Molluscicidal efficacy of gallic acid in combination with niclosamide

doi:10.12140/j.issn.1000-7423.2024.02.017

Abstract

Abstract:

Objective To investigate the combined snail killing effect and mechanism of traditional Chinese medicine monomers gallic acid and niclosamide, which have alternating oxidase (AOX) inhibitory activity. Methods Negative snails were collected from Gong’an County, Hubei Province and randomly divided into 7 groups: a blank control group (H₂O), experimental groups were treated with niclosamide (N1 group: 0.06 mg/L, N2 group: 0.1 mg/L) and gallic acid (G1 group: 0.8 g/L, G2 group: 1.6 g/L) alone or in combination (M1 group: 0.06 mg/L niclosamide + 0.8 g/L gallic acid, M2 group: 0.06 mg/L niclosamide + 1.6 g/L gallic acid). Detect the survival rates of each group of snails after drug immersion for 12, 24, and 48 hours. Slice and stain them after liquid nitrogen embedding, observe and quantitatively analyze them under an optical microscope. Measure the relative enzyme activity values of cytochrome C oxidase (CCO) and lactate dehydrogenase (LDH) in the soft sections of snails, expressed as average optical density values. After homogenizing the snail, centrifuge and take the supernatant. Incubate with DCFH-DA probe, determine the total protein content using a BCA protein quantification kit, detect fluorescence intensity (FI) using a multifunctional enzyme-linked immunosorbent assay (ELISA), and divide the ROS value by the FI measured in the sample by the protein concentration (μg). The value represents the determination of superoxide dismutase (SOD) levels using a total SOD activity detection kit. The difference analysis of snail mortality rate was conducted using χ² test. After determining the homogeneity of variance using Levene’s Test, the data on CCO activity and oxidative stress levels were compared pairwise between different groups using Tukey HSD’s multiple comparison method. Results The G1 and G2 groups did not show significant snail killing effects after immersion. The mortality rates of snails in the M1 and M2 groups after 48 h of immersion reached 70.0% (56/80) and 84.2% (101/120), respectively, higher than those in the N1 group (44.4%, 32/72) (χ² = 9.13, 32.52; P < 0.05); Compared with the N2 group, there was no statistically significant difference in snail mortality rate (χ² = 0.11, 2.58; P > 0.05). The LDH activity of snails in N1 and N2 groups showed a decreasing trend; The activities of CCO and LDH in the screw bodies of M1 and M2 groups decreased (P < 0.05). After 48 hours, the LDH activities in muscle tissue and liver of M1 group were 0.152 ± 0.002 and 0.172 ± 0.016, respectively, and the CCO activities were 0.180 ± 0.022 and 0.335 ± 0.014, respectively; The LDH activity of the M2 group was 0.166 ± 0.008 and 0.173 ± 0.022, and the CCO activity was 0.199 ± 0.009 and 0.294 ± 0.015, respectively. There was a statistically significant difference (P < 0.05) between the two groups and the blank control group (LDH activity was 0.229 ± 0.006 and 0.227 ± 0.010, and CCO activity was 0.259 ± 0.008 and 0.428 ± 0.024, respectively). The SOD activity of the M1 group at 24 hours after treatment was (5.56 ± 0.91) UI/g, which was higher than that of the blank control group at (5.26 ± 0.08) UI/g (P < 0.05); The SOD activity of the M2 group showed a trend of first increasing and then decreasing [(2.40 ± 0.45), (8.14 ± 0.15), (1.60 ± 0.21) UI/g] at 12, 24 and 48 h after treatment. Compared with the UI/g of the blank control group at the corresponding time points [(3.54 ± 0.94), (5.26 ± 0.08), (5.10 ± 0.87) UI/g], the trend of change was significant (P < 0.05). The ROS levels in the M1 group were (1 619.00 ± 168.25) FI/μg and (1 866.65 ± 211.79) FI/μg, respectively, at 24 and 48 h after treatment. The ROS levels in the M2 group reached (2 451.29 ± 195.91) FI/μg after 48 hours, which were higher than those in the blank control group at the corresponding time points [(802.37 ± 114.69), (1 393.81 ± 86.12) FI/μg] (P < 0.05). Conclusion Gallic acid significantly enhances the snail killing effect of low concentration chloramphenicol. By blocking the compensatory upregulation of AOX, it further exacerbates the energy metabolism imbalance and oxidative stress in snails. This may be an important mechanism by which gallic acid has an auxiliary snail killing effect.

Key words: Oncomelania hupehensis, Gallic acid, Niclosamide, Molluscicidal effects, Glucose metabolism enzyme activity, Oxidative stress levels

CLC Number:

R978.82

ZHENG Tao, LIU Jiahao, LI Bin, LI Jiashan, NIE Juan, XIONG Tao. Molluscicidal efficacy of gallic acid in combination with niclosamide[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(2): 251-258.

Figures/Tables 3

Table 1

Table 2

Effects of the experimental drug on the glycolytic enzyme activity of snails post-immersion in different time

组别Group		钉螺相对酶活力值 Relative enzyme activity value of snails
组别Group		12 h	24 h	48 h
肌肉组织中LDH	空白对照 Control	0.234 ± 0.004	0.215 ± 0.006	0.229 ± 0.006
LDH in muscle	G1	0.228 ± 0.010	0.201 ± 0.010	0.213 ± 0.016
	N1	0.161 ± 0.005^a	0.174 ± 0.006^a	0.150 ± 0.008^a
	M1	0.116 ± 0.008^a，b，c	0.180 ± 0.013^a	0.152 ± 0.002^a，c
	G2	0.220 ± 0.014	0.221 ± 0.007	0.197 ± 0.006^a
	N2	0.191 ± 0.002^a	0.187 ± 0.016	0.143 ± 0.011^a
	M2	0.174 ± 0.014^a，c	0.197 ± 0.010	0.166 ± 0.008^a，c
肝脏中LDH	空白对照 Control	0.224 ± 0.006	0.240 ± 0.014	0.227 ± 0.010
LDH in liver	G1	0.227 ± 0.017	0.224 ± 0.018	0.255 ± 0.011
	N1	0.164 ± 0.014^a	0.257 ± 0.013	0.155 ± 0.003^a
	M1	0.202 ± 0.008^a	0.207 ± 0.005^b	0.172 ± 0.016^a，c
	G2	0.234 ± 0.007	0.256 ± 0.005	0.230 ± 0.018
	N2	0.162 ± 0.007^a	0.227 ± 0.010	0.154 ± 0.020^a
	M2	0.206 ± 0.018^b	0.210 ± 0.015^c	0.173 ± 0.022^a，c
肌肉组织中CCO	空白对照 Control	0.264 ± 0.008	0.304 ± 0.013	0.259 ± 0.008
CCO in muscle	G1	0.312 ± 0.014^a	0.315 ± 0.011	0.222 ± 0.025
	N1	0.234 ± 0.018	0.279 ± 0.013	0.156 ± 0.014^a
	M1	0.179 ± 0.011^a，b，c	0.223 ± 0.016^a，b，c	0.180 ± 0.022^a
	G2	0.269 ± 0.005	0.259 ± 0.010	0.236 ± 0.015
	N2	0.228 ± 0.004^a	0.215 ± 0.014^a	0.202 ± 0.010
	M2	0.218 ± 0.013^a，c	0.240 ± 0.017^a	0.199 ± 0.009^a
肝脏中CCO	空白对照 Control	0.453 ± 0.014	0.422 ± 0.026	0.428 ± 0.024
CCO in liver	G1	0.411 ± 0.023	0.428 ± 0.009	0.441 ± 0.014
	N1	0.287 ± 0.011^a	0.389 ± 0.031	0.310 ± 0.024^a
	M1	0.349 ± 0.016^a，b，c	0.305 ± 0.020^a，b，c	0.335 ± 0.014^a，b
	G2	0.421 ± 0.021	0.420 ± 0.016	0.390 ± 0.007
	N2	0.241 ± 0.014^a	0.265 ± 0.027^a	0.274 ± 0.022^a
	M2	0.298 ± 0.015^a，b，c	0.295 ± 0.021^a，b	0.294 ± 0.015^a，b

Table 2

Table 3

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组别Group	钉螺死亡率/%（比值） Snail mortality rate/% (Ratio)
组别Group	12 h	24 h	48 h
空白对照 Control	0（0/36）	5.0（2/40）	5.3（2/38）
N1	22.2（8/36）^a	21.1（8/38）	44.4（32/72）^a
G1	0（0/38）	5.0（2/40）	15.0（6/40）
M1	35.0（14/40）^a	50.0（20/40）^a，b	70.0（56/80）^a，b
N2	20.0（8/40）^a	37.5（12/32）^a	73.7（56/76）^a，b
G2	0（0/38）	10.0（4/40）	10.5（4/38）
M2	30.0（12/40）^a	31.6（12/38）^a	84.2（101/120）^a，b

组别Group	软体组织匀浆中SOD/UI·g^-1SOD in soft tissue homogenate/UI·g^-1			软体组织匀浆中ROS/FI·μg^-1 ROS in soft tissue homogenate/FI·μg^-1
组别Group	12 h	24 h	48 h	12 h	24 h	48 h
空白对照 Control	3.54 ± 0.94	5.26 ± 0.08	5.10 ± 0.87	792.89 ± 50.73	802.37 ± 114.69	1 393.81 ± 86.12
G1	7.61 ± 0.54^a	10.09 ± 0.36^a	6.14 ± 0.92	853.39 ± 105.43	2 000.45 ± 472.21^a	2 662.99 ± 50.88^a
N1	4.53 ± 0.65	10.92 ± 1.24^a	6.16 ± 1.11	834.22 ± 104.89	1 300.01 ± 258.40	1 298.69 ± 43.89
M1	2.26 ± 0.75^b，c	5.56 ± 0.91^b，c	7.78 ± 0.11	838.71 ± 72.45	1 619.00 ± 168.25^a	1 866.65 ± 211.79^a，b，c
G2	8.40 ± 0.33^a	9.64 ± 1.27^a	4.88 ± 0.51	879.66 ± 105.79	1 430.35 ± 214.02	1 569.13 ± 128.93
N2	3.14 ± 0.29	9.77 ± 0.90^a	3.80 ± 0.94	534.01 ± 38.75^a	1 109.16 ± 62.23	1 998.05 ± 151.13^a
M2	2.40 ± 0.45^a，c	8.14 ± 0.15	1.60 ± 0.21^a，c	949.15 ± 84.78^b	1 272.73 ± 83.52	2 451.29 ± 195.91^a，c