(Asteraceae) Extracts in the Control of Spodoptera Litura Fabricus (Noctuidae: Lepidoptera) Under

J Biochem Tech (2012) 3(5):S167-S169 ISSN: 0974-2328

Bioefficacy of Calendula officinalis Linn. (Asteraceae) extracts in the control of Spodoptera litura Fabricus (Noctuidae: Lepidoptera) under

laboratory conditions

Medhini N, Divakara Y G, Prabha D, Manjulakumari D*

Received: 22 August 2012 / Received in revised form: 9 September 2012, Accepted: 11 December 2012, Published online: 18 April 2013

Abstract

The effect of Calendula officinalis L on the nutritional physiological their larvicidal, ovicidal, repellent and oviposition deterrent effects parameters of Spodoptera litura Fab larvae were studied by treating in Liriomyza helianthi Spencer Callosobruchus maculates, with leaf and flower extracts in various solvents. Protease and Brevicoryne brassicae L., Pieris spp., Mamestra brassicae and amylase enzyme activities along with their respective substrates Plutella xylostella. Intercropping with Pot Marigold was the most were assayed in hemolymph and midgut of treated and untreated effective pest control on cabbage. Its phytoconstituents include larvae. Irrespective of tissues, all the extracts showed significant triterpenoid saponins, sesquiterpene lactones, flavanol glycosides, reduction in the biochemical profile compared to controls. The most phenolic acids that may affect one or the other physiological significant change was observed in chloroform and petroleum ether parameters of the insects. This plant has been widely used to cure leaf and flower extracts treated larvae compared to other extracts human ailments due to its antimicrobial, antiviral, cytotoxic, anti- suggesting their components affect nutritional physiology of inflammatory, anti-oxidant properties. However, their actual role in S.litura. Hence, Calendula extracts can not only be used to treat the insect physiology has to be explored yet. Preliminary studies in human ailments but also as an effective botanical pesticide. our laboratory have indicated its antifeedant effect on the larvae of Spodoptera litura thereby affecting their consumption-utilization Key words: Calendula officinalis, digestive enzymes, proteins, indices (Medhini et al., 2009). The current investigation has been sugars, Spodoptera litura. carried out to further track down the parameters of nutritional physiology that are affected by the plant extracts. Introduction. Methods Plant derivatives are highly toxic to many insect species without phytotoxic properties. Of the different plant families, Asteraceae, is Laboratory Mass Culture of Spodoptera litura. known to possess such behavior-modifying chemicals that can be explored to be used as botanical pesticides. Tobacco cutworm, Spodoptera litura larvae were obtained from the National Bureau of Spodoptera litura Fab. is a devastating pest, of more than 150 Agriculturally Important Insects, (NBAII), Bengaluru and reared in species of plants of economic importance, which voraciously feed the laboratory on castor leaves until pupation and adult emergence. on leaves causing extensive damage by defoliation. Majority of the The mass culture and the experiments were carried out at 25 ±2oC, strains of S .litura have exhibited high resistant levels to various 70±5% relative humidity with 12:12 L: D cycle. organic pesticides and conventional synthetic pyrethroids Henceforth, the use of insecticides for controlling this pest is on the Preparation of Extracts rise and has the ability to develop resistance to many more insecticides. In the present study, Calendula officinalis L. About 500g of powdered shade dried flowers and leaves of C. (Asteraceae), a widely cultivated garden plant for ornamental and officinalis were subjected to Soxhlet extraction using petroleum medicinal purposes, is chosen to test against a phytophagous ether, benzene, chloroform, methanol and water. The crude extract lepidopteran pest Spodoptera litura Fabricus. Its pungent odor has obtained was dissolved in minimal quantity of the solvent that been used as an effective pesticide and insect repellant to deter served as a stock. The required dilution from the stock were white flies. Recent research by several workers has demonstrated prepared in acetone and emulsified with few drops of sandovit.

Medhini N, Divakara YG, Prabha D, Manjulakumari D* Bioassay and Treatments

Department of Biotechnology & Microbiology, Bangalore Bioassays were performed with fourth instar larvae of S. litura using

University, Bengaluru-560056, Karnataka, India. 10% concentration of extracts of C. officinalis leaves and flower.

Positive control leaves were treated with acetone and the feed of

Ph: 0091 80 22961564; Fax: 0091 80 22961461; untreated leaves served as negative control. A minimum of 30 larvae

*E-mail: [email protected] per treatment were used and the experiments were replicated thrice.

S168 J Biochem Tech (2012) 3(5):S167-S169

Fresh castor leaves were sprayed with 10% concentration of were not significant compared with one another while the midgut of different extracts of leaves and flowers of C. officinalis and allowed the larvae treated with chloroform leaf, flower and petroleum ether to air dry. The freshly moulted fourth instar larvae were starved for flower extracts showed significant reduction compared to that of the 4 h and then fed with treated and untreated leaves for 48 h. The controls. However, the reduction caused by the benzene, methanol uneaten leaves were removed every 24 h, and the larvae were fed and aqueous extracts were significant with that of the control but with fresh treated leaves. After 48 h of feeding, larvae were were insignificant among themselves and also with chloroform and sacrificed to estimate protease activity (Snell and Snell, 1949), petroleum ether extracts. amylase activity (Bernfield, 1955), total protein content (Lowry et al., 1951) and total sugar content (Dubois et al., 1956) in Table 2: Total Protein Content (μg/mg) in different tissues of S.litura after hemolymph and midgut of both treated and untreated larvae. treatment with Calendula extracts. Treatments Hemolymph Midgut Leaf Flower Leaf Flower Preparation of Enzyme extracts for Biochemical Analysis b a b a Hemolymph(HL) Petroleum 0.38±0.04 0.25±0.04 0.044±0.1 0.019±0.07 ether Benzene 0.4±0.05b 0.23±0.07a 0.068±0.07c 0.079±0.07c Hemolymph samples of treated fourth instar larvae of S. litura were Chloroform 0.15±0.05a 0.35±0.1b 0.013±0.05a 0.034±0.05b collected by puncturing the proleg and drawing the exudate into Methanol 0.48±0.05c 0.58±0.08c 0.071±0.06c 0.076±0.08c insect Ringer’s solution containing a few crystals of sodium thio- Aqueous 0.72±0.03d 0.91±0.06a 0.099±0.08d 0.15±0.07d sulphate to prevent melanization. The hemolymph sample was Negative 1.33±0.06 1.68±0.07 centrifuged at 10,000 rpm for 30 min at 4C and the supernatant was Control used as a source for biochemical analysis. Positive 1.24±0.08 1.76±0.08 Control (Acetone) Midgut Tissue (MG) Values are mean ± S.D (n=30 larvae) in each trial. Values not sharing a common superscript (a-d) differ significantly at p<0.05 (Tukey’s test). o The larvae were frozen at -21 C and the entire digestive tract dissected out in ice-cold insect Ringer’s solution. The Malphigian Earlier studies on Xanthogaleruca luteola Mull. and Eurygaster tubules, adhering tissues and gut contents were removed. The gut integriceps Puton have shown similar reduction in protease and its was homogenized for 3min at 4 °C in ice-cold citrate-phosphate substrate levels of hemolymph and midgut tissue due to the effect of buffer (pH 6.8) using a tissue grinder. The homogenate was Artemesia annua L. extracts (Zibaee and Bandani 2010). centrifuged at 500 rpm for 15 min and the supernatant was the Comparable results have been observed in S.litura upon treatment source for assays. with azadirachtin extracts. The physiology of digestion involves conversion of macromolecules into micromolecules to generate Statistical Analysis energy and provide metabolites that aide in growth, development and other vital functions and is mediated by digestive enzymes. In Data from different biochemical assays were subjected to analysis of general higher enzyme activity in the tissues is due to consumption variance (ANOVA). Differences between the treatments were and utilization of large quantities of food as seen in case of control determined by Tukey’s multiple range test (p<0.05). insects. In treated insects probably imbalance in enzyme–substrate complex and inhibition of peristaltic movement of the gut might Results and Discussion have inhibited the enzyme activity as observed by Hori (1969). Decreased levels of digestive enzymes with botanical extracts may Table 1: Protease Activity (μg/mg/min) in different tissues of S.litura after also be attributed to reduced phosphorous liberation for energy treatment with Calendula extracts metabolism, and decreased rate of metabolism, decreased rate of Treatments Hemolymph Midgut transport of metabolites and may be due to the direct effects of Leaf Flower Leaf Flower extracts on enzyme regulation. Alexander V. Konarev et.al, (2002) Petroleum 14.5±6.9b 12.6±0.0b 51.1±1.9b 47.8±6.9a has reported the presence of serine proteinase inhibitors (subtilisin ether Benzene 15.5±1.9b 10.0±0.0a 57.8±1.9b 77.8±1.9b a b a a Table 3: Amylase Activity (μg/mg/min) in different tissues of S.litura after Chloroform 6.6 ± 0.0 13.3±0.0 44.4±1.9 51.1±1.9 treatment with Calendula extracts Methanol 15.5 ±1.9b 13.3±0.0b 78.9±1.9b 76.7±0.0b b c c c Treatments Hemolymph Midgut Aqueous 20.0 ±0.0 16.6±0.0 107.8±1.9 95.6±1.9 Leaf Flower Leaf Flower Negative 153.3±3.3 174.4±1.9 Petroleum 3.4±1.9a 2.2±1.9a 20.0±1.0a 23.3±0.9b Control ether Positive 145.5±3.8 151.1±1.9 Benzene 7.8±1.9c 6.3±1.9b 30.0±0.7b 36.7±0.9c Control Chloroform 3.3 ± 1.2a 3.2±1.9ab 20.0±1.2a 16.7±1.4a (Acetone) Methanol 4.5 ±1.9b 4.2±1.9b 26.7±1.4b 30.0±1.7bc Values are mean ± S.D (n=30 larvae) in each trial. Values not sharing a Aqueous 4.8 ±1.4b 4.3±1.5b 40.0±1.3c 43.3±1.2d common superscript (a-c) differ significantly at p<0.05 (Tukey’s test) Negative 9.4±1.9 51.1±1.9 Control Effect of the extracts on Protease activity and total proteins Positive 8.4±1.9 53.3±1.9 Control The protease activity and the total protein content were significantly (Acetone) decreased in hemolymph and midgut of larvae irrespective of the Values are mean ± S.D (n=30 larvae) in each trial. Values not sharing a extracts when compared to the positive and negative controls as common superscript (a-d) differ significantly at p<0.05 (Tukey’s test). shown in Table 1 & 2. In the hemolymph, among all the extracts, significant decrease in protease activity as well as protein content and trypsin and/or chymotrypsin (T/C/SI) in C. officinalis L. These was observed in chloroform leaf and benzene flower extracts. The inhibitors would block the protease thereby lowering its activity a leaf and flower extracts of methanol, water and petroleum ether possible reason for the reduced protease activity observed in S. though showed drastic reduction when compared to the controls, but litura. The level of protein content is dependent upon rate of

J Biochem Tech (2012) 3(5):S167-S169 S169

synthesis and breakdown of proteins and water movement between insects. In most of the herbivore insects (invertebrates) the tissues and hemolymph can also account for changes in protein digestion of leaf material is mediated by several symbiotic level. Under stress conditions an increased rate of protein microorganisms that reside in the hindgut. Any compound that kills catabolism has been reported in insects. It is likely that the insect off a reasonable amount of these supporting bacteria could reduce degrades proteins to resultant amino acids in order to let them enter the insect’s digestive capabilities is another possibility for the into the TCA cycle as a keto acid for compensation for the lower reduction in the nutrient level. When saponins are eaten by an energy caused by stress. S. litura possibly employs this mechanism insect, the hydrolytic conditions in the gut will activate the to detoxify the active principles present in the plant extracts degradation of saponin from an inactive to an active form which resulting in the reduced levels of proteins. In addition to this, could influence the microflora and/or various digestion and antifeedancy induced by several phenolic compounds like absorption processes. The antimicrobial activity of C. officinalis flavanoids and tannins present in C. officinalis may affect protein (Chakraborthy et al 2008) may act against the gut microflora of the biosynthesis and accumulation, a possible explanation for the larvae thereby contributing for the reduced digestive capacity as in reduced protein level in spite of decreased enzyme activity. These case of cotton leafworm S. littoralis (Adel et al., 2000). phyto-components probably act as metabolic inhibitors of proteins (Etebari et al., 2007). Hence reduction in protein level may not Conclusion necessarily a resultant of increased catabolic activity of enzyme. In the present investigation it has been observed that among all the Effect of the extracts on the amylase activity and total sugars extracts tested the chloroform and petroleum ether extracts of leaves and flowers of Calendula officinalis L. showed significant impact The amylase activity and the total sugar content of hemolymph and on the nutritional physiology and reduced the digestive efficiency midgut significantly decreased irrespective of the extracts when irrespective of the tissues tested in the pest. This shows the potency compared to the positive and negative controls as represented in of Calendula to be used as a natural pesticide. Further investigations Table 3 and 4. In the hemolymph, among all the leaf and flower may be focused on investigating the detailed mode of action of each extracts tested, the amylase activity and its substrate levels of the phytoconstituents present in this plant for a better drastically reduced in petroleum ether flower, chloroform leaf and understanding of their structure-activity relationship and specificity flower extracts treated larvae when compared to benzene, methanol with respect to different nutrients and respective enzymes. and aqueous extracts whose effect was not significant when compared among themselves. In case of midgut instead of References petroleum ether flower, the leaf extracts showed significant reduction in both the enzyme activity as well as the total sugars Adel MM, Sehnal F, Jurzysta M (2000) Effect of alfalfa saponins content. Here too chloroform leaf and flower extracts had similar on the mouth Spodoptera littoralis. J Chem Eco 26:1065-1078 effect as in case of hemolymph. Amylase is an enzyme that Konareva Alexander V, Irina N Anisimovab, Gavrilovab VA et al hydrolyses the alpha-linked polysaccharides, such as starch and (2002) Serine proteinase inhibitors in the Compositae: glycogen to yield glucose and maltose. The present study clearly distribution, polymorphism and properties Phytochem 59:279– depicts the enzyme’s lower activity following treatment with 291 Calendula extracts. Larval feeding on petroleum ether extract from Etebari K, Bizhannia AR, Sorati R, Matindoost L (2006) Ammi majus L. and Apium graveolens L., acetone extract from Biochemical changes in haemolymph of silkworm larvae due to Melia azedarach L. and water extract from Catharanthus roseus pyriproxyphen residue. Pest Biochem and Physiol 88:14–19 Linn. significantly reduced the amylase activity as well as the total Abo El-Ghar GES, Khalil ME, Eid TM (1996) Some biochemical sugar content (Abo-El-Ghar and Khalil, 1996). effects of plant extracts in the black cutworm, Agrotis ipsilon (Hufnagel) (Lep., Noctuidae). J Appl Entomol 120 (1-5): 477– Table 4: Total Sugars (μg/mg) in different tissues of S.litura after treatment 482 with Calendula extracts Treatments Hemolymph Midgut Chakraborthy GS (2008) Antimicrobial activity of the leaf extracts Leaf Flower Leaf Flower of Calendula officinalis (Linn). J Herb Med and Toxic 2 Petroleum 0.25 ±0.04a 0.12±0.04a 0.16±0.07a 0.18±0.04b (2):65-66. ether Hori K (1969) Effect of various activators on the salivary amylase Benzene 0.43±0.10c 0.39±0.08c 0.27±0.04b 0.25±0.02c of the bug Lygus disponsi. J Ins Physiol 15:2305-2317 Chloroform 0.21±0.05a 0.15±0.04a 0.14±0.06a 0.11±0.04a Medhini.N, Palakshaprabhu, Divakar YG, Kuntal Das, Methanol 0.35±0.08b 0.27±0.04b 0.19±0.05a 0.2±0.04bc b b b d Manjulakumari D (2009) Effect of Calendula officinalis Aqueous 0.38±0.1 0.28±0.06 0.29±0.04 0.33±0.09 extracts on food consumption and utilization of Spodoptera Negative 0.55±0.25 0.84±0.05 litura. Kar J Agri Sci 22( 3-Spl.Issue):621-623 Control Positive 8.4±1.9 53.3±1.9 Zibaeea, AR Bandania (2010) Effects of Artemisia annua L. Control (Asteraceae) on the digestive enzymatic profiles and the cellular (Acetone) immune reactions of the Sunn pest, Eurygaster integriceps Values are mean ± S.D (n=30 larvae) in each trial. Values not sharing a (Heteroptera: Scutellaridae), against Beauveria bassiana, Bull common superscript (a-d) differ significantly at p<0.05 (Tukey’s test). Entomol Res 100:185-196

Secondary metabolites like tannins can bind a wide variety of compounds including carbohydrates that might attribute for the lowered sugar levels in the tissues. Phytoconstituents like triterpenoid saponins, flavonol glycosides and phenolic acids reduce the feeding efficiency inducing stress in the insects which in turn reduces some of the vital components in the body. Under such stress conditions, the nutrients get catabolized to meet the high energy demand especially carbohydrates which are the primary source of energy. Reduced energy metabolism may result in death of the