Evaluation of larvicidal activity and brine shrimp toxicity of rhizome extracts of Zingiber zerumbet ( L . )

Introduction: In this study, we used dichloromethane (DCM) and methanol (MeOH) extracts of the Zingiber zerumbet rhizome to evaluate brine shrimp lethality and larvicidal activity on Aedes aegypti and Anopheles nuneztovari mosquitoes. Methods: Bioassays were performed by exposing third-instar larvae of each mosquito species to the DCM or MeOH extracts. Results: Probit analysis with DCM and MeOH extracts demonstrated effi cient larvicidal activity against A. aegypti and A. nuneztovari larvae. Conclusions: The DCM and MeOH extracts showed higher activity against A. nuneztovari larvae than against A. aegypti larvae, suggesting that the extracts have species-specifi c activity.

Zingiber zerumbet (L.) Smith, popularly known as gengibre amargo (bitter ginger), is an Asiatic plant introduced into the Amazon region that has long been used in Asian popular medicine for treating a number of illnesses.The literature shows that some compounds isolated from the essential oil of Z. zerumbet, such as zerumbone, humulene, zederone, and camprene, possess anti-infl ammatory, antiviral, antitumor, antioxidant, antiallergic, and antimicrobial activities 1,2 .Zederone, a sesquiterpene compound, has been suggested to contribute to the larvicidal activity of the ethanol extract 3 .
Tropical diseases like dengue and malaria still represent a major public health concern, mainly in developing countries 4 .Recently, to treat these diseases, botanical and microbial insecticides have been increasingly used for mosquito control because of their effi cacy and documented non-toxic effects on non-target organisms 5 .Mosquito larval control may prove to be an effective tool that can be incorporated into integrated vector management strategies for reducing malaria transmission 6 .
The promising larvicidal potential of Z. zerumbet essential oils has been recently reported in literature 2 .The aim of the present investigation was to evaluate the brine shrimp lethality and larvicidal potential of Z. zerumbet rhizome extracts.The larvicidal assay was performed against larvae of 2 mosquito species (Aedes aegypti and Anopheles nuneztovari).This plant was selected because it has been used for cancer treatment and it also exhibits antimicrobial activity 1 , while only a few studies on its larvicidal activity have been reported 7,8 .
Rhizomes of Z. zerumbet were collected from the Tarumã River region, Manaus, AM, Brazil (03º00'05′′S and 60º05′01′′W).The rhizomes were crushed, dried, macerated with dichloromethane (DCM) for 72h, and then fi ltered.The plant material was dried, extracted with methanol (MeOH), for 72h, and fi ltered.The extracts obtained were concentrated in a low-pressure rotary evaporator to remove excess solvent.The extracts were redissolved in dimethylsulfoxide (DMSO) for further tests.A voucher specimen was deposited in the herbarium of Instituto Nacional de Pesquisas da Amazônia (INPA) (Number 186913).
Both mosquito species were maintained for oviposition in the insectary at 26 ± 2°C, with a photoperiod of 12:12 (L/D) and a relative humidity of 80-90%, according to the criteria of Scarpassa and Tadei 9 .
Aedes aegypti Linnaeus, 1762: Eggs were obtained from the colonies of the Malaria and Dengue Laboratory (INPA) and they were kept in insectaria cages.
Anopheles nuneztovari Gabaldón, 1940: Species collections of the genus Anopheles were carried out at Natan Farm in the east region of Manaus City, State of Amazonas, Brazil (03º04′10′′S and 59º51′40′′W).Catches were carried out in cattle pens, and only fed females were selected.Samples were collected using an entomological manual captor between 18 and 21h.
The larvicidal bioassay was carried out using 5 different doses of the DCM and MeOH extracts of Z. zerumbet rhizomes.Ten third-instar larvae of each mosquito species were used and 50μL of liquid food added containing DCM or MeOH extracts at concentrations of 100, 200, 300, 400, or 500μg/mL.In each case, 4 replicates of each concentration were assayed.The negative control received only DMSO at the same concentration and a 10% mortality rate and a 95% confi dence interval were set as  the limits.Readings were collected at 24, 48, and 72h, recording the number of live and dead larvae at each concentration.The larvae were considered dead if they were immobile and unable to reach the water surface.
The extracts were evaluated for lethality to brine shrimp larvae (Artemia salina Leach) according to the procedure described by Meyer et al. 10 with some modifi cations.Briefl y, dried brine shrimp eggs were bred in saline medium (Instant Ocean®).After 48h, a few shrimps hatched and were ready for testing.One-day-old larvae (10 per vial) were transferred into 5-mL vials containing saline solution along with 25, 50, 100, 250, 500, or 1,000μg/mL of each DCM and MeOH extract dissolved in DMSO and diluted serially in saline water.In each case, 4 replicates of each concentration were assayed.After 24h, the survivors were counted and the percentage mortality at each dose was recorded.A saline solution containing DMSO (1%) was used as the negative control (LC 50 > 1,000μg/mL), while colchicine (LC 50 = 25μg/mL) was used as the positive control 11 .
For statistical analysis, the lethal concentration (LC 50 and LC 90 ) was calculated using Probit analysis.The percentage mortality was calculated and mortality corrections when necessary were carried out using Abbot's formula.
The brine shrimp lethality assay is considered a useful tool for preliminary toxicity assessment, to screen medicinal plants popularly used for several purposes, and for monitoring the isolation of a great variety of biologically active compounds 12 .The method is rapid, simple, reproducible, and economical.This in vivo test has been successfully employed for bioassayguide fractionation of active cytotoxic and antitumor agents 1 .
Furthermore, positive correlations have been demonstrated between the antimicrobial 12 and larvicidal 13 activities, and lethality to brine shrimp and the corresponding lethal dose of medicinal plants.
The results of Artemia salina testing are summarized in Table 1 (mortality % and LC 50 -LC 90 ).The results revealed that Z. zerumbet extracts showed signifi cant toxicity against Artemia salina for up to 48h, with an LC 50 of 30.9μg/mL for the DCM extract, and an LC 50 of 64.0μg/mL for the MeOH extract.These results are consistent with the results of Déciga-Campos et al 11 , who reported LC 50 values ranging from 37 to 227μg/mL, and of Bastos et al 12 , who reported an LC 50 of 29.55μg/mL for hexane acid and an LC 50 of 398.05μg/mL for a 1:1 mixture of hexane-CHCl 3 (Table 1).
Larvicide activity involves applying chemicals to habitats to kill pre-adult mosquitoes.This experiment validates and reveals the effi cacy of the DCM and MeOH extracts of Z. zerumbet against A. aegypti and A. nuneztovari larvae.Furthermore, a positive correlation was observed between the concentration of the extract and the mortality percentage (Tables 1 and 2), with the mortality rate being directly proportional to concentration.Bioassays showed that the DCM extract was more toxic than the MeOH extract to the larvae of both mosquito species, and that A. nuneztovari (CL 50 < 70μg/mL) was more susceptible to the DCM extract than A. aegypti (CL 50 < 300μg/mL) in both treatments (Table 2).Zerumbone is the main common component (31.7%) in the rhizome oil of the Asian species of Z. zerumbet 8 , and 97% pure zerumbone was obtained from the essential oil of the rhizomes of the Brazilian species 14 .Based on

Bücker A et
al -Larvicidal activity and brine shrimp toxicity of rhizome extracts of Z. zerumbet

TABLE 1 -
Percentage mortality of brine shrimp (Artemia salina), and Aedes aegypti and Anopheles nuneztovari larvae, at different time intervals and with different Zingiber zerumbet dichloromethane and methanol extract concentrations.