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

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-inflammatory, 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 extract³.

Tropical diseases like dengue and malaria still represent a major public health concern, mainly in developing countries⁴. Recently, to treat these diseases, botanical and microbial insecticides have been increasingly used for mosquito control because of their efficacy and documented non-toxic effects on non-target organisms⁵. Mosquito larval control may prove to be an effective tool that can be incorporated into integrated vector management strategies for reducing malaria transmission⁶.

The promising larvicidal potential of Z. zerumbet essential oils has been recently reported in literature². 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 activity¹, 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 filtered. The plant material was dried, extracted with methanol (MeOH), for 72h, and filtered. 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 ± 2ºC, with a photoperiod of 12:12 (L/D) and a relative humidity of 80-90%, according to the criteria of Scarpassa and Tadei⁹.

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% confidence 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 modifications. Briefly, 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 (LC₅₀ > 1,000µg/mL), while colchicine (LC₅₀ = 25µg/mL) was used as the positive control¹¹.

For statistical analysis, the lethal concentration (LC₅₀ and LC₉₀) 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 compounds¹². The method is rapid, simple, reproducible, and economical. This in vivotest has been successfully employed for bioassay-guide fractionation of active cytotoxic and antitumor agents¹. Furthermore, positive correlations have been demonstrated between the antimicrobial¹² and larvicidal¹³ 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 LC₅₀–LC₉₀). The results revealed that Z. zerumbet extracts showed significant toxicity against Artemia salina for up to 48h, with an LC₅₀ of 30.9µg/mL for the DCM extract, and an LC₅₀ of 64.0µg/mL for the MeOH extract. These results are consistent with the results of Déciga-Campos et al¹¹, who reported LC₅₀ values ranging from 37 to 227µg/mL, and of Bastos et al¹², who reported an LC₅₀ of 29.55µg/mL for hexane acid and an LC₅₀ of 398.05µg/mL for a 1:1 mixture of hexane-CHCl₃ (Table 1).

Larvicide activity involves applying chemicals to habitats to kill pre-adult mosquitoes. This experiment validates and reveals the efficacy 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 (CL₅₀ < 70µg/mL) was more susceptible to the DCM extract than A. aegypti (CL₅₀ < 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. zerumbet⁸, and 97% pure zerumbone was obtained from the essential oil of the rhizomes of the Brazilian species¹⁴. Based on these results, the authors suggest that the high activity present in the DCM extract might be associated with a considerable amount of the active principal of Z. zerumbet, which would cause the greater susceptibility of the tested organisms (brine shrimp and mosquito larvae) (Table 2). Rahuman et al.¹⁵ evaluated the larvicidal activity of the petroleum ether extract of Z. officinalis, and their results show that the most effective compound against A. aegypti (4.25ppm) and Culex quinquefasciatus (5.52ppm) was 4-gingerol. Sutthanont et al.⁸ have reported that the essential oils of Z. zerumbet and Kaempferia galanga, two Zingiberaceae, were found to be larvicidal against pyrethroid-susceptible A. aegypti, with LC₅₀ values of 48.88 and 53.64ppm, respectively. Tewtrakul et al.⁷ reported the effective toxicity of the ethanol extract of Z. zerumbet against Anopheline larvae, with an LD₅₀ of 18.9µg/mL. However, the biological activity of Z. zerumbet as a larvicide has not been studied so far.

In conclusion, the present findings support the use of Z. zerumbet as an alternative to combat mosquito vectors of diseases. The results reported here pave the way for further investigations into the larvicidal properties of natural product extracts. We are currently testing the zerumbone component from the rhizomes of Z. zerumbet¹⁴.