2008) 17–24. [8] Z.X. Qing, P. Cheng, X.B. Liu, Y.S. Liu, J.G. Zeng, W. Wang, Structural speculation and identification of alkaloids in Macleaya cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening procedure, Rapid Commun. Mass Spectrom. 28 (2014) 1033–1044. [9] C.M. Sai, N.B. Qin, C.C. Jia, D.H. Li, K.B. Wang, Y.H. Pei, J. Bai, Z.L. Li, H.M. Hua, Macleayine, a new alkaloid from Macleaya cordata, Chin. Chem. Lett. 27 (2016) 1717–1720 (in Chinese).
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