Evaluation of the chemical compositions, larvicidal and antimicrobial efficacies of Zingiber castaneum and Zingiber nitens essential oils

In this paper, we report the chemical constituents, larvicidal and antimicrobial activities of essential oils from Zingiber castaneum Škorničk. & and Zingiber nitens M.F. growing in Vietnam. The main constituents of Z. castaneum leaf were bicyclogermacrene (24.8%), germacrene D (12.9%), cis -β-elemene (11.2%) and β-pinene (10.3%), while the pseudo-stem contained bicyclogermacrene (15.8%), cis -β-elemene (9.8%) and germacrene D (9.2%). The significant compound of the rhizome oil was sabinene (22.9%), along with α-pinene (7.8%), β-pinene (6.5%), bornyl acetate (6.1%) and γ-terpinene (5.5%). However, β-pinene (45.8%), α-pinene (10.7%) and bicyclogermacrene (7.8%) were the dominant compounds in the leaf oil of Z. nitens . Terpinen-4-ol (77.9%) occurred as the compound occurring in higher amount in the rhizome oil. The rhizome oil of Z. castaneum exhibited 100% mortality towards Ae. aegypti (concentration, 200 µg/mL; 24 h and 48 h) and Ae. albopictus (concentration, 100 µg/mL; 24 h and 48 h). However, mortality of 81.3% was observed against Cx. quinquefasciatus at 48 h (concentration 100 µg/mL). The leaf also exhibited 100% mortality against Ae. aegypti (concentration, 100 µg/mL; 24 h and 48 h) and Cx. quinquefasciatus (concentration, 150 µg/mL; 24 h and 48 h). The rhizome oil displayed a minimum lethal concentration LC 50 of 121.43 µg/mL and 110.31 µg/mL Overall results in this study showed that essential oils hydrodisitlled from the leaf and rhizome of Z. castaneum and Z. nitens exhibited good mortality and larvicidal activity on Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus larvae. The observed larvicidal action of Z. castaneum and Z. nitens in this study was comparable with findings from Zingiber plants analysed for their larvicidal activity from Vietnam and other parts of the world. The essential oil of Z. collinsii from Vietnam displayed larvicidal action against Ae. albopictus (LC 50 = 25.51 μg/mL; LC 90 = 40.22 μg/mL) and Cx. quinquefasciatus (LC 50 = 50.11 μg/mL and LC 90 = 71.53 μg/mL) after 24 h [6]. Z. zerumbet oil showed potent larvicidal activity against Cx. quinquefasciatus with LC 50 of 33.28 mg/mL and 21.81 mg/mL respectively after 24 h and 48 h test period. Moreover, the oil exhibited significant larvicidal action against Ae. albopictus within the 24 h and 48 h tested period having LC 50 of 55.75 µg/mL and 36.22 µg/mL respectively [7]. The 24 h mosquito larvicidal activity of the rhizome oil of Z. montanum from Vietnam [8] was Ae. albopictus (LC 50 = 35.17 μg/mL; LC 90 = 56.02 μg/mL), Ae. aegypti (LC 50 = 32.20 μg/mL; LC 90 = 45.64 μg/mL) and Cx. quinquefasciatus (LC 50 = 31.12 μg/mL; LC 90 = 52.25 μg/mL). The essential oil of Z. zerumbet from Malaysia displayed lower

Zingiber castaneum Škorničk. & Q.B. Nguyễn is easily recognized among other terminally flowering species by its upright inflorescence with reflex bracts. The plant is also a rhizomatous herb forming small clumps. The creeping aromatic rhizome which grows up to 1.5 cm in diameter is externally light brown and internally cream white [3]. The translucent light green leaves are glabrous.
Till moment, nothing is known about their medicinal uses as well as the biological effects of essential oils from both species. In the present paper, the compounds identified in the essential oils from various parts of both Z. nitens and Z. castaneum grown in Vietnam were reported. In addition, the larvicidal and antimicrobial efficacies of the essential oils were also described. Previously, the chemical compounds identified in the essential oils of some other Zingiber plant grown in Vietnam [5][6][7], their larvicidal potentials [6][7][8] and antimicrobial activities [7] were reported by us. Although, terpene compounds were prominent in these essential oils, the identities of the compounds differ from one species to another [2,[4][5][6][7][8].
Vietnam is classified as a hyperendemic dengue country with present throughout the year and dengue fever epidemics have increased in frequency [9]. Mosquitoes have been and continue to be the most deadly creatures on earth. Aedes albopictus (Skuse) (Diptera Culicidae) is ranked among the most invasive mosquito species in the world [10]. Besides it's aggressive daytime biting behaviour, the medical importance of Ae. albopictus is due to its ability to transmit many human pathogens and parasites (e.g. yellow fever, dengue fever, West Nile, Japanese encephalitis, St. Louis encephalitis, chikungunya viruses, filarial nematodes). Culex quinquefasciatus Say, commonly known as the southern house mosquito, is a medium-sized brown mosquito that exists throughout the tropics. It is a vector of many pathogens of humans, domestic and wild animals. Viruses transmitted by this species include lymphatic filariasis, West Nile virus, St. Louis encephalitis virus, Western equine encephalitis virus and Zika virus [11]. The yellow fever mosquito, Aedes aegypti (Linn), has been a nuisance species for centuries. Aedes aegypti is the primary vector of yellow fever, a disease that is prevalent in tropical South America and Africa, and often emerges in temperate regions during summer months. All four dengue viruses are spread primarily through the bite of an infected Aedes species (Ae. aegypti and Ae. albopictus) mosquito [12].
The control of adult mosquitoes commonly relies on the use of synthetic insecticides and repellents, but treatments with such chemicals are expensive, show scarce efficacy and have a strong environmental impact associated to relevant human 6 health risks. For these reasons, alternative natural insecticides and repellents are now very appreciated by consumers. Essential oils of aromatic plants are considered among the most promising alternative to synthetic chemicals [13]. Essential oils are generally recognized as environmental friendly, easily biodegradable, minimally toxic to mammals and have shown repellent activities against different mosquito ±species.
As part of our ongoing research aimed at the identification of the chemical constituents, larvicidal and antimicrobial potentials of essential oils from plant (especially Zingiber species) grown in Vietnam [2,[4][5][6][7][8], we have obtained essential oils from Z. nitens and Z. castaneum and hereby report the compounds present therein. In addition, we examined their mosquito larvicidal and antimicrobial activities and present the report for the first time. The authors are aware that there were no previous investigations on the larvicidal and antimicrobial activities of the studied essential oils.

castaneum.
The essential oils from the leaf and rhizome of Z. nitens were obtained in yields of 0.27% and 0.54% (v/w) respectively. From the GC and GC/MS analysis, it was discovered that monoterpene hydrocarbons (59.0%) and sesquiterpene hydrocarbons (36.3%) constitute the bulk of the oil sample (Table 1)  Thus, higher inhibition of mosquito larvae was observed as concentration increases.   [19].
Since the WHO has not established a standard criterion for determining the larvicidal activity of natural products, several authors have developed individual criteria to characterize the potency of mosquito larvicides developed from natural products [21][22]. For example, considered products showing LC 50 ≤ 50 mg/L to be active, 50 mg/L < LC 50 ≤ 100 mg/L to be moderately active, 100 mg/L < LC 50 ≤ 750 mg/L to be effective, and LC 50 > 750 mg/L to be inactive [21]. Likewise, considered compounds with LC 50 < 100 mg/L to exhibit a significant larvicidal effect [22]. It should be stressed that these criteria must be directly correlated with the time of exposure and the origin of larvae, which are variables that can alter the LC 50 values. The results obtained in this study showed that the essential oils of Z.
castaneum and Z. nitens had promising effects, according to the criterion established previously [21,22]. In summary, Z. castaneum and Z. nitens essential oils from Vietnam revealed important toxicity and larvicidal properties on Ae.
Albopictus, Ae. aegypti and Cx. quinquefasciatus larvae and stands as a promising tool to manage the phenomenon of insecticides resistant vectors in malaria endemic regions.
The variations in toxicity of essential oils against different species of mosquitoes are common, due to qualitative and quantitative variations of chemical constituents.
Interestingly, the active larvicidal compounds in these works, including, α-pinene, βpinene, sabinene, limonene, p-cymene, 1,8-cineole, terpinen-4-ol, β-caryophyllene, bicyclogermacrene and germacrene [23][24][25]. The isolation and purification of active compound which could be responsible for the larvicidal activity against mosquito vectors of would be an important step in the development of novel mosquitocidal agents. Production of larvicides from the locally available plants, could be a new acceptable alternative to employ which may lead to decreasing dependence on imported synthetic insecticides and be beneficial for developing countries such as Vietnam.

Antimicrobial test
The essential oil from pseudo-stem of Z. castaneum showed stronger inhibitory effect on P. aeruginosa with MIC of 12.5 μg/mL and MIC value of 50 μg/mL against A.
niger and F. oxysporum. In addition, all the other tested oil samples inhibited the growth of P. aeruginosa with MIC value of 50 μg/mL (Table 4). In this test experiment, activity was presumed to occur with MIC £ 50 μg/mL while MIC > 50 μg/mL is considered inactive towards the tested microorganism. No previous information exists on the antimicrobial activity of essential oils from Z. castaneum and Z. nitens. The present data represent the first report on the antimicrobial action of the studied essential oils. The observed antimicrobial results of Z. castaneum and Z. nitens oils were in agreement with information that Zingiber oil samples posses' antimicrobial action.
The antimicrobial activity of essential oils of some Zingiber species was reported.
The essential oil of Z. zerumbet was shown to inhibited the growth of A. niger [26] as wll as C. albicans, S. auries, Salmonella typhi, several species of Trichophyton, Streptococcus mutans [27]. Z. officinale and Z. zerumbet essential oils were considered potential therapeutic agents against bacterial several infections [28].
Essential oil of Z.officinale was efficient against three positive strains of bacteria (S. aureus, B. cereus and L. monocytogenes), with a minimum concentration to inhibit B. cereus and L. monocytogenes of 6.25 mg/mL [29]. The observed antimicrobial activity of Z. castaneum and Z. nitens essential oils can be related to the compounds present in it. For example, essential oil constituents such as α-pinene, β-pinene, sabinene, 1,8-cineole, terpinen-4-ol, β-caryophyllene, bicyclogermacrene and germacrene were previously reported to inhibit significantly the growth and cell viability of potential infectious of broad spectrum microorganisms [7,14].

Preparation of samples
In the course of preparation for hydrodisitillation process, the leaf, pseudo-stem and rhizome were air-dried (22 0 C) under laboratory shade for two weeks to reduce the moisture contents. Moreover, unwanted materials were also removed by handpicking. Afterwards, samples were pulverized to coarse powder using a locally made grinder.

Hydrodistillation procedure
A total of 1000 g of each of the pulverized samples were used for the experiment at different time. Known weight of samples was separately and carefully introduced into a 5 L flask and distilled water was added until it covers the sample completely.
Essential oils were obtained hydrodistillation which was carried out in an all glass Clevenger-type distillation unit designed according to Vietnamese Pharmacopoeia 26 [30] as described previously [2,[4][5][6][7][8]. The distillation time was 3 h and conducted at normal pressure. The volatile oils distilled over water and were collected by running through the tap in the receiver arm of the apparatus into clean and previously weighed sample bottles. The oils were kept under refrigeration (4 o C) until the moment of analyses as described previously [2,[4][5][6][7][8].

Gas chromatography (GC) analysis
Gas chromatography (GC) analysis was performed on an Agilent Technologies HP as those used for gas chromatography analysis as described previously [2,[4][5][6][7]. The GC conditions were the same as described above with He (1 mL/min) as carrier gas.
The MS conditions were as follows: ionization voltage 70eV; emission current 40 mA; acquisitions scan mass range of 35-350 amu at a sampling rate of 1.0 scan/s.

27
The identification of constituents from the GC/MS spectra of Z.nitens and Z.
castaneum was performed on the basis of retention indices (RI) determined with reference to a homologous series of n -alkanes (C 4 -C 40 ), under identical experimental conditions. In some cases, co-injection with known compounds or standards (Sigma-Aldrich, St. Louis, MO, USA) under the same GC conditions was employed. The mass spectral (MS) fragmentation patterns were checked with those of other essential oils of known composition [31] and with those in the literature as described previously [2,[4][5][6][7][8].

Mosquito larvae
Adults of Culex quinquefasciatus, Aedes aegypti and Aedes albopictus collected in Hoa Khanh Nam ward, Lien Chieu district, Da Nang city(16°03'14.9"N, 108°09'31.2"E). Adult mosquitoes were maintained in entomological cages (40 x 40 x 40 cm) and fed a 10% sucrose solution and were allowed to blood feed on mice.
Eggs hatching were induced with tap water. Larvae were reared in plastic trays (24×35×5 cm). The larvae were fed on dog biscuits and yeast powder in the 3:1 ratio.

Statistical analysis
The data obtained were subjected to log-probit analysis [33] to obtain LC 50 values, LC 90 values, 95% confidence limits, and chi square values using XLSTAT v. 2018.5 (Addinsoft, Paris, France).

Microdilution broth susceptibility assay
The Minimum inhibitory concentration (MIC) values were measured by the 29 microdilution broth susceptibility assay [34,35]. For the assays, the essential oil was diluted with DMSO and loaded into the microtiter plate with each of the microbial strains. The plate was then incubated overnight at 37 o C. One hundred microlitre of microbial culture of an approximate inoculums size of 1.0 x 10 6