Biphasic Dose–Response of Components From Coptis chinensis on Feeding and Detoxification Enzymes of Spodoptera litura Larvae

Abstract

Due to long-term coevolution, secondary metabolites present in plants apparently function as chemical defense against insect feeding, while various detoxification enzymes in insects are adaptively induced as a prosurvival mechanism. Coptis chinensis, a medicinal plant used in traditional Chinese medicine for a thousand years, was found to be less prey to insects in our earlier field observations. Herein, 4 crude extracts obtained from sequential partition of aqueous extract of Rhizoma coptidis with petroleum ether, ethyl acetate, and n-butanol exhibited antifeedant activity against Spodoptera litura (Fabricius) larvae at high doses and inducing activity at low doses. Furthermore, a similar biphasic dose–response of the antifeedant activity against S litura larvae was also observed for jateorhizine, palmatine, and obakunone in Coptis chinensis. Notably, the enzyme activities of glutathione-S-transferase and carboxyl esterase in S litura larvae affected by the different components (jateorhizine, palmatine, obakunone, berberine, and coptisine) of C chinensis also showed a biphasic dose–response with an increasing trend at low doses and a decreasing trend at high doses. Together, our study suggests that the components of C chinensis may play a chemical defensive role against S litura larvae in a hormetic manner.

Introduction

In the natural environment, plants are subjected to various stresses such as insect feeding. Although plants cannot simply change location to escape from disadvantages, they have evolved to synthesize a large number of phytochemicals to play a chemical defense role. ¹ Insects have evolved to feed on plants at least 4 billion years ago, thereby establishing a close coevolutionary relationship. ² As an adaptive stress response, insects have accordingly evolved diverse metabolic enzymes to detoxify the phytochemicals. ³ Accumulating evidence indicates that the effect of phytochemicals on insects may fit into a general concept known as hormesis, ^4,5 which refers to a process in which exposure to a low-dose stressor that is damaging at higher doses induces an adaptive beneficial effect on the cell or organism. ^6,7 Hormesis is characterized by biphasic dose responses (stimulation at low dose and inhibition at high dose) with an inverted U-shaped or J-shaped dose–response curve. ^7,8

Rhizoma coptidis, also known as Huang Lian, is the dried rhizome of Coptis chinensis Franch, C deltoidea C. Y. Cheng et Hsiao, or C teeta Wall (Ranunculaceae). In traditional Chinese medicine, R coptidis has been used to clear heat, eliminate dampness, and purge fire and detoxification for a 1000 years. ^9,10 Currently, there are over 100 chemical constituents isolated and structurally identified from R coptidis, which can be assigned to several classes: alkaloids, organic acids, coumarins, phenylpropanoids, quinones, and other chemical components. The major bioactive component in R Coptidis is alkaloids, such as berberine, palmatine, coptisine, jatrorrhizine, and epiberberine. ^{9 -11} Botanical insecticides, that is, extracts or compounds derived from Chinese herbal medicine, have long been touted as potential alternatives to conventional synthetic insecticides. ^12,13 In the screening studies of agrochemicals, the chloroform fraction isolated from C chinensis exhibited the insecticidal activity against Myzus persicae (Sulzer), and 2 active compounds were identified as 2-methoxy-4-vinylphenol and aniline. ¹⁴ The methanol extract of C chinensis roots showed potent antifeedant activity against the third instar larvae of Ostrinia furnacalis. ¹⁵ In the preliminary field observation of C chinensis in Shizhu County of Chongqing City in China, we found that C chinensis was less affected by pests, such as feeding of Spodoptera litura (Fabricius) larvae, compared to the plants in the same area. There was no serious insect infestation observed in the field of C chinensis. Thus, it is extremely interesting for us to explore the chemical defense role and the mode of action of the components derived from C chinensis against S litura larvae.

In this study, the antifeedant activity of the components derived from C chinensis against S litura larvae was evaluated. Moreover, influence of these components on the activities of detoxification enzymes in . litura larvae was further examined.

Materials and Methods

Plant Material and Chemicals

Rhizoma coptidis were purchased from Wanglong Coptis Company (Chongqing, China). Palmatine, obakunone, jateorhizine, coptisine, and berberine with a purity greater than 98% were purchased from Chroma-Biotechnology Co (Chengdu, China), dissolved in 50% ethanol (Sigma-Aldrich, St Louis, Missouri), and stored at −20°C.

Preparation of Crude Extracts

The dried slices of R Coptidis were pulverized to a fine powder (granule size 20 μm). One hundred twenty grams of the powder was subjected to reflux extraction with 95% ethanol. Then, the extract was concentrated to remove ethanol using a rotary evaporator (Büchi, Essen, Germany) under vacuum and dissolved in distilled water. Subsequently, the aqueous extract was extracted with petroleum ether (B.R. 40°C-60°C) followed by ethyl acetate and water saturated n-butanol. The solvents were finally removed using a rotary evaporator (Büchi) under vacuum to get the petroleum ether extract (RC-PE) 1.16 g, the ethyl acetate extract (RC-EA) 1.1 g, n-butanol extract (RC-B) 6.56 g, and aqueous phase (RC-A) 6.38 g.

Insect Rearing

The rearing was carried out in our laboratory under constant conditions: 27°C ± 1°C, relative humidity 60%-70%, and 12 light:12 dark photoperiod. To maintain the generation of insects, female and male moths with the same number were caged in a 10-cm-diameter beaker supplied with honey solution (10%) and permitted to copulate for 48 hours. Eggs were then laid by female moths on wax paper. Upon egg hatching, S litura larvae were fed on standard artificial diet. ¹⁶

Antifeedant Activity

The selective antifeedant activity was determined with a leaf disk choice test with the third instar S litura larvae. Disks (1 cm in diameter) were punched from cabbage leaves and soaked in the crude extracts of different concentrations for 5 seconds. The control was soaked in distilled water containing equal volume of ethanol. The leaf disks were air-dried before feeding the larvae. The treated leaf disks and the control leaf disks were simultaneously placed into each Petri dish with 3-cm distance. Each larva that already starved for 4 hours was introduced into a Petri dish with 3 leaf disks and allowed to feed. Each treatment used 10 larvae with 3 replicates. The number of larvae within 1-cm range of the leaf disks was counted within 72 hours after the introduction of larvae. The selective antifeedant rate (%) is calculated as: (C − T)/(C + T) × 100, where C refers to the average larvae number of the control group and T refers to the average larvae number of the treated group.

Determination of Glutathione-S-Transferase and Carboxyl Esterase Activities

The fifth instar larvae were exposed to the treated cabbage leaf disks or the control leaf disks for 48 hours. Midguts separated from larvae of each treatment were homogenized in liquid nitrogen and then added with 5 volumes of 0.1 M phosphate buffer (pH 7.0). Each sample was centrifuged at 8000g for 10 minutes at 4°C. Then, the activities of glutathione-S-transferase (GST) and carboxyl esterase (CarE) were further determined with the respective commercial kits (Nanjing Jiancheng Biotechnology, Nanjing, China), following the instructions provided by the manufacturer.

Statistical Analysis

The data in this study were reported as means ± standard deviation for triplicate. A t test or 1-way analysis of variance was applied to evaluate for significant differences between the 2 groups. A P value of <.05 was considered to be statistically significant.

Results

Antifeedant Activity of Crude Extracts of R Coptidis

To determine the antifeedant activity of the crude extracts derived from R Coptidis, S litura larvae were provided with the leaf disks treated with different crude extracts and the leaf disks treated with vehicle only. As shown in Figure 1A, S litura larvae exhibited even higher selectivity to leaf disks treated with 0.1 mg·mL⁻¹ of RC-PE than those with vehicle. Similar observation was also obtained in Figure 1B to D, where S litura larvae showed higher selectivity to leaf disks treated with 0.1 and 0.5 mg·mL⁻¹ of RC-EA, 0.1 mg·mL⁻¹ of RC-B, and 0.1 mg·mL⁻¹ of RC-A. However, the selective antifeedant rate remarkably increased to 43.02% in S litura larvae exposed to 0.5 mg·mL⁻¹ of RC-PE and then showed a decreased trend from 1.0 to 5 mg·mL⁻¹ of RC-PE. Figure 1B to D shows that S litura larvae exhibited an obvious selective antifeedant behavior from 0.5 to 5 mg·mL⁻¹ of RC-B and RC-A and 1 to 5 mg·mL⁻¹ of RC-EA. These results indicate that high doses of crude extracts of R Coptidis may exhibit antifeedant activity to S litura larvae, while low dose may show an inducing activity to S litura larvae.

Figure 1.

The selective antifeedant rate of crude extracts derived from Rhizoma coptidis against Spodoptera litura larvae. (A) Petroleum ether extract (RC-PE); (B) ethyl acetate extract (RC-EA); (C) n-butanol extract (RC-B); (D) aqueous phase (RC-A). Replicates = 3 and n = 10 larvae in each replicate. Values are presented as means ± standard deviation (SD).

Antifeedant Activity of Representative Compounds in C chinensis

To further evaluate the antifeedant activity of the representative compounds derived from C chinensis, S litura larvae were provided with the leaf disks treated with vehicle and the leaf disks treated with one of the representative compounds (jateorhizine, palmatine, obakunone, coptisine, and berberine). As shown in Figure 2A, 0.05 mg·mL⁻¹ of jateorhizine exhibited a little inducing activity to S litura larvae, while a dose-dependent increase in antifeedant activity to S litura larvae was observed in 0.1 to 10 mg·mL⁻¹ of jateorhizine. Notably, 10 mg·mL⁻¹ of jateorhizine showed an antifeedant activity of 67.9%. Figure 2B showed that both 0.05 and 0.1 mg·mL⁻¹ of palmatine exhibited the inducing activity to S litura larvae, and almost 30% of the inducing activity was observed in 0.1 mg·mL⁻¹ of palmatine. However, 0.5 to 10 mg·mL⁻¹ palmatine showed obvious antifeedant activity to S litura larvae. Similar trend was observed in Figure 2C, where 0.05 and 0.1 mg·mL⁻¹ of obakunone showed the inducing activity to S litura larvae and 0.5 to 10 mg·mL⁻¹ obakunone showed the antifeedant activity to S litura larvae. There was irregular inducing or antifeedant activity to S litura larvae for coptisine and berberine (data not shown).

Figure 2.

The selective antifeedant rate of the different compounds in Coptis chinensis against Spodoptera litura larvae. (A) Jateorhizine; (B) palmatine; (C) obakunone. Replicates = 3 and n = 10 larvae in each replicate. Values are presented as means ± standard deviation (SD).

Effect of Representative Compounds in Coptis chinensis on GST Activity

To determine the effect on GST activity, the fifth instar larvae were exposed to the leaf disks treated with the representative compounds derived from C chinensis or vehicle for 48 hours and then the enzyme activity of GST in the midgut was evaluated. As shown in Figure 3A, 0.05 to 0.5 mg·g⁻¹ of berberine significantly increased the GST activity compared to vehicle (P < .05). However, 1 to 10 mg·g⁻¹ of berberine remarkably decreased the GST activity compared with vehicle (P < .01 for 1 mg·g⁻¹ of berberine and P < .001 for 5 to 10 mg·g⁻¹ of berberine). Figure 3B demonstrates that 0.05 and 0.5 mg·g⁻¹ of coptisine significantly enhanced the GST activity compared to vehicle (P < .05), while 5 and 10 mg·g⁻¹ of coptisine dramatically reduced the GST activity (P < .001). Similar trend of phasic dose–response in the change of GST activity was also observed in S litura larvae exposed to other compounds derived from R coptidis. There was an increasing trend of GST activity in S litura larvae, respectively, exposed to 0.05 to 0.5 mg·g⁻¹ of jateorhizine, palmatine, and obakunone, although only each 0.5 mg·g⁻¹ significantly enhanced the GST activity in S litura larvae compared to the vehicle (P < .05). However, GST activity dramatically decreased for 1 or 5 to 10 mg·g⁻¹ of these 3 compounds compared to vehicle (P < .001; Figure 3C-E).

Figure 3.

Effect of the different compounds in Coptis chinensis on the activity of detoxification enzyme glutathione-S-transferase (GST). The GST activity was determined as mentioned under Materials and Methods section. (A) berberine; (B) coptisine; (C) jateorhizine; (D) palmatine; (E) obakunone. Replicates = 3 and n = 10 larvae in each replicate. Values are presented as means ± standard deviation (SD). *P < .05. **P < .01. ***P < .001.

Effect of Representative Compounds in Coptis chinensis on CarE Activity

The CarE activity in the midgut of S litura larvae was also determined after exposed to the representative compounds derived from C chinensis or vehicle for 48 hours. Interestingly, the effect of all these compounds on CarE activity exhibited biphasic dose responses with an increasing trend from 0.05 or 0.1 to 0.5 or 1 mg·g⁻¹ and a decreasing trend from 1 or 5 to 10 mg·g⁻¹ (Figure 4A-E). Among them, berberine exhibited the greatest impact on the CarE activity. As shown in Figure 4A, all 0.05 to 1 mg·g⁻¹ of berberine significantly increased the CarE activity (P < .05), while both 5 and 10 mg·g⁻¹ of berberine remarkably decreased the CarE activity (P < .01). Figure 4B shows that 0.1 to 1 mg·g⁻¹ of coptisine significantly increased the CarE activity (P < .05) and only 10 mg·g⁻¹ of coptisine remarkably decreased the CarE activity (P < .01). Figure 4C and D shows that only 0.5 mg·g⁻¹ jateorhizine and 1 mg·g⁻¹ palmatine significantly enhanced the CarE activity (P < .05), while 10 mg·g⁻¹ jateorhizine and 5 and 10 mg·g⁻¹ palmatine remarkably decreased the CarE activity. There was no dose of obakunone exhibited statistical significance on increasing the CarE activity, although an increasing trend was observed in 0.05 to 1 mg·g⁻¹ of obakunone. However, 5 (P < .05) and 10 mg·g⁻¹ (P < .01) of obakunone decreased the CarE activity (P < .01; Figure 4E).

Figure 4.

Effect of the different compounds in Coptis chinensis on the activity of detoxification enzyme carboxyl esterase (CarE). The CarE activity was determined as mentioned under Materials and Methods section. (A) berberine; (B) coptisine; (C) jateorhizine; (D) palmatine; (E) obakunone. Replicates = 3 and n = 10 larvae in each replicate. Values are presented as means ± standard deviation (SD). *P < .05. **P < .01.

Discussion

Botanical pesticides have emerged as attractive alternatives to synthetic pesticides due to the environmentally friendly and safe characteristics. ¹² There is growing evidence indicates that phytochemicals derived from stressed natural plants may interact with insects in a hormesis way owing to the long period of coevolution. ³ In our preliminary field observation, we found that C chinensis was less fed by S litura larvae, one of regular polyphagous and damaging insects, while other plants in the same area suffered serious insect infestation. Thus, the current study aimed to figure out whether the components derived from R coptidis play a defense role against S litura larvae.

In our study, we first evaluated the antifeedant activity of the crude extracts (RC-PE, RC-EA, RC-B, and RC-A) isolated from R coptidis with systematic solvent extraction. Our results demonstrated that high doses (0.5-5 mg·mL⁻¹) of all these crude extracts of R coptidis exhibited antifeedant activity to S litura larvae, while low dose (0.1 mg·mL⁻¹) may show an inducing activity to S litura larvae. It is suggested from this biphasic dose–response that the effect of the crude extracts derived from R coptidis may follow the hormesis model. Recent studies have highlighted that many ingredients from natural plants exhibited bifunction in model organisms did so by mediating hormesis. For example, rutin, which is rich in citrus fruits and buckwheat seeds, at 200 and 400 μM significantly extended median lifespan of Drosophila melanogaster, while rutin with a dose beyond 400 μM dramatically reduced longevity. ⁸ In our previous study, Z-ligustilide in Ligusticum chuanxiong Hort. exhibited potent antifeedant activity against S litura larvae within the dose range of 0.25 to 5 mg·g⁻¹ and 0.1 mg·g⁻¹ of Z-ligustilide showed an inducing activity to S litura larvae. ¹⁷ Furthermore, we also examined the larvicidal activity of these crude extracts against S litura larvae. However, only weak larvicidal activity (<20%) was observed for all the crude extracts of R coptidis. Moreover, there was almost no statistical difference for the larvicidal activity among the different groups (data not shown). Consistent with our results, Zhou et al reported that methanol extract of R coptidis exhibited potent antifeedant activity and weak larvicidal activity against the third instar larvae of Ostrinia furnacalis. ¹⁵ It is believed that the mixture of phytochemicals may be deterrent to insects and herbivores for longer period than single compound. ¹² Our study revealed that all the 4 different crude extracts exhibited antifeedant activity against S litura larvae under certain doses, indicating that the antifeedant activity of C chinensis may be derived from the mixture of different kinds of chemical components and not just 1 or 2 single compounds.

Palmatine, coptisine, berberine, and jatrorrhizine belong to protoberberine-type alkaloids, which are the most abundant components and considered as the main active ingredients in C chinensis, and obakunone is a representative triterpenoid in C chinensis. ^{9 -11} Then, we used these 5 compounds as an example to determine the antifeedant activity and the mode of action of the single compound derived from C chinensis. Similar with crude extracts of R coptidis, our results revealed that the biphasic dose–response of the antifeedant activity was observed for jateorhizine, palmatine, and obakunone to S litura larvae. Moreover, there was also only weak and unregular larvicidal activity against S litura larvae observed for these components (data not shown). Previous study showed that the methanol extract of Phellodendron amurense bark exhibited a strong antifeedant activity against Reticulitermes speratus and further bioguided fractionation led to the identification of obacunone and kihadanin A in the chloroform fraction and berberine and palmatine iodide as the bioactive components. ¹⁸ Berberine, palmatine, and coptisine identified in Coptis japonica roots exhibited strong antifeedant activity against Hyphantria cunea (Lepidoptera: Arctiidae) and Agelastica coerulea (Coleoptera: Galerucinae). ¹⁹

Insect GSTs and CarE belong to a superfamily of detoxification enzymes. It is reported that 40, 35, 13, 23, and 32 isoforms of GSTs were identified in Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively. ⁸ Glutathione-S-transferases play a protective role in detoxification through catalyzing the conjugation of reduced glutathione with electrophilic endogenous and xenobiotic compounds, as well as end products of lipid peroxidation, such as 4-hydroxynonenal and malonaldehyde. ^20,21 CarE is a polyprotein belonging to the serine hydrolase family. It hydrolyzes endogenous and exogenous substances such as esters and thioesters, participates in the metabolic detoxification of many toxic substances, and reduces toxicity. ²² Notably, our study demonstrated that the enzyme activities of GST and CarE in S litura larvae affected by the different components of C chinensis also showed a biphasic dose–response with an increase at low dose and decrease at high dose. These results suggest that detoxification enzymes may be adaptively activated in S litura larvae as a prosurvival mechanism in response to low dose of the components in C chinensis, whereas they were directly inhibited under the insult of high dose of these components, which further confirms the hormetic role of the components in C chinensis. Similarly, our previous report indicates that detoxification enzymes in S litura larvae was also modulated by Z-ligustilide in Ligusticum chuanxiong Hort. in a hormesis way. ¹⁷ Although berberine and coptisine showed greater impact on GST and CarE enzyme activities compared to other 3 compounds (palmatine, jatrorrhizine, and obakunone), no typical biphasic dose–response for the antifeedant activity of berberine and coptisine was observed in our study. This suggests that there may be other unknown mechanisms, which deserves further in-depth research in our future study, in addition to these 2 enzymes.

Conclusion

Our study demonstrated that a biphasic dose–response was observed in the antifeedant activity of the crude extracts and bioactive compounds in C chinensis as well as the modulation of detoxification enzymes of GST and CarE by those bioactive compounds. These results suggest that the components in C chinensis may function via a hormesis way in the chemical defense against S litura larvae.