Effect of Drought Condition of North Region of Saudi Arabia on Accumulation of Chemical Compounds, Antimicrobial and Larvicidal Activity of Thuja Orientalis  

Introduction

Desert plants are in general did  not find the required international interest for many reasons; they are not a potential source  for food due to their  low density in this harsh conditions,  its location in arid zones high in temperature and drought which decrease the interest on them, they low diversity among the vegetation compared with other environments rich in all growth parameters such as water, soil fertility and good climate. However, regarding drugs, desert plants could be unexploded source for bioactive compounds of medicinal importance, this is because desert plants are rich  in this  bioactive compounds to  protect them from this harsh environmental  conditions, challenge and competition from soil  microorganisms, herbivores and insects which feed on them, also  competition between different plant species in desert and tolerance against ecological conditions, drought and high temperature. All these factors lead to production of unique compounds helps in survival of these arid environment.

Spodoptera littoralis (Lepidoptera: Noctuidae), which infests a wide range of economic crops causes critical injuries and loss to cotton, soybean, groundnut, chilli, tobacco, caster, bhendy, tomato and potato.¹ This pest is widely distributed in Middle East countries and temperate zones in Asia and Africa²

Several  methods have been performed to control S. littoralis chemically using synthetic chemical pesticides. Due to the residual toxic effect of these chemicals in the environment, the need for the development of products that not hazardous to the environment can be an alternative strategy for the control of S. littoralis.^3,4 Botanical pesticides hat not hazardous to the environment, are highly effective, and ecologically acceptable.⁵

Thuja orientalis, Cupressaceae family is an evergreen and monoecious tree or shrub evergreen tree, up to 10-20 m high which grows in south and east of Europe, west part of Asia including various parts of Iran. Thuja orientalis has been used in the different activity that is, antipyretic, antitussive, astringent, diuretic, refrigerant and stomachic.⁶ (Yeung, 1985). The phyto-constituents of T. orientalis such as flavonoids and terpenoids, coumarine  showed high biological activities.⁷

Comparative study between Thuja orientalis plant growing in KSA and Egypt and evaluated its antioxidant and cytotoxic activity, against HCT116, MCF7, PC3, A549 and Hep-G2 cell lines. The essential oil  extract from Saudi plant exhibited higher antioxidant activity and cytotoxic activity against deferent cell lines. than the Egyptian plant oil extracts, which is correlated with its high content of some compounds which found in Saudi plant and absence in Egyptian plant.⁸

Thuja occidentalis  cones extract contain high levels of bioactive phenolics, flavonoids and other free radical scavengers that can help to control lipid oxidation. This study showed that extract  had effective antioxidant activity in raw ground chicken meat during refrigerated storage because use of these extracts inhibited the formation of lipid peroxide and thiobarbituric acid reactive substances in ground chicken meat.⁹

Traditional Plants considered a good source for novel drugs, Owing to their popular use as remedies for many infectious diseases, searches for substances with antimicrobial and insecticidal activity from botanicals source are the main goal for many researchers. This study aimed to select some species form these plants (Thuja orientalis ), dominated in the Northern Region according to plant’s information and literature, then study It  through chemical analyzing and knowing some of its  antimicrobial properties and  the the bioactivity of plant extract against the larva of S. littoralis.  Which provide basic necessary information to explore the properties of this plant in this area, which are  not studied adequately, and give a picture about  the role of environment on the accumulation of chemical compounds to adapt the desert condition.

Materials and Method

Plant Material

Plant Thuja orientalis were collected from wild population from Arar region, Northern Region, Saudi Arabia in March 2018 the Authentic sample was identified and deposited in Faculty of science girl section.

Extraction

The air-dried powdered leaves (500 g) of Thuja orientalis were subjected to successive extraction with CHCl₃ using a Soxhlet apparatus. The CHCl3 extract (38.00 g) was dissolved in a suitable amount of hot distilled H₂O–MeOH (95:5 v/v, 200 mL), then partitioned between petroleum ether and CHCl3. The petroleum ether (20.50 g){C1) and CHCl3 (17.10 g,C2) fractions were individually concentrated under reduced pressure and kept for biological analysis.

Fractionation of the CHCl₃ Fraction

The CHCl₃ fraction was subjected to silica gel  column chromatography (550 g, mesh size 0.063–0.200 mm, Merck) (150 cm × 3 cm i.d.) eluted with n-hexane, gradually increasing the polarity with EtOAc, which resulted in the isolation of terpenoid  fraction (C2)which were subjected to analyzed with GC-MS.

GC-MS Analysis

The compounds were analyzed using a Thermo GC-Trace ultra system (Thermo Co. USA), they were separated on 30m X0.25 mm X 0.25 µm Elite-5MS  column (Thermo Scientific GC Column).The column temperature was increased from 40°C to 220°C at a rate of 4°C/min; injector  temperature, 250°C; injection volume, 1 µl; helium carrier gas flow rate 20ml/min; transfer temperature, 280°C. MS parameters were as follows: EI mode, with ionization voltage 70 ev, ion source temperature, 180°C; scan range, 50-600 Da. The peaks were tentatively identified based on library search using NIST and Wiley Registry 8 Edition .

Insects

laboratory strain of S. littoralis was reared in the laboratory at 26± 2°C and 65±5 % R.H., with 8:16 L:D h photoperiod. Larvae were fed on fresh castor leaves, Ricinus communius. The experiments were performed on the 4th instar larvae.

Insecticidal Bioassay

The determination of the lethal concentration values (LC30; LC50; LC90) of the Thuja orentalis against the 4th larval instar of S. littoralis was evaluated by leaf dipping technique method. Five concentrations (10, 15, 20, 30, and 40%) of the Thuja orentalis were used. Equal discs of fresh castor bean leaves were dipped in each tested concentrations of the extract for a while and left to dry. Ten starved larvae were transferred into each cup and allowed to feed on the treated and untreated leaves. Five replicates for each concentration were performed. Mortality counts were evaluated 24 h post-treatment and corrected according to Abbott (1925).¹⁰

Antibacterial Activity

The antibacterial potential of the leaves of Thuja orientalis was evaluated using the disc diffusion method as mentioned in, (Elsharkawy et al, 2018),⁸ with slit modifications. Dr. Fiaz Ahmed generously provided pathogenic bacterial strains (2 gram-positive and 3 gram-negative bacteria) from the pathology lab, Al-Rass Hospital, Saudi Arabia. Bacterial strains were sub-cultured in blood agar plates for 24 hours and 37°C. the plant extract was reconstituted in absolute methanol (not lethal for bacteria) to get a concentration 500 mg/ml. For disc diffusion method, a single colony was transferred to a sterile tube containing 100 µl normal saline (0.9%) and diluted to get a turbidity equivalent to 0.5 McFarland standard (1 × 10⁸ cfu/mL). 100 µl of that suspension was spread over previously prepared Mueller Hinton agar plate, then, covered and left for a while. Sterile paper discs (size 6 mm) were cut from Whatman filter paper (No.1) and immersed in the reconstituted extract and then loaded over the inoculated  plates. Another paper disc saturated with chloramphenicol solution (5mg/ml) was loaded on the plate to act as a positive control. All plates were incubated at 37°C for up to 24 h. The diameter of inhibition zones (in mm) was measured and the mean was calculated from two replicates.

Results and Discussion

GC-MS analysis of chloroform fraction of plant Thuja orientalis revealed the existence of diterpene compounds, Ferruginol, Andrographolide, Torulosol, dihydro, 18-Oxo-kauran-17-yl acetate and of ent-16-Hydroxy-17-acetoxy-19-kauranal, Other non terpenoid compound also detected in chloroform fraction, Cymarin and K-Strophanthin, cardic glycoside type.  the results are  shown in Table 1.

The antibacterial of active diterpene types isolated from Chamaecyparis lawsoninana have modulator activity against multi drug resistance of Staphylococcus aureus, S. aureus strains and two epidemic methicillin-resistant, clinical isolates. ferruginol, pisiferol and its epimer 5-epipisiferol, formosanoxide, trans-communic acid and torulosal, Some of these compounds also exhibited modulatory activity in potentiating antibiotic activity against effluxing strains and ferruginol, used at a sub-inhibitory concentration, resulted in an 80-fold potentiation of oxacillin activity against strain EMRSA-15. An efflux inhibition assay using an S. aureus strain possessing the MDR NorA efflux pump resulted in 40% inhibition of ethidium bromide efflux at 10 micro M ferruginol (2.86 microg/ml).¹¹ (Smith et al ., 2007), this come in harmony with current study where ferruginol and torusolal are detected in diterpene fraction of the plant in high amount.

Table 1: Major compounds of chloroform fraction of leaves of Thuja orientalis.

	Compounds	%	RI	%
1	Andrographolide	1.6	1005	C20H30O5
2	ent-16a-Hydroxy-17-acetoxy-19-kauranal	1.4	1934	C22H34O4
3	3α-Acetyloxy-5α-pregnan-20-one	0.15	1050	C23H36O3
4	alpha.-D-Glucopyranose	0.24	1404	C6H12O6
5	18-Oxo-kauran-17-yl acetate	0.35	1980	C22H34O3
6	Torulosol, dihydro	2.5	1200	C20H36O2
7	Ferruginol	5.5	2225	C20H30O
8	Benzoic acid	5.20	1697	C7H6O2
9	Dihydro-coumarone	0.30	1036	C8H8O
10	Chromone-hydroxy6,7,8-trimethoxy-2,3-dimethyl	0.11	2250	C14H16O6
11	Card-20(22)-enolide, 3-[(2,6-dideoxy-3-O-methyl-.beta.-D-ribo-hexopyranosyl)oxy]-5,14-dihydroxy-19-oxo-, [Cymarin]	26.94	4191	C30H44O9
12	Card-20(22)-enolide, 3-[(2,6-dideoxy-4-O-.beta.-D-glucopyranosyl-3-O-methyl-.beta.-D-ribo-hexopyranosyl)oxy]-5,14-dihydroxy-19-oxo-[ K-Strophanthin]	2.25	5558	C36H54O14

 

Antibacterial Properties

The evaluation of the antibacterial activity of the chloroform fraction of methanolic extract of Thuja orental leaves are represented in Figures 1,2 and Table 2. As shown from the results,  only the gram-positive were susceptible to the plant extract while the gram-negative bacteria showed very weak or no susceptibility against the extract. The tested gram-positive bacteria recorded 13.5±0.7mm for Staphylococcus epidermidis and 11.0±0.0 mm for Staphylococcus aureus, respectively.  Whereas, the tested gram-negative bacteria recorded 6.5±0.7mm for Klebsiella pneumoniae and Escherichia coli and 6.0±0.0mm for Acinitobacter baumannii, putting in consideration that the blank disc diameter is 6.0 mm. The antibiotic, chloramphenicol (5mg/ml) was used as a positive control. The noticeable antibacterial activity of the methanol extract of the leaves of Thuja orental against Staphylococcus epidermidis and Staphylococcus aurerus in the current study is attributed to some bioactive phytochemical constituents present in this aromatic plant. Plants are rich source of alternative antibacterial copounds able to combat the growing phenomenon of antibiotics resistant bacteria.¹² The finding of the current study is in agreement with previous studies on the antibacterial activity, in general. Duhan et al., 2013,¹³ cited that the methanol, acetone and ethyl acetate extracts of Thuja orental leaves were found effective against various gram-negative and gram-positive bacteria (i.e. Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Alcaligenes faecalis and Klebsiella pneumoniae) and were mostly competitor to some antibiotics. Kshirsagar et al.,¹⁴ mentioned that, the acetone, ethyl acetate and methanol extracts of Thuja orentalis leaves where tested against different bacterial strains. Only methanol and acetone extracts recorded highest antibacterial efficacy, particularly against Bacillus subtilis. Moreover, Jain and Grag 1997,¹⁵ reported significant antibacterial activity of the essential oils of Thuja orental against all tested gram-positive and gram-negative bacteria, especially S. typhi. Accordingly, more future studies should be conducted on this plant particularly its aromatic compounds, which could be used in the formulation of new natural antibacterial agents.

Table 2: Antibacterial activity of chloroform fraction of Thuja orentalis leaves compared with the antibiotic (Chloramphenicol).

Tested	Zone of Inhibition (mm)
	Sa	Se	Ab	Kp	Ec
Extract (500mg/ml)	11.0±0.0	13.5±0.7	6.0±0.0	6.5±0.7	6.5±0.7
Chloramphenicol (5mg/ml)	25.5±0.7	27.0±0.0	21.0±1.4	26.5±0.7	29.5±0.7

 

Sa=Staphylococcus aureus, Se= Staphylococcus epidermidis, Ab=Acinitobacter baumannii, Ec=Escherichia coli, Kp=Klebsiella pneumonia Zone diameter equal 6 mm= no inhibition, mean ± standard deviation.

Figure 1: Representative photo showing susceptibility of S. epidermidis and S. aureus to the chloroform fraction. Click here to view figure

Figure 2: Antibacterial activity of the chloroform fraction of Thuja orentalis leaves using disc diffusion test. Click here to view figure

 

Insecticidal Activity

Insecticidal activity of Thuja orentalis was calculated based on larval mortality after treatment. According to the LC₅₀ values of the tested products, the present results indicated that Thuja orentalis have larvicidal activity against 4^th instar larvae of S. littoralis.

Little know about the effects of plant metabolites on the biology of S. littoralis. terpenoid and cardiac glycoside, considered as secondary metabolites synthesized by desert plants with physical and biochemical properties. Hexane extract of was found to have antimicrobial and larvicidal properties against Aedes aegypti larva, chromatographic separation of the extract leading to the isolation of five Kaurane diterpene as the major compounds, this agree with present study, where the diterpene fraction contain 18-Oxo-kauran-17-yl acetate and kuran 19-ol-acetate, two type of kauran.¹⁶

Andrographolide compound show antifedent activity against  fourth instar larvae of Papilo demoleus at concentration of 200 ppm and it was recommended to use as a pest control against Papilo demoleus.¹⁷ The use of plants as larvicidals agents are very useful and can be used as substitute against pesticides.

Table 3: Susceptibility of the 4th instar larvae of S. littoralis to Thuja orentalis.

Plant	Lethal concentrations (%)		95 % confidence limits for conc.		Slope	χ2 (d.f)
			Lower	Upper
Thuja orentalis	LC30	22.74	21.37	24.04	0.9	χ2 (2) =1.49
	LC50	27.63	26.17	29.26
	LC90	44.48	40.74	49.86

 

Conclusion

Plant Thuja orentalis is medicinal plant used for antipyretic, antitussive, astringent, diuretic current study was aimed to study the role of plant as a herbal insecticide and bactericide instead of synthetic drugs, and role of chemical composition of the plant on these biological activity, Gc-Ms  analysis reflect the presence of diterpene compounds as major compound in chloroform fraction, many promising compound,  which have antimicrobial and larvicidal activity was detected, Ferruginol,  Torulosol, dihydro, 18-Oxo-kauran-17-yl acetate and Andrographolide. Insecticidal activity of Thuja orentalis was calculated based on larval mortality after treatment. According to the LC₅₀ values of the tested products, the present results indicated that Thuja orentalis have larvicidal activity against 4^th instar larvae of S. littoralis, also have good  antimicrobial activity against gram-positive bacteria while all the tested gram-negative bacteria recorded weak or no susceptibility. we can concluded that plant Thuja orentalis have a potent activity as a larvicidal and microbial activity.

Acknowledgement

The author gratefully acknowledgment the approval and support of this research by the grant no: SCI- 2017-1-8-F-7417  from the deanship of Scientific Research of Northern Border University, Arar, Saudi Arabia.

Conflict of Interest

There is no conflict of interest.

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