Larvicidal potential of Thuja orientalis leaves and fruits extracts against Culex pipiens (Diptera: Culicidae)

Hanan Abo El-Kassem Bosly

doi:10.1016/j.jksus.2022.102396

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Original article

01 2022

:35;

102396

doi:

10.1016/j.jksus.2022.102396

Larvicidal potential of Thuja orientalis leaves and fruits extracts against Culex pipiens (Diptera: Culicidae)

Hanan Abo El-Kassem Bosly

Associate Prof. of Entomology - Biology Department, Faculty of Science, Jazan University, PO Box 2097, Jizan 45142, Saudi Arabia

Received: 2022-4-24, Accepted: 2022-10-20,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Botanical pesticides targeted to avoid the pesticide resistance to synthetic ones that conventionally affecting ecosystems diversity. The study designed to evaluate the larvicidal potentials of leaves and fruits extracts (methanol, acetone, hexane, and aqueous) from Thuja orientalis (Pinales: Cupressaceae) against Culex pipiens 3rd instar larvae and to identify the extracts compounds by gas chromatography-mass spectrometry (GC–MS) method of analysis. Leaves and fruits extracts used in concentrations, 25, 50, 100, 200, and 400 ppm against the 3rd larval instar of Cx. pipiens in five replicates and larval mortalities recorded after 24 and 48 h post exposure. The larvicidal potentials of extracts showed concentration dependent and varied between leaves and fruits extracts. At 400 ppm concentration, leaves extracts showed 100 % larval mortality except in hexane extract exhibited 98 %. Acetone, methanol, aqueous and hexane leaves extracts recorded LC₅₀ values, 58.04, 70.20, 77.19 and 84.25 ppm, respectively. Fruits extracts by hexane and methanol exhibited 100 % larval mortality and LC₅₀, 68.26 and 83.21 ppm, respectively, while, acetone and aqueous fruits extracts showed 98 % and 96 % larval mortality and LC₅₀, 92.81 and 102.97 ppm, respectively. Terpenoids and sesquiterpenoids, fatty acid esters mainly identified in both extracts.

The present study showed that extracts from Thuja orientalis leaves and fruits acquired promising larvicidal potential for the control of Cx. pipiens larvae with the role of their chemical constituents.

Keywords

Thuja orientalis

Methanol

Acetone

Hexane

Aqueous

Culex pipiens

Larvicidal

GC–MS

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1 Introduction

Mosquitoes considered as a burden to human health by transmitting diseases including malaria, filariasis, dengue, and leishmaniasis (Wilson et al., 2020). Culex pipiens mosquitoes is common house mosquito and one of the most widely distributed mosquitoes worldwide due to its adaptation to human environments and its mode of feeding on birds and mammals reflected by its role in transmission of the West Nile virus and other pathogens (Farajollahi et al., 2011) that urged researches of its control.

Vector control is the main way to reduce public concerns about mosquito-borne diseases including filariasis, dengue, malaria, and leishmaniasis (Wilson et al., 2020). Control of mosquito larvae in aquatic phases is an effective method for reducing mosquito-spread (WHO, 2013). Excessive use of synthetic insecticides, with a complete lack of awareness of the strategy of changing the pesticides, led to resistance to pesticides along with environmental pollution and health risks to humans and non-target biota. Therefore, the search for environmentally friendly alternatives as plants or oils rich in secondary metabolites is a recent trend since they are more efficient, less toxic, biodegradable, and capable of insect decrease plant resistance to these natural compounds (Mouden et al., 2017; Ahmed et al., 2021) besides serving as larvicides, adult pesticides, insect repellents and deterrents (Govindarajan et al., 2016) they destroy only the insects they are meant to kill, leaving no residue on food or in the environment.

Thuja orientalis is a dense, evergreen and coniferous tree belongs to the family Cupressaceae growing in Saudi Arabia (Elsharkawy et al., 2017; Elsharkawy and Ali, 2019). In folk medicine T. orientalis used as herbal medicine for treatment of psoriasis, amenorrhea, enuresis, rheumatism, cystitis, bronchial catarrh and uterine carcinomas (Srivastava et al., 2012). Studies evaluated different activities of T. orientalis like, antimicrobial (Choi et al., 2021), antifungal activity (Caruntu et al., 2020), antioxidant (Nizam and Mushfiq, 2007), anticancer (Elsharkawy et al., 2017), anti-inflammatory (Darwish et al., 2021; Shin et al., 2015), hair growth promotion (Zhang et al., 2013).

The present study designed to investigate the larvicidal activities of methanol, hexane and acetone and aqueous extracts from leaves and fruits of Thuja orientalis plant against Cx. pipiens third instar larvae after exposure to extracts for 24 and 48 h. As well, identification of the chemical composition of the tested extracts by the aid of gas chromatography-mass spectrometry (GC–MS) method of analysis.

2 Materials and methods

2.1

2.1 Plants materials

Thuja orientalis L. (Pinales: Cupressaceae) leaves and fruits were obtained from Arar region, at Northern Region of Saudi Arabia, where growing wild in March 2022 as previously recorded (Elsharkawy and Ali, 2019). The plant was identified by a taxonomist, prof. Dr. Yahya Masrahi, from the Department of Biology, Faculty of Science, Jazan University, Saudi Arabia.

2.2

2.2 Culex pipiens colony

Mosquitoes (Cx. pipiens) were obtained from the Center for Environmental Research and Studies at Jazan University. Rearing was performed under controlled conditions (27 ± 2 °C, relative humidity at 70 % ± 10 %, and 12:12 h light:dark regime). Mosquito larvae were reared in round enamel plates (25 × 20 × 10 cm) filled with 2 L de-chlorinated water and fed with fish food daily. The third instar Cx. pipiens larvae were used for the larvicidal examination.

2.3

2.3 Plant extracts.

Leaves and fruits were dried in shade for 7 days at laboratory temperatures (27–29 °C). The dried leaves (40 g) and fruits (25 g) were powdered using a commercial electrical stainless-steel blender and extracted for each solvent including methanol, acetone, hexane, and aqueous using Soxhlet apparatus for 6–8 h according to the solvent type. The leaves and fruits extracts were filtered with Whatman number 1 filter paper through a Buchner funnel. The filtrates then dried using a rotary evaporator under vacuum at 40 °C. The plant leaves extract yields 3.7, 3.2, 2.8, and 1.7 g for methanol, acetone, hexane, and aqueous solvents and yields 2.1, 1.5, 2.3 and 1.1 g for plant fruits, respectively.

2.4

2.4 Larvicidal assay

The larvicidal activity was determined for the extracts in concentrations that prepared as 25, 50, 100, 200, and 400 ppm based on 1 g/1L (1000 ppm) from each extract stock solution against the 3rd larval instar of Cx. pipiens (WHO, 2005). Twenty Cx. pipiens third instar larvae of were subjected to each extract concentration in 250 mL glass beakers containing 150 mL de-chlorinated water (aqueous suspension) at 27 ± 2 °C, 70 ± 10 % RH, and a 12:12 h (L/D) photoperiod. Five replicates per concentration per extract and control were conducted. Larval mortalities were recorded after 24 and 48 h of exposure.

2.5

2.5 Identification of chemical compounds in extracts by gas chromatography-mass spectrometer.

Extracts from promising plant, Thuja orientalis L. were analyzed to investigate their chemical constituents by GC–MS using the Trace GC-TSQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) through TG–5MS direct capillary column (30 m × 0.25 mm × 0.25 m thickness of the film) where the column oven temperature initially maintained at 50 °C, then rate was increased 5 °C/min up to 250 °C, for 2 min, and then increased 30 °C/min to 300 °C. The lines for the injector and MS transfer were suspended at 280 °C and 260 °C, respectively, helium was the carrier gas at the rate of 1 mL/min. The solvent delay was 3 min and 2 µl samples were injected automatically using Autosampler AS1310 coupled with GC in the splitless mode. In full scanning mode, electrospray ionization (EI) mass spectra were obtained covering the range 50–650 m/s at an ionization voltage of 70 V. The ion source temperature was fixed at 200 °C. The chemical constituents were identified from the Total Ion Chromatogram (TIC), where the chemical compounds were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral databases.

2.6

2.6 Data analysis

The percentage mortalities were determined according to Abbott, (1925). The larval control results did not need correction, as the mortality was less than 5 %, according to the WHO guidelines (WHO, 2005) (no larval control mortality recorded throughout the study). Mortality data from all the replicates were performed using one-way analysis of variance (ANOVA) to find the differences among the activity between each plant extract concentrations using the least significant difference (LSD) test. Also, data from all the replicates were subjected to analysis to determine the larval LC₅₀, LC₉₀, and LC₉₅ as well as chi-square values within confidence limits at 95 % (lower confidence limit (LCL) and upper confidence limit (UCL) by using probit analysis and regression between log extract concentration and probit values. Data analysis was performed using SPSS software (IBM SPSS Statistics v22 – 64 bit), and p < 0.05 was considered significant.

3 Results

3.1

3.1 Larvicidal activity

The data about the larvicidal activities of the tested Thuja orientalis leaves extracts against the third instar larvae of Cx. pipiens after 24 h are summarized in Table 1, and after 48 h are summarized in Table 2. The analysis of responses of the leaves extracts revealed that the larvicidal activities after 24 h showed 100 % mortalities at 400 ppm of methanol, acetone and aqueous extracts. While hexane leaves extract at 400 ppm showed 98 % larval mortality. Acetone leaves extract showed the highest efficacy by inducing 100 % mortality (LC₅₀, 58.04, LC₉₀, 169.27, and LC₉₅, 229.27 ppm), followed by methanol extract by 100 % mortality (LC₅₀, 70.20, LC₉₀, 221.66, and LC₉₅, 307.07 ppm), followed by aqueous extract by 100 % mortality (LC₅₀, 77.19, LC₉₀, 244.26, and LC₉₅, 338.95 ppm). While, hexane extract showed the least efficacy as compared to others, inducing 98.00 % mortality (LC₅₀, 84.25, LC₉₀, 283.47, and LC₉₅, 399.84 ppm) (Table 1). The larvicidal activities of the leaves extracts after 48 h showed 100 % mortalities at 400 ppm for all tested extracts. Acetone leaves extract showed the highest efficacy (LC₅₀, 43.40, LC₉₀, 102.62, and LC₉₅, 130.98 ppm), followed by methanol extract (LC₅₀, 54.75, LC₉₀, 156.00, and LC₉₅, 209.92 ppm), followed by the aqueous extract (LC₅₀, 63.76, LC₉₀, 198.50, and LC₉₅, 273.89 ppm). Then, hexane showed (LC₅₀, 69.02, LC₉₀, 225.14, and LC₉₅, 314.78 ppm) (Table 2).

Table 1 The larvicidal effects of leaves extracts of Thuja orientalis (Pinales: Cupressaceae) against the third instar larvae of Culex pipiens at 24 h post-treatment.

Leaves extract	Conc. ppm	Mortality% (Mean ± SE)	LC₅₀ (LCL–UCL.)	LC₉₀ (LCL–UCL.)	LC₉₅ (LCL–UCL.)	Chi (Sig)	Regression equation	R²
Methanol	0.0	0.00 ± 0.0^a	70.20 (61.92–79.19)	221.66 (185.53–278.05)	307.07 (248.31–404.83)	4.417 (0.220^a)	Y = −4.33 + 2.33*x	0.997
	25	15.00 ± 2.24^b
	50	35.00 ± 3.16^c
	100	61.00 ± 4.30^d
	200	86.00 ± 2.45^e
	400	100.00 ± 0.00^f

Acetone	0.0	0.00 ± 0.0^a	58.04 (51.25–65.24)	169.27 (143.49–208.94)	229.27 (188.22–296.63)	1.884 (0.597^a)	Y = −4.68 + 2.65*x	0.997
	25	18.00 ± 2.55^b
	50	41.00 ± 2.92^c
	100	72.00 ± 2.55^d
	200	93.00 ± 2.55^e
	400	100.00 ± 0.00^f

Hexane	0.0	0.00 ± 0.0^a	84.25 (74.21–95.43)	283.47 (234.18–361.87)	399.84 (318.35–537.98)	3.348 (0.341^a)	Y = −4.95 + 2.6*x	0.976
	25	12.00 ± 2.00^b
	50	29.00 ± 3.32^c
	100	54.00 ± 2.45^d
	200	79.00 ± 2.45^e
	400	98.00 ± 1.22^f

Aqueous	0.0	0.00 ± 0.0^a	77.19 (68.22–87.06)	244.26 (204.00–307.18)	338.95 (273.22–447.30)	5.630 (0.131^a)	Y = −4.32 + 2.27*x	0.999
	25	13.00 ± 2.00^b
	50	31.00 ± 4.00^c
	100	58.00 ± 2.55^d
	200	82.00 ± 1.22^e
	400	100.00 ± 0.00^f

Significance at 0.05 level between different superscripts. (a) In Chi-Square Tests, no heterogeneity factor was used in the calculation of confidence limits because the significance level was greater than 0.05.

Table 2 The larvicidal effects of leaves extracts of Thuja orientalis (Pinales: Cupressaceae) against the third instar larvae of Culex pipiens at 48 h post-treatment.

Leaves extract	Conc. ppm	Mortality % (Mean ± SE)	LC₅₀ (LCL–UCL.)	LC₉₀ (LCL–UCL.)	LC₉₅ (LCL–UCL.)	Chi (Sig)	Regression equation	R²
Methanol	0.0	0.00 ± 0.0^a	54.75 (48.63–61.48)	156.00 (132.59–192.02)	209.92 (172.87–270.73)	2.260 (0.520^a)	Y = −4.8 + 2.77*x	0.992
	25	20.00 ± 1.58^b
	50	42.00 ± 4.90^c
	100	75.00 ± 4.18^d
	200	95.00 ± 3.16^e
	400	100.00 ± 0.00^e

Acetone	0.0	0.00 ± 0.0^a	43.40 (38.72–48.22)	102.62 (88.81–123.99)	130.98 (110.22–165.22)	3.112 (0.375^a)	Y = −5.26 + 3.21*x	0.988
	25	24.00 ± 2.45^b
	50	53.00 ± 4.06^c
	100	89.00 ± 1.87^d
	200	100.00 ± 0.00^e
	400	100.00 ± 0.00^e

Hexane	0.0	0.00 ± 0.0^a	69.02 (60.68–78.06)	225.14 (187.68–284.10)	314.78 (253.15–418.36)	4.622 (0.202^a)	Y = −4.15 + 2.24*x	0.999
	25	16.00 ± 1.87^b
	50	36.00 ± 1.87^c
	100	62.00 ± 2.55^d
	200	85.00 ± 2.24^e
	400	100.00 ± 0.00^f

Aqueous	0.0	0.00 ± 0.0^a	63.76 (56.17–71.92)	198.50 (166.68–248.05)	273.89 (222.26–359.70)	4.041 (0.257^a)	Y = −4.38 + 2.43*x	0.991
	25	18.00 ± 2.55^b
	50	37.00 ± 2.00^c
	100	65.00 ± 4.18^d
	200	90.00 ± 4.18^e
	400	100.00 ± 0.00^f

The results about the larvicidal activities of the tested T. orientalis fruits extracts against the third instar larvae of Cx. pipiens after 24 h are summarized in Table 3 and after 48 h are summarized in Table 4. The analysis of fruits extracts data revealed that the larvicidal activities after 24 h showed 100 % mortalities at 400 ppm of methanol and hexane extracts. While acetone and aqueous fruits extract at 400 ppm showed 98 % and 96 % larval mortalities, respectively. Hexane fruits extract showed the highest efficacy by inducing 100 % mortality (LC₅₀, 68.26, LC₉₀, 201.48, and LC₉₅, 273.84 ppm), followed by methanol extract by 100 % mortality (LC₅₀, 83.21, LC₉₀, 253.85, and LC₉₅, 348.25 ppm). The acetone extract induced 98 % mortality (LC₅₀, 92.81, LC₉₀, 296.34, and LC₉₅, 411.83 ppm). While, the aqueous extract showed the least efficacy as compared to others, inducing 96 % mortality (LC₅₀, 102.97, LC₉₀, 333.60, and LC₉₅, 465.54 ppm) (Table 3). The larvicidal activities of the fruits extracts after 48 h showed 100 % mortalities at 400 ppm for all tested extracts. Hexane extract showed the highest efficacy (LC₅₀, 50.60, LC₉₀, 119.30, and LC₉₅, 152.14 ppm), followed by methanol extract (LC₅₀, 68.62, LC₉₀, 205.88, and LC₉₅, 281.12 ppm), followed by the acetone extract (LC₅₀, 76.43, LC₉₀, 236.40, and LC_{95 ()}, 325.58 ppm). While, aqueous showed (LC₅₀, 81.85, LC₉₀, 253.48, and LC₉₅, 349.23 ppm) (Table 4).

Table 3 The larvicidal effects of fruits extracts of Thuja orientalis (Pinales: Cupressaceae) against the third instar larvae of Culex pipiens at 24 h post-treatment.

Fruit extract	Conc. ppm	Mortality% (Mean ± SE)	LC₅₀ (LCL–UCL.)	LC₉₀ (LCL–UCL.)	LC₉₅ (LCL–UCL.)	Chi (Sig)	Regression equation	R²
Methanol	0.0	0.00 ± 0.0^a	83.21 (73.84–93.61)	253.85 (212.80–317.49)	348.25 (282.48–456.44)	5.980 (0.113^a)	Y = −4.55 + 2.34*x	1.000
	25	10.00 ± 1.58^b
	50	29.00 ± 2.92^c
	100	55.00 ± 4.74^d
	200	80.00 ± 3.16^e
	400	100.00 ± 0.00^f

Acetone	0.0	0.00 ± 0.0^a	92.81 (82.18–104.78)	296.34 (246.13–375.30)	411.83 (330.40–547.84)	3.894 (0.273^a)	Y = −5.27 + 2.71*x	0.974
	25	9.00 ± 1.00^b
	50	25.00 ± 1.58^c
	100	51.00 ± 3.32^d
	200	76.00 ± 2.92^e
	400	98.00 ± 1.22^f

Hexane	0.0	0.00 ± 0.0^a	68.26 (60.49–76.66)	201.48 (170.32–249.29)	273.84 (224.24–354.73)	2.659 (0.447^a)	Y = −4.68 + 2.54*x	1.000
	25	13.00 ± 2.55^b
	50	36.00 ± 1.00^c
	100	65.00 ± 4.18^d
	200	88.00 ± 3.00^e
	400	100.00 ± 0.00^f

Aqueous	0.0	0.00 ± 0.0^a	102.97 (91.14–116.45)	333.60 (275.53–425.82)	465.54 (371.08–624.83)	2.596 (0.458^a)	Y = −5.18 + 2.59*x	0.987
	25	7.00 ± 1.22^b
	50	23.00 ± 2.00^c
	100	46.00 ± 4.30^d
	200	73.00 ± 2.00^e
	400	96.00 ± 1.87^f

Table 4 The larvicidal effects of fruits extracts of Thuja orientalis (Pinales: Cupressaceae) against the third instar larvae of Culex pipiens at 48 h post-treatment.

Fruits extract	Conc. ppm	Mortality% (Mean ± SE)	LC₅₀ (LCL–UCL.)	LC₉₀ (LCL–UCL.)	LC₉₅ (LCL–UCL.)	Chi (Sig)	Regression equation	R²
Methanol	0.0	0.00 ± 0.0^a	68.62 (60.74–77.16)	205.88 (173.67–255.45)	281.12 (229.59–365.45)	3.189 (0.363^a)	Y = −4.56 + 2.47*x	0.999
	25	14.00 ± 2.92^b
	50	34.00 ± 3.67^c
	100	66.00 ± 4.30^d
	200	87.00 ± 5.83^e
	400	100.00 ± 0.00^e

Acetone	0.0	0.00 ± 0.0^a	76.43 (67.66–86.07)	236.40 (198.09–295.92)	325.58 (263.83–427.56)	5.274 (0.153^a)	Y = −4.44 + 2.34*x	0.997
	25	13.00 ± 3.00^b
	50	31.00 ± 4.85^c
	100	57.00 ± 4.66^d
	200	84.00 ± 4.00^e
	400	100.00 ± 0.00^e

Hexane	0.0	0.00 ± 0.0^a	50.60 (45.43–56.07)	119.30 (103.29–143.64)	152.14 (128.27–190.61)	4.305 (0.230^a)	Y = −5.21 + 3.04*x	0.994
	25	18.00 ± 3.39^b
	50	45.00 ± 3.54^c
	100	82.00 ± 6.04^d
	200	100.00 ± 0.00^e
	400	100.00 ± 0.00^e

Aqueous	0.0	0.00 ± 0.0^a	81.85 (72.54–92.17)	253.48 (212.04–317.98)	349.23 (282.50–459.55)	6.258 (0.100^a)	Y = −4.46 + 2.3*x	0.998
	25	11.00 ± 1.87^b
	50	30.00 ± 2.24^c
	100	54.00 ± 2.92^d
	200	81.00 ± 3.32^e
	400	100.00 ± 0.00^f

3.2

3.2 Chemical analysis

The GC–MS analysis revealed that the main constituents of the T. orientalis leaves extracts were the sesquiterpenoids and terpenoids (Table 5) and (Fig. 1). The main % area for sesquiterpenoids compounds commonly detected in all extracts, were, cedrol (33.03, 35.77, 33.99 and 38.25 %), caryophyllene (18.49, 25.31, 21.68 and 20.67 %) and humulene (10.94, 15.22, 12.28 and 12.35 %) for methanol, hexane, acetone and aqueous leaves extracts, respectively. Followed by, germacrene D (2.80, 4.00, 3.60 and 3.35 %), allocedrol (2.26, 2.22, 2.16 and 2.43 %), α-Eudesmol (1.95, 1.92, 2.91 and 2.58 %), cadina-1(10),4-diene (1.29, 1.67, 1.30 and 1.58 %), selinene (1.27, 1.18, 1.32 and 1.59 %), γ-muurolene (1.17, 1.47, 1.08 and 1.26 %), caryophyllene oxide (1.16, 1.25, 1.39 and 1.29 %), cedrene (0.39, 2.66, 0.60 and 2.33 %) and γ-elemene (0.28, 1.54, 0.62 and 1.52 %) for methanol, hexane, acetone and aqueous leaves extracts, respectively. Also, fatty acid methyl ester, cis-4,7,10,13,16,19-docosahexaenoic acid, methyl ester (1.22, 1.31, 0.29 and 0.97 %) for the same aforementioned extracts, respectively. other compounds detected in three extracts only, pimara-7,15-dien-3-ol (6.04, 1.53 and 0.54 %), α-muurolene (0.29, 1.80 and 0.31 %), alloaromadendrene (1.11, 2.11 and 0.64 %) and butyl 4,7,10,13,16,19-docosahexaenoate (3.52, 1.43 and 3.00 %) where, were detected in methanol, acetone and aqueous leaves extracts, respectively. The retinoid compound 4,14-retro-retinol represented by 1.44, 1.25 and 1.25 area % in hexan, acetone and aqueous leaves extracts, respectively. The compounds, Bornyl acetate (0.39 and 0.44 %), α-terpinyl acetate (0.48 and 0.36 %), β-elemene (0.52 and 1.73 %), 1,7-Di-epi-β-cedrene (2.47 and 2.06 %), valencen (1.05 and 1.44 %), cedarn-8-ol (0.19 and 0.14 %) and α-acorenol (1.20 and 1.64 %) were recorded in methanol and acetone leaves extracts, respectively. Meanwhile, the terpenoid compound, ehydro-4-epiabietol represented by 0.73 and 1.03 area % and the sesquiterpenoid, γ-gurjunene represented by 0.33 and 1.10 area % in acetone and aqueous leaves extracts, respectively. There are compounds were detected only in the methanol leaves extract as 4-hydroxy-3,3′,4-trimethoxystilbene (3.95 %), sugiol (1.49 %), totarol (0.52 %) and copalol (0.51 %). β-Chamigrene and linolenic acid, methyl ester were detected in hexane leaves extract and represented by 1.81 and 1.26 area %, respectively.

Table 5 Chemical constituents of Thuja orientalis leaves methanol, hexane, acetone and aqueous extracts.

No	Molecular formula	Chemical compound	Methanol (%)	Hexan (%)	Acetone (%)	Aqueous (%)	Nature of compound
1	C₁₂H₂₀O₂	Bornyl acetate	0.39	–	0.44	–	Bicyclic monoterpenoid
2	C₁₂H₂₀O₂	α-Terpinyl acetate	0.48	–	0.36	–	Monoterpene ester monoterpenoid
3	C₁₇H₂₈O₂	Elemyl acetate	–	–	0.75	–	Monocyclic monoterpenoid
4	C₂₀H₃₂O	Pimara-7,15-dien-3-ol	6.04	–	1.53	0.54	Terpenoid
5	C₂₀H₃₄O	Copalol	0.51	–	–	–	Terpenoid
6	C₂₀H₃₀O	Sugiol	1.49	–	–	–	Terpenoid
7	C₂₀H₃₀O	Totarol	0.52	–	–	–	Terpenoid
8	C₂₀H₃₀O	Dehydro-4-epiabietol	–	–	0.73	1.03	Terpenoid
9	C₁₅H₂₄	β-Elemene	0.52	–	1.73	–	Sesquiterpenoid
10	C₁₅H₂₄	1,7-Di-epi-β-cedrene	2.47	–	2.06	–	Sesquiterpenoid
11	C₁₅H₂₄	Caryophyllene	18.49	25.31	21.68	20.67	Sesquiterpenoid
12	C₁₅H₂₄	α-Muurolene	0.29	–	1.80	0.31	Sesquiterpenoid
13	C₁₅H₂₄	γ-Elemene	0.28	1.54	0.62	1.52	Sesquiterpenoid
14	C₁₅H₂₄	Humulene	10.94	15.22	12.28	12.35	Sesquiterpenoid
15	C₁₅H₂₄	Cedrene	0.39	2.66	0.60	2.33	Sesquiterpenoid
16	C₁₅H₂₄	Selinene	1.27	1.18	1.32	1.59	Sesquiterpenoid
17	C₁₅H₂₄	Germacrene D	2.80	4.00	3.60	3.35	Sesquiterpenoid
18	C₁₅H₂₄	Valencen	1.05	–	1.44	–	Sesquiterpenoid
19	C₁₅H₂₄	γ-Muurolene	1.17	1.47	1.08	1.26	Sesquiterpenoid
20	C₁₅H₂₄	Cadina-1(10),4-diene	1.29	1.67	1.30	1.58	Sesquiterpenoid
21	C₁₅H₂₄	Alloaromadendrene	1.11	–	2.11	0.64	Sesquiterpenoid
22	C₁₅H₂₄	Cedrenol	–	–	–	0.20	Sesquiterpenoid
23	C₁₅H₂₄	Aromandendrene	–	–	–	1.65	Sesquiterpenoid
24	C₁₅H₂₄O	Caryophyllene oxide	1.16	1.25	1.39	1.29	Sesquiterpenoid
25	C₁₅H₂₆O	Allocedrol	2.26	2.22	2.16	2.43	Sesquiterpenoid
26	C₁₅H₂₆O	Cedrol	33.03	35.77	33.99	38.25	Sesquiterpenoid alcohol
27	C₁₅H₂₆O	Cedran-8-ol	0.19	–	0.14	–	Sesquiterpenoid
28	C₁₅H₂₆O	α -acorenol	1.20	–	1.64	–	Sesquiterpenoid
29	C₁₅H₂₆O	α-Eudesmol	1.95	1.92	2.91	2.58	Cycloeudesmane sesquiterpene
30	C₁₅H₂₄	β-Chamigrene	–	1.81	–	–	Sesquiterpenoid
31	C₁₅H₂₄	γ-Gurjunene	–	–	0.33	1.10	Sesquiterpenoid
32	C₁₉H₃₂O₂	Linolenic acid, methyl ester	–	1.26	–	–	Fatty acid methyl ester
33	C₂₃H₃₄O2	cis-4,7,10,13,16,19-Docosahexaenoic acid, methyl ester	1.22	1.31	0.29	0.97	very long-chain fatty acid methyl ester
34	C₂₆H₄₀O₂	Butyl 4,7,10,13,16,19-docosahexaenoate	3.52	–	1.43	3.00	very long-chain fatty acid
35	C₁₇H₁₈O₄	4-Hydroxy-3,3′,4-trimethoxystilbene	3.95				Stilbene polyphenol
36	C₂₀H₃₀O	4,14-retro-retinol	–	1.44	1.25	1.25	Retinoid

The TIC chromatograms of leaves, methanol, hexane, acetone and aqueous extracts from Thuja orientalis showing chemical constituents separation detected by GC–MS.

Elemyl acetate compound detected in acetone leaves extract only and represented by 0.75 area %. Cedrenol and aromandendrene compounds detected in aqueous leaves extract only and represented by 0.20 and 1.65 area %, respectively.

The GC–MS analysis revealed that the main constituents of the fruits extracts were represented in Table 6 and (Fig. 2). The main commonly sesquiterpenoids compounds in all extracts were, cedrol (21.26, 25.11, 18.55 and 36.66 %) and caryophyllene (17.06, 19.93, 15.21 and 17.04 %) for methanol, hexane, acetone and aqueous fruits extracts, respectively. Followed by the compounds, humuline (9.05, 10.93, 8.62 and 9.06 %), germacrene D (4.93, 5.76, 5.16 and 4.44 %), elemol (4.20, 4.57, 3.42 and 4.05 %), bornyl acetate (3.14, 3.08, 2.48 and 3.35 %), terpinolene (1.14, 2.75, 1.77 and 1.62 %), α-terpinyl acetate (1.10, 0.93, 1.10 and 1.22 %), cedrene (1.45, 1.36, 1.22 and 1.44 %), allocedrol (1.48, 1.18, 1.26 and 1.84 %), β-elemene (1.15, 1.28, 0.86 and 1.07 %), caryophyllene oxide (1.08, 1.14, 1.04 and 1.62 %), selinene (0.60, 0.59, 0.56 and 0.65 %) for methanol, hexane, acetone and aqueous fruits extracts, respectively. Two compounds were detected in three extracts only, labda-8(20),12,14-triene (4.64, 1.80 and 8.02 %) and trachyloban (0.81, 0.63 and 0.52 %), in methanol, hexan and acetone fruits extracts, respectively. Alloaromadendrene recorded in methanol and acetone fruits extracts by 0.48 and 0.53 area%, respectively. While, γ-elemene detected in methanol, hexane and aqueous extracts by 0.51, 0.53 and 0.42 area %, respectively. Isopimaral and olic acid detected in hexan and acetone extracts by 5.64, 5.64 and 2.02 and 0.76 area %, respectively. cis-5,8,11,14,17-eicosapentaenoic acid, methyl ester represented by 7.11 and 3.48 area %, i-Propyl 7,10,13,16,19-docosapentaenoate represented by 2.26 and 2.26 area %, arachidonic acid methyl ester represented by 0.58 and 1.13 area %, 6,9,12,15-docosatetraenoic acid, methyl ester represented by 0.63 and 1.89 area %, dodecanoic acid, 2,3-bis(acetyloxy)propyl ester represented by 0.76 and 0.42 area %, and in methanol and aqueous extracts, respectively. β-Eudesmol compound detected in hexane and aqueous fruits extracts and represented by by 0.80 and 0.70 area %, respectively.

Table 6 Chemical constituents of Thuja orientalis fruits methanol, hexane, acetone and aqueous extracts.

No	Molecular formula	Chemical compound	Methanol (%)	Hexan (%)	Acetone (%)	Aqueous (%)	Nature of compound
1	C₁₀H₁₆	Terpinolene	1.14	2.75	1.77	1.62	Menthane monoterpenoid
2	C₁₀H₁₆	2-Carene	1.00	–	–	–	Bicyclic monoterpenoid
3	C₂₀H₃₂	Labda-8(20),12,14-triene	4.64	1.80	8.02		Terpenoid
4	C₂₁H₃₂O₂	Labda-8(20) 12 14-triene-19-oic acid methyl ester (z)-	–	2.95	–	–	Terpenoid
5	C₂₁H₃₄O	Labda-8(20),14-dien-13-ol, (13S)-	–	–	–	1.62	Terpenoid
6	C₂₀H₃₂	(-)-Atisirene	0.42	–	–	–	Terpenoid
7	C₂₀H₃₂	Trachyloban	0.81	0.63	0.52	–	Terpenoid
8	C₁₂H₂₀O₂	Bornyl acetate	3.14	3.08	2.48	3.35	Bicyclic monoterpenoid
9	C₁₂H₂₀O₂	α-Terpinyl acetate	1.10	0.93	1.10	1.22	Monoterpene ester
10	C₂₀H₂₈O₆	16-Hydroxyingenol	0.45	–	–	–	Terpenoid
11	C₂₀H₃₀O₂	Communic Acid	0.93	–	–	–	Terpenoid
12	C₂₀H₃₂O	Pimara-7,15-dien-3-ol	–	–	1.46	–	Terpenoid
13	C₂₃H₃₈O₂Si	Pimaric acid TMS derivative	–	–	8.43	–	Terpenoid
14	C₂₃H₃₈O₂Si	Isopimaric acid TMS ester	–	–	–	–	Terpenoid
15	C₂₀H₃₀O	Isopimaral	–	5.64	5.64	–	Terpenoid
16	C₂₀H₃₄O	Copalol	–	1.05	–	–	Terpenoid
17	C₂₀H₃₀O	Totarol	–	–	–	–	Terpenoid
18	C₂₀H₃₀O	Dehydro-4-epiabietol	–	–	–	–	Terpenoid
19	C₂₁H₃₂O₂	Isopimaric acid, methyl ester	–	1.00	–	–	Terpenoid
20	C₁₅H₂₄	β-Elemene	1.15	1.28	0.86	1.07	Sesquiterpenoid
21	C₁₅H₂₄	γ-Elemene	0.51	0.53	–	0.42	Sesquiterpenoid
22	C₁₅H₂₄	δ-EIemene	0.68	–	–	–	Sesquiterpenoid
23	C₁₅H₂₄	1,7-Di-epi-β-cedrene		–	–	–	Sesquiterpenoid
24	C₁₅H₂₄	Caryophyllene	17.06	19.93	15.21	17.04	Sesquiterpenoid
25	C₁₅H₂₄	Humulene	9.05	10.93	8.62	9.06	Sesquiterpenoid
26	C₁₅H₂₄	Cedrene	1.45	1.36	1.22	1.44	Sesquiterpenoid
27	C₁₅H₂₄	Selinene	0.60	0.59	0.56	0.65	Sesquiterpenoid
28	C₁₅H₂₄	Germacrene D	4.93	5.76	5.16	4.44	Sesquiterpenoid
29	C₁₅H₂₄	Isogermacrene D	0.31	–	–	–	Sesquiterpenoid
30	C₁₅H₂₄	Valencen	–	–	–	1.05	Sesquiterpenoid
31	C₁₅H₂₄	Cadina-1(10),4-diene	0.47	–	–	–	Sesquiterpenoid
32	C₁₅H₂₄	Alloaromadendrene	0.48	–	0.53	–	Sesquiterpenoid
33	C₁₅H₂₄	Chamigrene	–	0.61	–	–	Sesquiterpenoid
34	C₂₀H₃₂	Cubenol	–	–	–	1.48	Sesquiterpenoid
35	C₁₅H₂₄O	Caryophyllene oxide	1.08	1.14	1.04	1.62	Sesquiterpenoid
36	C₁₅H₂₆O	Allocedrol	1.48	1.18	1.26	1.84	Sesquiterpenoid
37	C₁₅H₂₆O	Cedrol	21.26	25.11	18.55	36.66	Sesquiterpenoid alcohol
38	C₁₅H₂₆O	α-Eudesmol	0.64	–	–	–	Cycloeudesmane sesquiterpene
39	C₁₅H₂₆O	β-Eudesmol	–	0.80	–	0.70	Cycloeudesmane sesquiterpene
40	C₁₅H₂₆O	Elemol	4.20	4.57	3.42	4.05	Sesquiterpenoid
41	C₁₇H₁₈O₄	1-Heptatriacotanol	0.95	–	–	–	Fatty alcohol
42	C₁₈H₃₄O₂	Oleic Acid	–	2.02	0.76	–	Fatty acid
43	C₁₈H₃₀O₂	10-Heptadecen-8-ynoic acid, methyl ester,(E)-	–	–	–	0.49	Fatty acid methyl ester
44	C₁₉H₃₄O₆	Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester	0.76	–	–	0.42	Lauric fatty acid ester with hydroxypropanediyl diacetate
45	C₁₉H₃₀O₂	13,16-Octadecadiynoic acid, methyl ester	3.24	–	–	–	Fatty acid methyl ester
46	C₁₉H₃₂O₂	6,9,12-Octadecatrienoic acid, methyl ester		–	–	0.62	Fatty acid methyl ester
47	C₂₁H₃₄O₂	Arachidonic acid methyl ester	0.58	–	–	1.13	Fatty acid methyl ester
48	C₂₁H₃₂O₂	cis-5,8,11,14,17-Eicosapentaenoic acid, methyl ester	7.11	–	–	3.48	Fatty acid methyl ester
49	C₂₁H₃₆O2	Linolenic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (Z,Z,Z)-	0.29	–	–	–	Fatty acid ethyl ester
50	C₂₃H₃₆O₂	7,10,13,16,19-docosapentaenoic acid, methyl ester	–	–	–	0.81	Fatty acid ethyl ester
51	C₂₂H₃₂O₂	Doconexent	1.42	–	–	–	very long-chain fatty acid
52	C₂₃H₃₄O2	cis-4,7,10,13,16,19-Docosahexaenoic acid	0.84	–	–	–	very long-chain fatty acid
53	C₂₅H₄₀O2	i-Propyl 7,10,13,16,19-docosapentaenoate	2.26	–	–	2.26	very long-chain fatty acid
54	C₂₃H₃₈O2	6,9,12,15-Docosatetraenoic acid, methyl ester	0.63	–	–	1.89	very long-chain fatty acid methyl ester
55	C₂₁H₃₆O₄	9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-	0.59	–	–	1.24	1-monoglyceride derives from α-linolenic acid
56	C₂₁H₃₈O₂Si	α-Linolenic acid, TMS derivative	–	–	12.23	–	Fatty acid trimethyl ester
57	C₂₃H₃₈O₂Si	Eicosapentaenoic acid, TMS derivative	–	–	1.81	–	Fatty acid trimethyl ester
58	C₂₁H₃₄	Androst-5-ene, 4,4-dimethyl-, (13à)-	1.85	–	–	–	Steroid
59	C₂₁H₃₄O₂	Androstan-17-one, 3-ethyl-3-hydroxy-, (5à)-	–	0.60	–	–	Steroid
60	C₂₇H₄₂O₃	Pseduosarsasapogenin-5,20-dien	0.54				Steroidal saponin
61	C₁₇H₁₈O₄	4-Hydroxy-3,3′,4-trimethoxystilbene					Stilbene polyphenol
62	C₂₀H₃₀O	4,14-retro-retinol		1.45	1.71		Retinoid
63	C₂₀H₂₈O₆	Dotriacontane		0.45			Alkane

The TIC chromatograms of fruits, methanol, hexane, acetone and aqueous extracts from Thuja orientalis showing chemical constituents separation detected by GC–MS.

There are 15 compounds detected only in methanol fruits extract only which were, 13,16-octadecadiynoic acid, methyl ester (3.24 %), Androst-5-ene, 4,4-dimethyl-, (13à)- (1.85), doconexent (1.42 %), 2-Carene (1.00 %), 1-heptatriacotanol (0.95 %), communic acid (0.93 %), cis-4,7,10,13,16,19-docosahexaenoic acid (0.84 %), δ-eIemene (0.68 %), α-eudesmol (0.64 %), Pseduosarsasapogenin-5,20-dien (0.54 %), Cadina-1(10),4-diene (0.47 %), 16-hydroxyingenol (0.45), (-)-Atisirene (0.42 %), isogermacrene D (0.31 %), linolenic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (Z,Z,Z)-(0.29 %). Six compounds detected only in hexane fruits extract which were, labda-8(20) 12 14-triene-19-oic acid methyl ester (z)-(2.95 %), copalol (1.05 %), isopimaric acid, methyl ester (1.00 %), chamigrene (0.61 %), Androstan-17-one, 3-ethyl-3-hydroxy-, (5à)-(0.60 %) and Dotriacontane (0.45 %). Four compounds detected only in acetone fruits extract were, α-linolenic acid, TMS derivative (12.23 %), pimaric acid TMS derivative (8.43 %), eicosapentaenoic acid, TMS derivative (1.81 %) and pimara-7,15-dien-3-ol (1.46 %). While, in aqueous fruits extract there are six compounds detected which were, labda-8(20),14-dien-13-ol, (13S)- (1.62 %), valencen (1.05 %), cubenol (1.48 %), 7,10,13,16,19-docosapentaenoic acid, methyl ester (0.81 %), 6,9,12-octadecatrienoic acid, methyl ester (0.62 %) and 10-heptadecen-8-ynoic acid, methyl ester,(E)- (0.49 %) (Table 6) and (Fig. 2).

4 Discussion

Generally the study reviled larvicidal activities of T. orientalis leaves and fruits extracts in a concentration dependent manner whatever the activities varied between leaves and fruits tested extracts. Leaves extracts ordered according to larval toxicity as acetone > methanol > aqueous > hexane. While, fruits extracts ordered as hexane > methanol > acetone > aqueous. Collectively acetone T. orientalis leaves extract showed throughout the tested extracts, the most effective larvicidal activity against C. pipiens larvae (100 % mortality) and (LC₅₀, 58.04 ppm LC₉₀, 169.27 ppm LC₅₀, 229.27 ppm) at concentration 400 ppm.

Different Plant extracts previously tested for their larvicidal activities against mosquitoes, El-Sheikh et al. (2012) tested the larval toxicity of ethanol, acetone and petroleum ether extracts of Tribulus terrestris leaves, against the third instar larvae Ae. aegypti and predicted dose dependent larvicidal activities for the tested extracts in addition to variation of larvicidal activities related to solvent used. Where, crude plant extracts showed previously more efficiency in controlling mosquitoes over the purified compounds toxicity, which in line with the present study results (Ghosh et al., 2012). Another study estimated larval toxicity of leaves aqueous extracts in three tested concentrations from Ricinus communis L. (0.06, 0.12 and 0.2 g/l), Daphne gnidium L (0.09, 0.18 and 0.3 g/l) and Thymus vulgaris L. (0.0225, 0.045 and 0.09 g/l), against early instar larvae of Cx. pipiens and Cs. Longiareolata and showed in line with the present study results that the larval mortality increased with exposure times (24, 48 and 72 h), and the LC₅₀ recorded values decreased in the same manner (Dahchar et al., 2016).

The chemical compounds detected in the extracts in line with that detected in previous studies, however, there were differences in the amount or number of the main components (Elsharkawy et al., 2017; Guleria et al., 2008; Nickavar et al., 2003; Ololade et al., 2014; Sanei-Dehkordi et al., 2018 ).

The common sesquiterpenoids highly represented as % areas in the chromatograms of all tested leaves and fruits extracts were, Cedrol, Caryophyllene, Humulene, Germacrene D and Elemol (common only in fruits extracts). The medium common ones were, γ-Elemene, Cedrene, Selinene, γ-Muurolene, Cadina-1(10),4-diene, Caryophyllene oxide, Allocedrol, α-Eudesmol and the fatty acid methyl ester, cis-4,7,10,13,16,19-Docosahexaenoic acid, methyl ester in leaves extracts and bornyl acetate, α-Terpinyl acetate, Cedrene, β-Elemene, Selinene and Caryophyllene oxide were medium common in fruits extracts. The observed larvicidal activities of the tested extracts may related to their chemical constituents synergistic actions, either the major or minor ones that may impact on the predicted larvicidal activities (Huong et al., 2020).

The essential oil P. orientalis L. (Family Cupressaceae) oil showed insecticide and molluscicidal activity (Hashemi and Safavi 2012; Ju-Hyun et al., 2005; Lei, et al., 2010 ). Thuja orientalis previously acquired cytotoxic principles and contained terpenoids including pimaric and isopimaric acids, fatty acids, aliphatic compounds like alkanes and bioflavonoids (Mehta et al., 1999). The essential oil extracted from T. orientalis leaves predicted larvicidal activity against Anopheles stephensi and Culex pipiens late third or young 4th instar larvae, where carene and cedrol were from the main constituents of the extract (Sanei-Dehkordi et al., 2018). Also extracts predicted diterpenes and labdane-type diterpenes in line with the previously recorded (Kim et al 2012; Kim et al., 2013). Cedrol the main constituent in the tested extracts was postulated as alternative to conventional synthetic acaricides for the black-legged ticks, Ixodes scapularis Say which is a human vector causing disease (Eller et al., 2014). Caryophyllene oxide and germacrene D individual compounds previously showed potent larvicidal activities against A. anthropophagus (Zhu and Tian, 2013). Also β-elemene and α-humulene showed larvicidal activities against A. subpictus, Ae albopictus, and Cx. tritaeniorhynchus (Govindarajan and Benelli, 2016). Jeon et al. (2005) showed that essential oils extracted from P. orientalis had strong activities on mosquito larvae Cx. pipiens and Ae. aegypti recording 100 % mortality at 400 ppm. Furthermore, Sanei-Dehkordi et al. (2018) verified the high efficiency of P. orientalis oil against A. stephensi (LC₅₀, 11.67 ppm) and Cx. pipiens (LC₅₀, 18.60 ppm). Ethanol and acetone leaves extracts of T. orientalis previously evaluated larvicidal activities against mosquitoes recording LC₅₀, 13.10 and 200.87 ppm after 24 h and 9.02 and 127.53 ppm after 48 h, against A. stephensi third instar larvae, respectively. Besides recording against C. quinquefasciatus third instar larvae, LC₅₀, 22.74 and 69.03 ppm after 24 h and 16.72 and 51.14 ppm after 48 h, respectively (Sharma et al., 2005).

In addition and in accordance with the present results behavior, hexane plant parts and seeds extracts of Physalis angulate, Peganum harmala, Tecrium polium and Thymus vulgaris were evaluated for their larvicidal potentials against the fourth instar larvae of Culex pipiens molestus, and showed that mortality increased with exposure times (24 and 48 h) and the LC₅₀ recorded values decreased with exposure times (Mekhlif and Muhammad, 2021). A study tested larvicidal activities leaves oils extracts against A. aegypti and recorded that the main components in leaves of G. blepharophylla was the caryophyllene oxide, while, in G. friesiana were α-,β- and γ-eudesmols and in G. hispida, was (E)-caryophyllene. According to the predicted results oil extracted from G. friesiana recorded the best larvicidal effect against A. aegypti and hypothesized that sesquiterpenes in oils can reflect more controlling activity as compared to monoterpenes (Aciole et al., 2011). The aforementioned study results may declare the predicted advantage of acetone leaves extract results where the extract acquired highest content of eudesmol as compared to other extracts.

5 Conclusion

The leaves and fruits methanol, hexane, acetone and aqueous extracts of T. orientalis reviled high larvicidal potential at 400 ppm concentration against the third instar larvae of Cx. pipiens with the advantage of leaves acetone extract that predicted the lowest effective larval toxicity concentration. Further studies about the mode of the larvicidal action of the extracts should be under investigation.

Authorship contribution

Author declare her acknowledgment to the Center for Environmental Research and Studies at Jazan University for the technical support.

Funding

This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics approval and consent to participate

Not applicable.

Acknowledgments

Author declare her acknowledgment to the Center for Environmental Research and Studies at Jazan University for the technical support.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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