Chemical Composition and Bioactivity of Vitex cofassus Reinw. Extracts on the Larval and Pupal Stages of Aedes aegypti.

AIMS AND
OBJECTIVES: This study sought to investigate the chemical composition, larvicidal, and cytotoxic potentials of the fruit extract of Vitex cofassus Reinw.
MATERIALS AND METHODS: The chemical composition was analyzed using the gas chromatography-mass spectrometry technique. Separately, mortality data were evaluated by probit analysis to determine the lethal concentration (LC)50 and LC90 values on brine shrimp and Aedes aegypti larvae. Moreover, the effects on different stages of Ae. aegypti were also examined.
RESULTS: Fourteen majority components representing carboxylic acid were identified. The extract was very toxic to both brine shrimp and Ae. aegypti larvae with LC50 values of 0.308 and 0.514 µg/mL, whereas the LC90 values were 4.317 and 1.921 µg/mL, respectively. The effect of the fruit extract of V. cofassus on different stages of Ae. aegypti indicated that high concentrations (2.00-4.00 µg/mL) promoted complete mortality. A concentration of 0.125 to 1.00 µg/mL inhibited larval metamorphosis.
CONCLUSION: To the best of our knowledge, this is the first study to evaluate the chemical composition and larvicidal effects of the fruit extract of V. cofassus. The results indicate that the extract may be a promising source of larvicidal compounds that could be useful for pharmaceutical applications. Copyright:

INTRODUCTION

Mosquitoes act as vectors for most tropical diseases such as dengue fever, Zika, chikungunya, malaria, yellow fever, filariasis, and encephalitis, often becoming endemic in tropical developing countries. Preventing disease proliferation by this route, improving the quality of life of patients, and ensuring public health are essential goals in mosquito control.[123]

Several strategies for mosquito control exist, including using structural barriers and applying synthetic insecticides (i.e., organochlorine and organophosphate compounds). Synthetic insecticides are the most effective of the available chemicals but still require more attention because of their harmful effects on human health; further, they are nonbiodegradable, with negative impacts on environmental sustainability.[45] In recent years, insecticides resistance has been reported worldwide in several mosquito populations, suggesting the need for alternative methods.[67]

One option for mitigating the growing insecticide resistance rates may be the use of bioinsecticides derived from plants, which would be renewable, easy to construct, and quickly degradable.[89] Among the more promising groups of plants used in Sulawesi is Vitex cofassus Reinw, a species of woody plants (Lamiaceae family) with the potential to be used in pharmacological activities. In Sulawesi, the V. cofassus main stem is often used as a primary raw material for building, woodworking, and boat construction as well as in warding off mosquitoes and insects. However, among the more than 250 species of Vitex, 24 to date have been investigated for phytoconstituents. Only terpenes, flavonoids, and lignans are the main constituents reported of the Vitex species. Various pharmacological activities such as cardioprotective,[10] anticancer,[11] gastroprotective,[12] antitermite,[13] antinociceptive,[14] antimicrobial,[15] and antifungal[16] abilities have been identified in this species. The first pharmacotoxicological study for V. cofassus was conducted in 1969.[17] More recently, Rasyid et al.[18] reported on a new clerodane diterpene, 16-hydroxy-pentadralactone acuminolide, as an anticancer agent against human umbilical vein endothelial cells (HUVECs) that was isolated from the methanol extract of V. cofassus.

The purpose of this study was to examine the potential larvicidal activity of the fruit extract of V. cofassus to elucidate its potential as a possible green bioinsecticide.

MATERIALS AND METHODS

Sample preparation

Fresh fruits of V. cofassus were collected in July 2016 from the Regency of Palopo in South Sulawesi, Indonesia. The fruits were subsequently dried and put through two consecutive maceration processes (each one lasting for two days) using methanol. A stock solution (500 µg/mL) containing 1% of dimethyl sulfoxide (Merck & Co, Kenilworth, NJ) was prepared. Three serials of each six concentration were placed in sea salt water for brine shrimp lethality bioassay and in distilled water for Ae. aegypti larvicidal bioassay.

Brine shrimp lethality bioassay

Brine shrimp eggs (Artemia salina) were obtained from the Indonesian Muslim University in Makassar, Indonesia. Filtered artificial seawater was prepared by dissolving 38 g of sea salt in 1 L of distilled water to hatch the shrimp eggs. The seawater was put in a small plastic container with a partition for dark (covered) and light areas. The eggs were added to the dark side of the chamber, whereas the lamp was above the other side (light) so as to attract the hatched shrimps away from the hatching area. Ten larvae were added to seven different concentrations of the extract in seawater (0.125, 0.250, 0.500, 0.750, 1.00, 2.00, and 4.00 µg/mL) and repeated three times (n = 3). The mortality rate was recorded after 24 h. The larvae were considered dead if they did not respond to a physical stimulus (i.e., prodding with a wooden stick). No food was provided to the larvae during bioassay.

The percentage mortality observed (%M) was corrected by the following formula[19]:

The dosage-mortality lines were estimated by computerized log-probit analysis. The 95% confidence intervals at the lethal concentrations of 50% and 90% (LD50 and LD90) were used to measure the plant potential.

Aedes aegypti larvicide

The test of the larvicidal bioassay fruit extract of V. cofassus against Ae. aegypti was carried out in accordance with the WHO standard method with some modifications and as per the previous method.[20] The third-instar larvae were placed in a glass beaker containing seven different concentrations of the extract (0.125, 0.250, 0.500, 0.750, 1.00, 2.00, and 4.00 µg/mL) and the mortality rate was recorded after 24 h. The larvae were considered dead if they did not respond to a physical stimulus (i.e., prodding with a wooden stick). The number of larvae that became mosquitos was recorded.

Pupicidal bioassays

There were three replicates, each containing 10 third-instar Ae. aegypti larvae and treated with seven different concentrations according to larvicidal bioassay. Daily monitoring involved reviewing verification of the larval stage; behavior changes; the presence of exuvia; adult emergence; possible mortality of the larvae, pupae, and adults; and the water temperature. The experiment was carried out until the last pupa or adult died or completely emerged.

Gas chromatography–mass spectrometry analysis

The phytochemical investigation was conducted using a piece of gas chromatography–mass spectrometry (GC-MS) equipment (Thermo Fisher Scientific, Waltham, MA, USA). The experimental conditions of the GC-MS system were TR 5-MS capillary standard with dimensions of 30 Mts, internal diameter (ID) of 0.25mm, and film thickness of 0.25 μm. The gasses as a mobile phase were set at 1.0mL/min. The sample dissolved in methanol ran fully at a range of 50 to 650 m/z and the results were compared using a certain database.

RESULTS

Phytochemistry

The results of GC-MS analysis of the extract led to the identification of a number of compounds. These compounds were identified through the mass spectrometry technique of the system. The various components present in the entire methanolic extract of V. cofassus detected by the GC-MS are shown in Table 1. 1,1-bibicyclo (2,,2,2) octane-4-carboxylic acid; 1,3-dioxolane-4-methanol; tetra decanoic acid; 9-hexadecenoic acid; hexadecenoic acid; ethyl docosanoate; pentacosane; hexadecanoic acid; (Z,Z)-methyl ester 9,12-Octadecadienoic acid; 9-octadecenoic acid (Z)-methyl ester; octadecanoic acid; 1-octadecanol; 1,2-benzenedicarboxylic acid; and 2,6,10,14,18,22-tetracosahexaene were present in the methanolic extracts of V. cofassus. The composition determined for this extract corresponded to 89.21% of the entire GC-MS chromatogram.

Table 1

Compounds identified in the methanolic extract during GC-MS

RT	Compound	Molecular formula	Molecular weight
3.03	1,1-bibicyclo (2,2,2) octane-4-carboxylic acid	C17H26O2	262
3.08	1,3-dioxolane-4-methanol	C6H12O3	132
15.45	Tetra decanoic acid	C15H30O2	242
17.67	9-hexadecenoic acid	C17H32O2	268
17.98	Hexadecenoic acid	C17H34O2	270
18.93	Ethyl docosanoate	C24H48O2	368
19.02	Pentacosane	C25H52	352
19.32	Hexadecanoic acid	C19H38O2	298
20.38	(Z,Z)-methyl ester 9,12-octadecadienoic acid	C19H34O2	294
20.47	9-Octadecenoic acid (Z)-methyl ester	C19H36O2	296
20.85	Octadecanoic acid	C19H38O2	298
21.88	1-Octadecanol	C18H38O	270
27.03	1,2-Benzenedicarboxylic acid	C24H38O4	390
31.13	2,6,10,14,18,22-tetracosahexaene	C30H50	410

RT: Retention time

Lethality bioassay

To investigate larvicidal toxicity, a brine shrimp (A. salina) and Ae. Aegypti larvae lethality test was used as a preliminary screening method. The extracts that triggered a rate of more than 90% larval mortality were regarded as highly toxic. As shown in Table 2, the serial increase in the concentration of the extracts from 0.125 to 4.00 µg/mL was followed by an increased number of larvae deaths, representing an indication of similarly rising toxicity.

Table 2

Larvicidal activity of the fruit extract of V. cofassus against 10 brine shrimp and third-instar Ae. aegypti larvae (mean ± standard deviation, n = 3)

Concentration (µg/mL)	Mortality of brine shrimp larvae (%)	Mortality of Ae. aegypti larvae (%)
Control	0 ± 0.00	0 ± 0.00
0.125	36.67 ± 7.64	6.67 ± 0.00
0.25	43.33 ± 7.64	30.00 ± 0.00
0.50	55.00 ± 5.00	46.67 ± 0.00
0.75	63.33 ± 7.64	56.67 ± 0.00
1.00	78.33 ± 18.93	80.00 ± 0.00
2.00	81.67 ± 16.07	100.00 ± 0.00
4.00	100.00 ± 0.00	100.00 ± .00
r2	0.937	0.959
LC50 (µg/mL)	0.308	0.514
LC90 (µg/mL)	4.317	1.921

The IC50 concentrations of the fruit extract of V. cofassus against brine shrimp and Ae. Aegypti larvae were 0.308 and 0.514 µg/mL, whereas those for IC90 were 4.317 and 1.921 µg/mL, respectively. The extract was found to be more toxic to brine shrimp than to Ae. aegypti, killing 50% of the organisms. Although there was a difference between the IC50 and IC90 values, all data included the very toxic activity (< 50 µg/mL), mainly because of differences in linearity (r2).

Larvicidal bioassay

The data in Table 3 report the biological activity of the test extract made from fruit of V. cofassus against the third-instar larvae of Ae. aegypti. The highest concentration (2.00–4.00 µg/mL) caused complete mortality, whereas the lowest transience (6.67%) was recorded at the lower most concentration (0.125 µg/mL), in comparison with 0% for the untreated insects. There was an 80% rate of pupation at the lowest concentration (0.125 µg/mL), whereas none was observed at the concentrations of 2.00 to 4.00 µg/mL, (as compared with a rate of 100% for the controls).

Table 3

Effects of various concentrations of the fruit extract of V. cofassus on different stages of Ae. aegypti with (n = summary of three replications)

Concentration (µg/mL)	Larvae mortality (%)	Malformed pupae (%)	Pupation (%)	Pupal mortality (%)	Adult emergency (%)	Adult age (days)
Control	0.00	0.00	100.00	0.00	100.00	7
0.125	6.67	6.67	80.00	0.00	80.00	6
0.25	30.00	10.00	60.00	6.67	53.33	5
0.50	46.67	0.00	53.33	10.00	43.33	4
0.75	56.67	0.00	43.33	10.00	33.33	2
1.00	80.00	0.00	20.00	10.00	10.00	2
2.00	100.00	0.00	0.00	0.00	0.00	0
4.00	100.00	0.00	0.00	0.00	0.00	0

The lethal effect of the extract was extended to the pupal stage at all concentrations (i.e., 0.125, 0.25, 0.50, 0.75, and 1.00 µg/mL), where the pupal mortality percentages were 0%, 6.67%, 10.00%, 10.00%, and 10.00%, respectively, versus 0% for the control.

There was a point reduction in the percentage of adult emergences from pupae produced by treating larvae with the extract. A 0% adult emergence percentage occurred at the concentrations of 2.00 to 4.00 µg/mL, though the percentage increased to 10.00%, 33.33%, 43.33%, 53.33%, 80.00%, and 100% at the concentrations of 1.00, 0.75, 0.50, 0.25, and 0.125 µg/mL, respectively, as compared with 100% in the control group. Of note, the extract had extended toxicity effects on the survival of adults which were treated as larvae at the two concentrations of 0.25 and 0.50 µg/mL, where the adult mortality was recorded as 53.33% and 43.33% as compared with 0% for the controls. The fresh fruit of V.cofassus was highly larvicidal and shortened the age of the adult mosquitos at high concentrations (1.00 µg/mL) to four days as compared with seven days in the controls.

DISCUSSION

Controlling the larval stage can be an effective way to regulate mosquitoes in man-made breathing habitats.[21] In this study, the percentage of emergence in most cases was less than the pupation rate, suggesting some pupae died. The emergence inhibition values for V. cofassus were much lower than the respective LC values, indicating that the growth disruption activity extended to the pupal stages.

Several previous studies on the activity of plant extracts on Ae. aegypti mainly examined larvicidal action, whereas few studies on the effect of natural products on the pupal stage have been carried out. To the best of our knowledge, no prior study in the literature involves the examination of this plant species and its effects on the pupal stage of Ae. aegypti. However, the pupicidal activities of other plant species were reported by Candido et al.,[22] where oils macerated from Cnidosculos phyllacanthus and Ricinus communis showed significant potential in the control of different developmental periods in the life cycle of the insect, with LC50 = 0.28 µL/mL and LC90 = 1.48 µL/mL and LC50 = 0.029 µL/mL and LC90 = 0.26 µL/mL, respectively. It is presumed that the secondary metabolites offering defense against mosquito larvae played an effective role in larvicidal activity in this study.

Ilyas et al.[23] investigated the toxicity effect of n-hexane, fraction, and pure compounds of the bark of V. cofassus with IC50 values of 74.079, 118.850, and 88.201 µg/mL achieved against A. salina. According to these authors, the steroid group was responsible for the effect. In addition, the methanol extract of this plant displayed an ability to inhibit cell growth in several human cell lines such as HUVECs stimulated by vascular endothelial growth factor.[18] Previous chemical studies involving V. cofassus indicated the presence of various types of compounds, including a novel clerodane diterpene with antiproliferative activities, against lung carcinoma (A549), epidermoid carcinoma (KB), vincristine-resistant KB subline (KB-VIN), triple-negative breast cancer (MDA-MB-231), and estrogen receptor–positive breast cancer (MCF-7), with IC50 values of 5.4 to 11.4 µM.[18]

CONCLUSION

In summary, the fruit extract of V. cofassus showed a high degree of toxicity against brine shrimp and Ae. aegypti larvae and inhibited metamorphosis. Further studies are required to identify the specific mechanism of action of the selective bioactive principle present in the fruit of V. cofassus responsible for larvicidal and cytotoxic activities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.