Antioxidant Activity and Biological Studies of Two Medicinal Plant Extracts on Spodoptera littoralis (Boisd)

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Alternative strategies have included the investigation for a new type of insecticides and re-evaluation traditional pest control agents.The adverse special effects of synthetic pesticides have enlarged the requisite for effective and bio-degradable pesticides.Because of the power of plant-insect interactions, the plant has a well-developed defense mechanism against herbivores and are excellent sources of new toxic substances for pests (Pickett, et.al. 2006).During recent years, intensive research has been carried out to control agricultural pests by using natural insecticides of plant origin to decrease hazards in the environment.oil extracts or isolated active compounds have been shown to act as potent acute or chronic insecticides (Abdel Aziz, et al. 2007;Colomaa, et al. 2006) Also the physiological and biochemical effect of some plant oil extracts on various insects were studied by Emara, et al. 2002.There are numerous researches on the efficiency of essential oils from Lamiaceae family (Rajendran and Sriranjini, 2008;Isman, et al., 2011;Ebadollahi and Ashouri, 2011).The advantage of using plant essential oils is that they are easily available and they have been used extensively for medicinal purposes, implying that they have low or no toxicity to human (Upadhyay 2013).Among various classes of natural substances that introduced as natural biopesticides are essential oils from aromatic plants (Isman andGrieneisen, 2014and Prakash, et al., 2014).
The deleterious effects of plant products on insects can be manifested in several manners including toxicity, mortality, antifeedant growth inhibitor, suppression of reproductive behavior and reduction of fecundity and fertility, growth inhibition, perturbation of reproductive behavior (reduction of fecundity and fertility) (Hernándezlambraño, et al., 2014).
Sage (Lamiaceae family) is a perennial low shrub native of the Mediterranean region and its family reported to include more than 900 species (Pierozan, et al. 2009;Ilkiu-Vidal, et al. 2010) Its essential oil is added to meat, sausage, poultry stuffing, fish, soups, canned foods and other food products.Sage essential oil protected liver pates from oxidation processes and could be used as an alternative option to synthetic antioxidants such BHT and was used in dry fermented buffalo sausage too (Estévez, et al. 2007;Salem & Ibrahim, 2010) Thyme (Lamiaceae family) is herbaceous plant of the platoon species, grows in mountainous areas, used as a beverage instead of or with tea, added to some food to give it an acceptable flavor, the plant is used in folk medicine frequently where it is prescribed to treat mouth infections, stomach, intestine and airways, coughing and gastroenteritis and expel intestinal worms, as well as to strengthen the heart (Mohamed, et al2013).Extracts from Thyme have been used in traditional medicine for the treatment of several respiratory diseases like asthma and bronchitis and for the treatment of other pathologies thanks to several properties such as antiseptic, antispasmodic, antitussive antimicrobial, antifungal, antioxidative, and antiviral (Ocana and Reglero 2012).
Control methods, which are used to limit the losses caused by S .littoralis,consist of treatments based on synthetic insecticides that are harmful as well for farmers, consumers and the environment.To seek for alternative ways to limit the use of these insecticides, essential oils and ethanol extract from the leaves of sage and thyme were tested in the laboratory.This work was designed to evaluate the biological and antioxidant activity effect of two medicinal plants extracts sage and Thyme treated on 4 th larval instar of Spodoptera littoralis

Chemicals and Reagents:
Pure ethanol, ether methanol were purchased from E. Merch Co. (Germany), and distilled before use.

Plants:
The selected plant for the study were garden Sage (Salvia officinalis) and thyme (Thymus vulgaris), (Lamiaceae),the plant leaves were collected from agricultural research center during may Month 2017, water rinsed then spread for drying in shade at room temperature about 2 -3 weeks for dryness, then the leaves were ground by high-speed grinder.

Essential Oil Extraction:
A100 grams of plant leaves were separately subjected to hydro-distillation for over 3hours using a modified Clevenger apparatus according to Santana, et al. (2013).The resulted oil was dehydrated over anhydrous sodium sulfate and stored at -20 ºC until use.

GC / MS / MS analysis of essential oil
The analysis was carried out using a GC (Agilent Technologies 7890A) interfaced with a mass-selective detector (MSD, Agilent 7000) equipped with a polar Agilent HP-5ms (5%-phenyl methyl polysiloxane) capillary column (30 m × 0.25 mm i. d. and 0.25 μm film thickness).The carrier gas was helium with the linear velocity of 1ml/min.The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature (Santana, et al., 2013).

Ethanolic Extraction:
A sample 50gof dried and ground leaves of each plant were macerated three times (48 hrs each) with300 mL ethyl alcohol70% at room temperature 25 ºC.The macerated samples were filtered and the ethanol was evaporated using rotary evaporator at 40ºC

Identification of Phenolic Compounds by HPL:
Separation and determination of phenolic compounds in ethanolic extracts were performed according to Belajova and Suhaj (2004) by HPLC (hp 1050, the used column was sprain SG X C-6 phenyl and the U.V. detector at 285 nm).The solvent system used was a gradient of A (CH3COOH 2.5%), B (CH3COOH 8%) and C (acetonitrile).The best separation was obtained with the following gradient: at 0min, 5% B; at 20 min, 10% B; at 50 min, 30% B; at 55 min, 50% B; at 60min, 100% B; at 100 min, 50% B and 50% C; at 110 min, 100% C until 120 min.The solvent flow rate was 1ml/min.and separation was performed at 35 °C.The volume injected was 10 ml.Peak identification was performed by comparing the relative retention time of each peak with those of known compounds.

2, 2 Diphenyl-1-picrylhydrazyl (DPPH) Radical Assay:
Scavenging effect of 2, 2 diphenyl-1-picrylhydrazyl (DPPH) radical was measured by the method of Brand-Williams, et al.(1995).Where, 0.1 ml of 1 mM methanol solution of DPPH was incubated with various concentrations of each plant extract (25, 50, 100 and 200 µg/ml).After 15 and 30 min incubation periods at room temperature, the absorbance of the resulting solution was recorded at 517 nm using a spectrophotometer (Jen way 6305 UV/Vis Spectrophotometer).In addition to measuring the color of tea extract without DPPH.DPPH radical scavenging activity was expressed as the inhibition percentage and was calculated as: Absorbance of control -absorbance of sample/absorbance of control × 100.Standard butylated hydroxyl toluene (BHT) was used for comparison.

Rearing of the Cotton Leaf Worm, S. littoralis:
The culture of the cotton leafworm was reared under laboratory conditions (27 ± 2°C and 60±5% RH), the egg masses were allowed to hatch in clean jars provided with castor bean leaves, Ricinus communis, the larvae continued their development till pupation.The pupae were collected in separated jars until adult emergence.Moths were fed on 10% sugar solution.The colony was reared for several generations before using the 4 th instar larvae in the bioassay experiments Bakr, et al., (2010).

Biological Studies:
Laboratory experiments were conducted to determine the efficiency of the concerned plant extracts, against 4th instar of S.littoralis larvae reared at 27 ± 2 °C and 55 -65% R.H.All experiments were carried out using dipping method, in which fresh castor leaves were dipped for 20 seconds in the different tested extracts.Then they were left in shad to dry.Two hundred and seventy larvae were divided into three replicates each of 10 larvae where they were kept in plastic cups.Larval mortality was daily recorded until pupation.Mortality percentage was determined according to (Meglla, 1984) as following: % Mortality of larvae = No. of dead larvae/ Total No. of larvae× 100 Larval malformation % = number of malformed larvae / number of tested larvae × 100 All malformed pupae were counted.The pupal malformation percent was calculated by using the following equation: Pupation %= No. of pupae / No. of tested larvae × 100 Pupal malformation % = No. of malformed pupae / Total No. of pupae × 100 A percentage of adult emergences were counted by using the following equation: Emergence of adults% = No. of emerged adults/ Total No. of pupae ×100 Adults malformation was estimated by any change in color, size, shape or failure to grown.All malformed Adults were counted and removed immediately.
Adults malformation % = No. of malformed Adults / Total No. of adults × 100 Some adults lay an egg to complete the life cycle, then counted egg hatchability% Egg hatchability % = No. of hatched egg / Total No. of egg × 100 Statistical Analysis: Data were subjected to an analysis of variance, and the means were compared using the "Least Significant Difference (LSD)" test at 0.01 levels, as recommended by Snedecor and Cochran (1982).

Table 1:
The relative percentage of essential oil compositions of Thyme and Sage plants (identified by GC / MS / MS).

Table 2.Phenolic compounds of Thymus and Salvia of ethanolic extract identified by HPLC analysis
Antioxidant Activity: The antioxidant activity of two plants of different extracts at different concentrations (25, 50, 100 and 200 ppm) was evaluated as free radical DPPH scavenging and their results are presented in Tables (3, 4).The results obtained show that the antioxidant activity of essential oil of Thymus and Salvia compared with ethanolic extract depends on which different compounds contain this oil.The results indicated that the DPPH scavenging activities (%) were increased significantly with increasing the concentration from 25 to 200 ppm essential oil (20.21, 36.39,30.33 and 34.14% Inhibition after 15 min respectively) of thymus.While, 11.18, 12.15, 24.01 and 36.88 respectively of salvia.While, 34.21, 47.30, 65.04 and 76.30 % Inhibition after 30 min respectively of thymus.But, 29.01, 40.32, 44.05 and 62.43respectively, of salvia.The data illustrated in this table revealed that relative percentages inhibition of the DPPH scavenging activity of ethanolic extract,(14.75,19.87, 27.21 and 30.21after 15 min respectively) of thymus.While, 3.18, 8.04, 18.01 and 25.84 respectively of sage.While,20.75,23.87,36.28 and 41.21after 30 min respectively of thymus.But,28.54,27.48,33.59and 54.65respectively, of salvia.Therefore, both extract and essential oil can be regarded as possessing moderate and weak antioxidant activities, in comparison to the synthetic BHA and the ethanol extract of sage, according to Kaur and Kapoor (2002).These results with the agreement with Rasmy et al., (2012) found that ethanolic extract were more efficient in scavenging free radicals.Because, it's contained the high level of total phenolic compounds in salvia.Also, Grigore, et al., (2010) and Miura, et al., (2002) showed that both sample of thymus a good antioxidant capacity, dose dependent, slightly higher for volatile oil fraction.Each value in the table was obtained by calculating average of three experiments a standard deviation, each row different letters mean significant differences (p<0.05).

Effect of Plant Extracts on 4 th instar Larvae of S. littoralis:
The experiments included the percentage of larval mortality, malformed larvae, pupation and malformed pupae.Also, percentages of adult emergence, malformed adult and egg hatchability.

Table 5: latent biological effect of S. officinalis (leaves) on developmental stages of S. littoralis
Means within the same column that have the same letters are not significantly different (P<0.001)using Least Significant Differences LSD

Effect of Age Leaves Extracts against S. littoralis:
Data in (Table 5) indicated the biological studies of ethanolic extract of sage leaves against the 4 th larvae of S.littoralis difference were difference significant between larval mortality%, malformed larvae%, pupation% and malformed pupae%.Also, percentages of adult emergence, malformed adult%, egg hatchability% compared with control, there was also are markable increase in the concentrations as it was 100ppm to become a percentage of larval mortality 85.3% comparing with control larvae.However, low concentration (25ppm) gave the highest percentage of malformation of larvae 15.8%.These results showed that pupation percentage was 20.8 , 14.7%, 62.6% and 77.4% at concentrations, 200, 100, 50 and 25ppm, respectively, compared with 97.9% control, while malformation of pupae was 0, 11.6% 23.7% and 21.6 at the same concentrations, comparing to zero of control.Data indicated that the percentage of adult emergence recorded 0, 3.1%, 38.9% and 55.8% for 200, 100, 50 and 25 ppm, respectively, compared with 100% of control.While the concentrations, 50 and 25ppm gave percentage of malformed adult 31.5% and 18.4%, respect.In addition, egg hatchability recorded 8.6% and 12.5% at previous same concentrations.
Data presented in (Table 5) show that the efficacy of essential oil of S.officinalis leaves against the 4 th instar larvae of S.littoralis, there was a significant difference between percentages of larvae mortality which was treated with different concentrations of essential oil compared with control.The increasing of the concentrations,200 ppm and 100 ppm recorded ( 32.3% and 25.5%) as larval mortality comparing to 2.1% of control larvae, while the concentrations, 50 ppm and 25ppm caused insignificant difference compared with the control of larval mortality.The essential oil was more effective on malformation of larvae (Fig 1) and caused 31.6, 30.4, 42.7 and 50.3% at concentrations, 200, 100, 50 and 25ppm, respectively.(lowconcentration gave highest percentage of malformed larvae).
The results showed that pupation percentages of S.littoralis treated with essential oil of S.officinalis were significantly different among the concentrations, where pupation percentages of S.littoralis recorded 36.1%, 44.1%, 57.3% and 49.7% at concentrations,200, 100, 50 and 25ppm .respectively,compared with control 100%, while malformation percentage of pupae was 18.5%, 25.6%, 43% and 37.5% at the same concentrations.The data showed that adult emergence percentage of S.littoralis was 17.6, 18.5, 14.3 and 12.3% at previous concentrations, compared with control 98.7%.But malformation percentage of adults was 5.1% and 4.5% at concentrations, 50 and 25 ppm.Also egg hatchability (%) recorded 24.1 and 14.5% at concentrations, 50 and 25 ppm.In conclusion, it was found that ethanolic extract of S.officinalis was more effective than essential oil on different stages of S.littoralis.

Table 6: latent biological effect of T. vulgaris (leaves) on developmental stages of S. littoralis
Means within the same column that have the same letters are not significantly different (P<0.001 ) using Least Significant Differences LSD.
Data presented in (Table 6) show the efficacy of essential oil of T. vulgaris leaves against the 4 th instar larvae of S.littoralis, there was significant between percentage of larvae mortality, malformation of larvae%, pupation%, malformation of pupae%, emergence of adults% malformation of Adults% and egg hatchability (%) compared with control (Fig. 3).The percentage of larval mortality recorded 31.6%, 13.8, 15.3 and 0%, at concentrations, 200, 100, 50 and 25ppm, respectively.The oil extracts were more effective on malformation of larvae, malformation of larvae percentage was 30.9, 47.2, 39.5 and 48.3% at the same concentrations, therefore low concentration gave the highest percentage of malformed larvae.
The results showed that pupation percentages of S.littoralis treated with essential oil of T. vulgaris were significant among the concentrations, where pupation percentages of S.littoralis was 37.5, 39.0, 45.2 and 51.7 at concentrations,200, 100, 50 and 25ppm, respect compared with control 100%, but malformation percentage of pupae recorded 27.1 and 19.1 only at the concentrations, 50 and 25ppm.The data showed that adult emergence percentage of S.littoralis was 15.7, 12.9, 18.1 and 32.6at concentrations, 200, 100, 50 and 25ppm, respectively compared with control 98.7%.but malformation percentage of adults recorded 23.1, 15.7, 0 and 21.4% at the same concentrations, Also egg hatchability (%) was 10.8and 26.7%at concentrations, 50 and 25 ppm.In conclusion, it was found that ethanolic extract of T. vulgaris was more effective than essential oil different stages of S.littoralis.
The foregoing results indicate that the tested plant extracts have properties which cause feeding deterrence, larval mortality, retardation in the developmental stages, pupal and adult morphogenesis, reduction in fecundity and viability of S. liioralis and persistent on cotton plants and this may be correlated to the chemical constituents of these plants under studies.
These results are in agreement with those reported by Abdel Fattah et al., (2009) revealed that the botanical volatile oils used, had morphogenic effects against S. nudiseta stages.These include larval-pupal intermediates, pupal-adult intermediates, deformed adults with crumpled wings and/ or deformed thorax and abdomen.Also some adults couldn't emerge and remained in their puparia.Also, Sahayaraj, et al., (2010) proofed that plant extracts extended the larval, pupal and adult periods; reduced the pupal weight, pupal and adult emergence and caused larval-pupal deformities.In addition to there is a significant reduction in adult emergence of S.littoralis as compared with control as a result of using wild plant extracts by Shadia and Azza. (2007).
It is evident that these chemicals as constituents of the tested plants have properties which inhibit feeding and cause retardation in the larval development, pupal and adult morphogenesis of S. littoralis.
Therefore, it can be concluded that, S.officinalis and T. vulgaris extracts were effective in suppressing the population size of S. littoralis either directly through their acute toxic effects on the larvae and egg masses or indirectly through their delayed effects on the pupae and adults and minimizing the cotton infestation by the cotton leafworm S. littoralis at the vegetative growth stage.

Fig. 1 :Fig. 2 :
Fig. 1: Normal and abnormal of different stages of S. littoralis treated with S.officinalis extracts (Ethanolic and essential oil)

Table 3 :
DPPH scavenging activity of essential oil and ethanolic extract of Thymus and Salvia Each value in the table was obtained by calculating average of three experiments a standard deviation , each different letters mean significant differences (p<0.05).

Table 4 :
DPPH scavenging activity of essential oil and ethanolic extract of Thymus and Salvia