Larvicidal Potential and Phytochemical Analysis of Garcinia mangostana Extracts on Controlling of Culex pipiens Larvae

P.O


INTRODUCTION
M osquito vectors are responsible for the transmission of many vector-borne diseases (Rosenberg et al., 2018). The recent global burden of the diseases showed about 229 million cases of malaria with 90 percent occurring in Sub Saharan Africa (WHO, 2020), 5.2 million cases of dengue (WHO, 2021a), 200,000 cases of yellow fever (WHO, 2021c), 117 633 cases of Chikungunya fever with majority from Brazil, India, Guatemala, Malaysia and Belize (ECDC, 2021), and 51 million people with lymphatic filariasis (WHO, 2021b).
Culex pipiens is the most widely distributed species around the globe and considered as important vector for west Nile and Usutu viruses, and also for filarial worms and avian malarial parasite (Huijben et al., 2007;Martinet et al., 2019). Females of Cx. pipiens feed on various vertebrate hosts and may therefore contribute to the spread of disease among birds, humans, and other mammals (Martinet et al., 2019).
Insecticides have been used in mosquito control for decades. The excessive use of these compounds has led to insecticides resistance and affect non-target organisms (Fernandes et al., 2016). The need for substitutes of new safe insecticides to the environment encourages investigation and development of new active ingredients of natural origin. Plants have been used for this purpose due to their rich diversity of safe compounds with insecticidal activities against wide range of different insect pest species. They are target-specific, readily available, affordable, non-toxic to environment, and biodegradable (Ghosh et al., 2012). Tremendous efforts are made to encourage environmentally friendly new active ingredients, using different tactics to replace or to improve insecticidal control methods (Tiawsirisup et al., 2007).
Garcinia mangostana L. (Clusiaceae family) is a tropical tree that grows in Southeast Asia. Its peels and seeds have been used in traditional medicine to treat gastrointestinal and urinary tract infections, and also used as an anti-scorbutic, anti-fever agent, and a laxative (Ovalle-Magallanes et al., 2017). Many studies have been published showing that G. mangostana can induce multiple

Plant material
The Garcinia mangostana was purchased from a fruit shop in Riyadh, Saudi Arabia. The fruits were washed with distilled water, and 70 g of the fresh pericarp (rind) was grounded using a commercial blender (SF stardust, Japan). The powder was extracted using 500 mL of hexane (Honeywell, Germany) in the sonicator (Wise Clean, China) for 30 min at 40ºC. The extract was filtered using Whatman filter paper (Grade No. 1, England) and evaporated under reduced pressure (Heidolph, Germany) at 40ºC. The extract was reconstituted in Dimethyl sulfoxide (DMSO) (VDR, France) and used for both phytochemical screening and larval bioassay.

Alkaloids test (Dragendorff's test)
Two milliliters of methanol containing 50 mg of the extract was mixed with 1mL of dragendorff's reagent and mixed. The result of an orange-red precipitate indicates a positive result.

Phenols test (Ferric chloride test)
The dried extract (50 mg) was dissolved in 5 mL of 5% ferric chloride solution. The development of bluishblack color reveals the presence of phenolic compounds.

Sterols test (Salkowski test)
The dried extract (50 mg) was dissolved in 2 mL of methanol. A few drops (50 µL) of concentrated H 2 SO 4 were added, and the formation of reddish-brown color indicates a positive result.

Saponin test
The dried extract (50 mg) extract was mixed with water (5 mL), and shaken vigorously using a test tube. The persistent foam shows the presence of saponin.

Anthraquinones test (Borntrager's test)
The dried extract (50 mg) was dissolved in 2 mL of methanol. Then, 2.5 mL of ammonia (10%) solution was mixed and shaken. The development of rosette color indicates a positive result.

Gas chromatography-mass spectrometry (GC-MS) analysis
The phytochemical analysis of hexane extract was done on Perkin Elmer Clarus 600 gas chromatograph attached to a mass spectrometer (Turbomass, Perkin Elmer, USA). One microliter of the hexane extract was injected into the Elite-5MS column of 30 m, 0.25 µm film thickness, 0.25µm internal diameter column and processed as reported earlier (Hidayathulla et al., 2018). Compounds were identified using the spectra recorded in the WILEY and National Institute of Standard and Technology (NIST) libraries (Coates, 2000;Linstrom and Mallard, 2005).

Larvicidal activity
The Culex pipiens larvae used in the bioassay obtained from the colony maintained in the Bio-product Research Chair Insectary, King Saud University, Saudi Arabia. The stock solution was prepared by dissolving 50 mg of the dried extract in 1mL of DMSO (VDR, France) and used for preparing the concentrations. The first test solution was prepared by adding 80 µL of stock solution (50 mg/mL) to 15.920 mL of tap water and vortexed. Four concentrations (15.63, 31.25, 62.50, and 125 µg/mL) were prepared from the stock solution (50 mg/mL) by double dilution method in tap water using a 20 mL centrifuge tube (Nest, China). Four test tubes filled with 8 mL of tap water were used for dilution. Eight milliliter was transferred from the stock solution to the first test tube to make 250 µg/mL and vortexed (first two-fold dilution.). The second two-fold dilution were carried out with the same tip and continued until the last tube (concentration). Similarly, control was diluted as above, where the DMSO concentrations were 0.25, 0.125, 0.062 and 0.031%. Each treated water (tested concentration) was introduced into the wells of a 6-well plate (Bottom width: 85.30 mm, Bottom length: 127.50 mm, and height: 20 mm) (Nest, China). A batch of 20 third instar larvae of Cx. pipiens were then introduced into each well containing 8 mL of test water. Three replicates (total 60 larvae) were used for each concentration. Then plates were kept at 27±1 o C and photoperiod of 12 h. No larval food was added. After 24 h, the dead larvae were counted. Larvae were considered dead if they could not reach the surface or move when touched with a wooden stick (Al-Mekhlafi et al., 2021).

Histology of larvae
The procedure was carried out following the method of Al-Mekhlafi et al. (2021). Briefly, the control and extracttreated larvae were fixed for 24 h in 10 % formaldehyde, dehydrated in different concentrations of ethanol, cleared with xylene, and embedded in paraffin blocks. The blocks were sectioned (5 μm) using a rotary microtome (Leica, Germany) and stained with hematoxylin and eosin (PDH, UK). The glass slides were imaged using a light microscope (Olympus, Japan).

Cytotoxicity assay
Cell viability was investigated based on the potential of the active cell to reduce a yellow tetrazolium salt (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Invitrogen, USA) to purple formazan by mitochondria. In brief, Normal Human Umbilical Endothelial cells (HUV-EC) (ATCC, USA) were grown in 24-well plates (NEST, China) at 5 × 10 5 cells/ well and treated with 0.01% DMSO or different concentrations (250-12.5 µg/mL) of hexane extract for 24 h. MTT was then added to each well and incubated for 2 h at 37 o C. Crystals formed (Formazan) were solubilized using 0.01% isopropanol (WINLAB, UK) on a shaker (GFL, Germany) for 5 min. The absorbance (570 nm) was read using a plate reader (ChroMate, UK). IC 50 (concentration needed to inhibit 50% of cell growth relative to a control) value was calculated using OriginPro 8.5 (Origin Lab Corporation, USA).

Phytochemical screening
The qualitative phytochemical investigation of the hexane extract of G. mangostana revealed the existence of different phytometabolites such as alkaloids, sterols, and phenolic compounds.

GC-MS analysis
The GC-MS analysis of hexane extract of G. mangostana recorded a total of 5 peaks (Fig. 1, Table I) corresponding to the phytometabolites that were recognized by comparing their mass spectral fragmentation patterns to that of the compounds described by the WILEY and NIST libraries. Overall, the five phytocompounds identified in the hexane extract are shown in Table I, along with their retention time and area percentage. The major compound detected was α-Copaene that constituted 61.95% of the extract. Other miner compounds identified were transα-Bergamotene, (+)-β-Costol, selin-4, 7(11)-diene, and γ-cadinene.

Larvicidal activity
The larvicidal effect of the hexane extract of the G. mangostana was observed at 24 h, showing increased toxicity to the third instar larvae as concentration increased (Table II). The 125 µg/mL hexane extract exhibited 100% mortality after 24 of treatment. The LC 50 and LC 90 values correspond to 33.95 µg/mL and 64.12 µg/mL, respectively. No mortality was observed in the control test. Small letters indicate significant differences between concentrations. Significant differences were assessed using one-way ANOVA followed by Tukey's test, p < 0.05 indicates significant differences.

Gut-histological activity
The midgut cells of Cx. pipiens third instar larvae treated with hexane extract (33.95 µg/mL) and the control (0.01% DMSO) are shown in Figure 2. The sections of control of the third instar larvae midgut were normal with intact peritrophic membrane (Pm), microvilli (MV), and nuclei (N). Contrary, the midgut cells of treated larvae showed morphological alterations such as microvilli damage (DMV), loss of nuclei, epithelial cells degeneration (DE), and peritrophic membrane degeneration (DPM).

Cytotoxicity assay
The cells incubated with hexane extract showed morphological changes. Images captured by a light microscope revealed cell shrinkage and floating cells. Control cells (DMSO 0.01%) showed normal cell appearance (Fig. 3). MTT results indicated an increase in cell death with the increasing concentrations of the extract (Fig. 3). The IC 50 value was 19.8 µg/ml.
The ethanol pericarp extract of G. mangostana has shown larvicidal activity against A. aegypti larvae with LC 50 : 4.84 µg/mL, while the hexane extract exhibited an LC 50 value of 27.61 µg/mL (Torres et al., 2015). α-mangostin has been reported to be toxic against six mosquito species, including Cx. pipiens (Larson et al., 2014). Similarly, isolated α-mangostin showed larvicidal activity with LC 50 of 19.4 µg/mL against larvae of Aedes aegypti (Ee et al., 2006). In the present study, the hexane extract showed slightly higher LC 50 . This could be attributed to the different mosquito species tested.
The histological investigation showed changes in the midgut region of Cx pipiens due to the toxic effect of the hexane extract of G. mangostana. Similar effect has been shown also with Foeniculum vulgare and Matricaria chamomilla hexane extract (Al-Mekhlafi et al., 2021). The toxicity effect of Epaltes divaricate hexane extract on the midgut caused severe damage to gut tissues such as the peritrophic matrix, the epithelial layer, and the brush border (Amala et al., 2021). Several reports stated that the primary target of phytometabolites is the midgut regions, which alters several functions, such as osmoregulation, nutrition absorption, ion transport, digestion (Rohmah et al., 2020;Sina and Shukri, 2016), metamorphosis (Procopio et al., 2015), and chemical defense (Terra, 2001). Although the hexane extract possessed larvicidal action against Cx, pipiens, the extract also showed higher toxicity to normal HUVEC cells. Therefore, precautions should be taken into consideration when used as larvicidal agents. Nevertheless, the risk should be further evaluated using different animal models.
This data highlighted the importance of screening the larvicidal potential of G. mangostana as a source of active ingredients that can be subjected to more biological evolution and further product development.

Funding
This research was funded by Researchers Supporting Project number (RSP-2021/112), King Saud University, Riyadh, Saudi Arabia.

Statement of conflict of interest
The authors have declared no conflict of interest.