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Brazilian Archives of Biology and Technology

Print version ISSN 1516-8913

Braz. arch. biol. technol. vol.58 no.2 Curitiba Mar./Apr. 2015  Epub Oct 14, 2014

https://doi.org/10.1590/S1516-8913201400128 

Agriculture, Agribusiness and Biotechnology

Evaluation of fractions and 5,7-dihydroxy-4',6-dimethoxy-flavone fromClerodendrum phlomidis Linn. F. against Helicoverpa armigera Hub.

Veeramuthu Duraipandiyan 1  

Chelliah Muthu 2  

Kathirvelu Baskar 2   3   *  

Naif Abdullah Al-Dhabi 1  

Savarimuthu Ignacimuthu 2  

1Department of Botany and Microbiology; Addiriyah Chair for Environmental Studies, College of Science; King Saud University; Riyadh - Saudi Arabia

2Entomology Research Institute - Loyola College; Chennai - India

3Bioscience Research Foundation; Porur, Chennai-India


ABSTRACT

Twelve fractions from chloroform extract of Clerodendrum phlomidis and 5,7-dihydroxy- 4',6-dimethoxy-flavone (pectolinaringenin) were evaluated against Helicoverpa armigera. Maximum antifeedant (89.41%), larvicidal (83.77%) and ovicidal (69.25%) activities were observed in fraction 5. The least LC50 value for antifeedant (178.09 ppm) and larvicidal (198.23 ppm) were observed in fraction 5. No adult emergence was recorded in fractions 4-6 at 1000 ppm. The oviposition deterrent activity was 100% in fraction 5 at all the concentrations. Pectolinaringenin recorded maximum antifeedant (74.68%) and larvicidal (81.11%) activities at 100 ppm; it completely prevented the adult emergence of H. armigera at 100 ppm. Maximum ovicidal activity at 100 ppm concentration was 67.95%. The oviposition deterrent activity was 100% in 100 and 50 ppm concentrations. C. phlomidis could be effectively used to develop a new formulation to control the economically important pests.

Key words: Adult emergence; antifeedant; larvicidal; ovicidal; oviposition deterrent; pectolinaringenin

INTRODUCTION

Continuous use of chemical insecticides cause negative impact on the environment, health and development of resistance by the insects and affect the natural enemies. The negative impact of toxic chemical insecticides leads to increase the strict measures on environmental regulation of insecticides resulted in searching an alternative strategy to develop and use the plant-based pesticides for the management of pests by agrochemical companies (Isman 2000). Plant-based pesticides are not accumulated in the food chain as by the synthetic chemicals which are the major concern for the environmental pollution (Malau and James 2008). Further, plant based secondary chemicals are non-toxic, targeted for a wide range of pests and could be potent alternatives to synthetic pesticides (Leatemia and Isman 2004). Azima tetracanthaLam. (syn. Monetia barlerioides L'Her.) derived friedelin did not shows any toxicity against Cyprinus carpio (Baskar et al. 2014). There are different kinds of insect pests which are causing extensive damage to crops and thereby reducing the productivity. David (2008) reported that some plants in nature possess secondary metabolites which act as antifeedants, oviposition deterrents, larvicides and insect growth regulators.

Many plants extracts, their fractions and isolated compounds present in them possess various pest control properties against many pests such as antifeedants (Raja et al. 2005), larvicides (Muthu et al. 2012a,b), ovicidal and oviposition deterrent activities (Muthu et al. 2013) and insect growth regulators (Baskar and Ignacimuthu 2012a; Munoz et al. 2013). The different flavonoids in the plants have diverse functions that include providing much of the colour to flowers and fruits, pollinator attraction mechanism, symbiotic relationship with N2-fixing rhizobia, protection from UV, pathogens and insects, allelopathy and inhibition of auxin transport (Morimoto and Komai 2000; Buer et al. 2010). The presence of various groups of flavonoids such as flavones, flavonols, flavanones, anthocyanins and chalcones in the plants play a significant role in insect-plant interactions (Harborne and Grayer 1994; Simmonds 2003; Green et al. 2003). Gahukar (2010) reviewed the allelochemicals from some plants for their pesticide activities. Flavonoids from Vitex negundo (Lamiaceae), Melia azedarach (Meliaceae) and Anacardium occidentale had antifeedant, oviposition deterrent and pesticidal properties. 5,7-dihydroxyflavanone (pinocembrine) fromTeloxys graveolens (Amaranthaceae) exhibited fasciolicide, ovicide and larvicidal activities against Ascaridi galli andStomoxys calcitrans (Camacho et al. 1991).

Helicoverpa armigera Hub. is a polyphagous pest causing extensive damage to numerous crops worldwide. It has the potential to migrate long distance, facultative diapause, and high egg laying capacity, developing resistance to many conventional pesticides (Kranthi et al. 2002; Nimbalkar et al. 2009). It is a cosmopolitan and agronomically important pest affecting more than 300 plants worldwide (Pawar et al. 1986; Rajapakse and Walter 2007). The damage caused by H. armigera on cotton and pulses alone is estimated to US$ 300-500 million in India (King 1994). Estimation have revealed that annual loss caused by H. armigera alone was estimated at approximately US$ 5 billion on different crops worldwide and India and China spent nearly 50% of the total agricultural pesticides to control H. armigera (Lammers and MacLeod 2007).

Clerodendrum phlomidis (Lamiaceae) is a medicinal plant used traditionally for various ailments around the world. Juice of leaves is alterative and given in neglected syphilitic complaints (Shafi et al. 2001). Bitter tonic from the root is used as antidote, analgesic, anti-asthmatic and to treat inflammatory and rheumatic diseases (Katewa et al. 2004). The aim of this work was to evaluate the chloroform extract derived fractions from the leaves of C. phlomidis and a compound isolated from it against H. armigera for their antifeedant, larvicidal, adult emergence, ovicidal and oviposition deterrent activities since the chloroform crude extract had shown maximum antifeedant and larvicidal activities against H. armigera(Vendan et al. 2008) andEarias vittella (Muthu et al. 2012b). To the best of our knowledge, this is the first report on the effect of fractions and an isolated compound, pectolinaringenin isolated from C. phlomidis were studied againstH. armigera for various biological activities.

MATERIAL AND METHODS

Extraction, fractionation and isolation of pectolinaringenin

Extraction, fractionation and isolation of pectolinaringenin from C. phlomidis were similar as reported by Muthu et al. (2012a).

Identification of Compound

Melting points of the compound were determined by an open capillary method on a heating block apparatus. The compound was subjected to UV-Visible Spectrophotometer (Model - Hitachi 2010), IR spectrum (Perkin-Elmer FT- IR instrument on KBr disc), 1HNMR spectrum (500MHz) and 13C NMR (75MHz) spectrum on a JEOL AL-300 instrument in CDCl3. EI-MS was taken on SHIMADZU instrument at 70ev by the direct inlet method. All the chemicals and solvents used were of analytical grade (Muthu et al. 2012a).

Collection and rearing of H. armigera

Larvae of H. armigera Hub. were collected from the field in Melakondayar village in Tiruvallur district of Tamil Nadu and the collected larvae were reared individually in a plastic container (vials) and fed regularly with Abelmoschus esculentus L. (Malvaceae) till the larvae became pupae under the laboratory conditions (27±2ºC and 75±5% relative humidity). Sterilized soil was used for pupation. After pupation, the pupae were collected from the soil and placed inside the cage for the emergence of adults. Cotton soaked with 10% honey solution mixed with a few drops of multivitamins was provided for adult feeding to increase the fecundity. Potted cotton plant was kept inside the adult emergence cage for egg laying. After hatching, the larvae were collected from the cage and fed with standard artificial diet. Laboratory reared insect cultures was used for bioassay.

Antifeedant activity

Antifeedant activity of the chloroform extract fractions of theC. phlomidis and pectolinaringenin from fraction 5 was studied using leaf disc no choice method. Fresh cotton leaves were used. Leaf discs of 4.0 cm diameter were punched using cork borer and were dipped individually in 1000, 500, 250 and 125 ppm for fractions and 100, 50, 25, 12.5 ppm for pectolinaringenin. The leaf discs dipped in acetone + Tween 80 were used as negative control since they were used to dissolve the fractions and pectolinaringenin. In each plastic Petri dish (1.5cm x 9.0 cm) wet filter paper was placed to avoid early drying of the tested leaves. Single larva ofH. armigera was introduced in each treated and control Petri dish. The consumption of leaf disc in the treated and control byH. armigera larvae after 24 h of the experiment was measured using Leaf Area Meter (Delta-T Devices, Serial No. 15736 F 96, U.K). Leaf discs consumed by the larvae in the treatment were corrected from the negative control. Five replicates were maintained for each treatment with 10 larvae per replicate (total, n = 50). The experiment was conducted at laboratory condition (27±2ºC) with 14:10 h light and dark photoperiod and 75 ± 5% relative humidity (Baskar and Ignacimuthu 2012a). Antifeedant activity was calculated according to the formula of Bentley et al. (1984).

Larvicidal bioassay

Larvicidal activity was studied using leaf disc no choice method.Cotton leaf discs (Gossypium sp.) were used; they were dipped in different concentrations of fractions and compound as used for the antifeedant bioassay. After 24 h treatment, the larvae were continuously maintained on untreated fresh cotton leaves. Diet was changed every 24 h. Larval mortality was recorded up to 96 h of treatment. The number of larvae, replicates used and laboratory conditions were same as antifeedant experiment. Per cent mortality was calculated using Abbott's formula.

Growth inhibitory activity

The treated larvae that attained pupae at different concentrations were kept in the adult emergence cage. Growth inhibitory activities like larval-pupal intermediates, malformation in adults and pupicidal were observed. The adults emerged with normal condition was recorded and was calculated using the following formula:

Adult emergence = No. of Emerged adults/Number of pupae X 100.

Oviposition deterrent activity

Oviposition deterrent activity was studied according to Raja et al. (2004). The oviposition deterrent activity was studied at the concentrations of 1000, 500, 250, 125, 100, 50, 25 and 12.5 ppm for fractions and compound, respectively. The treated materials were sprayed on fresh cotton plants (cultured in the pots) along with selected controls and placed inside the cage (60 cm X45 cm X 45cm) and covered with mosquito net. Ten pairs of adult H. armigera moths were introduced into a cage and were fed with 10% (w/v) sucrose solution with multivitamin drops. After 48 h, the number of eggs laid on the treated and control leaves were recorded and the percentage of oviposition deterrent was calculated. Five replicates were maintained for all the experiments.

The oviposition deterrent activity was calculated using the formula of Williams et al. (1986):

Ovicidal activity

Twenty individual eggs of H. armigera were separated and dipped in 125, 250, 500 and 1000 ppm concentrations of fractions and 100, 50, 25 and 12.5 ppm of pectolinaringenin. Five replicates were maintained (n=100). Number of eggs hatched in the control and treatments were recorded. The laboratory conditions were the same as in the antifeedant experiment. Per cent ovicidal activity was calculated according to Abbott (1925).

Statistical Analysis

All the biological data were subjected to analysis of variance (ANOVA). Significant differences between treatments were determined by Tukey's multiple range tests (p ≤ 0.05). LC50 value was calculated using Probit Analysis (Finney 1971). All the data were analysed using SPSS package version 11.5.

RESULTS

Fractions

Antifeedant activity

Antifeedant activity of 12 fractions isolated from the chloroform extract ofC. phlomidis against H. armigera is presented in Table 1. Fraction 5 recorded maximum antifeedant activity of 89.41% followed by fractions 6 and 4 which recorded 86.15 and 85.22%, respectively, at 1000 ppm concentration. Fractions 5, 6 and 4 exhibited statistically similar antifeedant activity in 500 and 1000 ppm concentrations. More than 60% antifeedant activity was observed in fractions 4, 5, 6 and 7 at 1000ppm concentration. Less than 20% antifeedant activity was observed in fraction 2 and 12 at 1000 ppm concentration. Fraction 5 showed the least LC50 and LC90 values of 178.09 and 946.13 ppm followed by fractions 6 and 4 which showed LC50 and LC90 values of 236.39 and 273.48 ppm and 1049.46 and 1083.07 ppm, respectively. The highest LC50 value was recorded in fraction 12. All the fractions recorded statistically significant Chi-square values except fractions 1, 11 and 12.

Table 1  Antifeedant activity (%) with LC50 and LC90 of different fractions isolated from chloroform extract of Clerodendrum phlomidis against Helicoverpa armigera. 

Fractions Concentrations (ppm) Effective concentration (ppm) Χ2
125 250 500 1000 LC50 LC50
1 12.26±3.12bcd 20.31±2.92bc 21.38±2.94b 27.40±3.69bc 2136.57 4677.23 18.44
2 7.41±1.34ab 15.18±2.89b 18.66±2.05b 19.32±5.35b 2755.51 5562.21 29.07*
3 15.63±3.28cd 25.62±3.87cd 43.62±5.66d 59.18±1.60e 761.73 1731.19 31.14*
4 34.24±3.45ef 52.93±5.00f 68.68±3.46f 85.22±2.77f 273.48 1083.07 30.53*
5 42.10±4.40g 56.12±4.36f 75.85±2.70f 89.41±5.13f 178.09 946.13 35.17*
6 37.38±2.65fg 53.60±5.36f 71.45±3.26f 86.15±3.15f 236.39 1049.46 29.59*
7 27.78±4.45e 37.54±4.41e 59.78±2.04e 63.29±4.58e 541.92 1799.57 50.43*
8 18.78±4.65d 31.23±3.57de 36.40±3.74cd 43.80±4.37d 1148.72 3029.64 29.32*
9 17.38±2.05d 25.67±3.88cd 33.52±4.87c 36.18±5.92cd 1507.45 3773.80 29.10*
10 13.95±2.83bcd 22.51±5.74bcd 33.02±1.24c 39.28±5.45d 1243.81 2834.54 33.32*
11 15.23±1.07cd 18.43±3.04bc 23.99±4.11b 27.20±4.08bc 2280.88 5155.99 13.39
12 9.89±1.93bc 14.00±3.84b 16.64±3.36b 18.43±3.68b 3372.13 6898.15 17.94
Control 2.72±0.64a

Values represent Mean ± SD of five replicates; Similar alphabets in a column do not differ significantly using Tukey's test (P≤0.05);

* Chi-square values are significant at P≤0.05.

Larvicidal activity

Results of the larvicidal activity of different fractions against H. armigera and are presented in Table 2. Statistically significant larvicidal activity of 83.77% was recorded in fraction 5 followed by fractions 4 and 6 which recorded 75.55 and 73.55%, respectively at 1000 ppm concentration. Fraction 5 exhibited more than 55% larvicidal activity in all the concentrations except 125 ppm which recorded 38.66%. All the other fractions recorded less than 50% larvicidal activity. Fractions 1, 2, 9, 10 and 11 showed very low larvicidal activity.

Table 2  Larvicidal activity (%) with LC50 and LC90 of different fractions isolated from chloroform extract of Clerodendrum phlomidis against Helicoverpa armigera. 

Fractions Concentrations (ppm) Lethal concentration (ppm) χ2
125 250 500 1000 LC50 LC90
1 6.22±5.69ab 10.22±0.49b 14.22±5.29bc 18.44±4.81bc 2387.93 4460.89 39.20*
2 00±00a 00±00a 6.22±5.69ab 10.22±0.49b 1817.01 2700.91 79.58*
3 14.22±5.29cd 20.44±0.99cd 40.88±1.98d 49.11±5.63e 930.73 2060.39 44.11*
4 22.44±4.33e 42.88±4.41e 55.11±5.01e 75.55±5.15fg 480.49 1343.93 43.66*
5 38.66±2.98g 55.11±5.15f 75.77±8.51f 83.77±5.18g 198.23 1094.23 64.17*
6 30.66±1.49f 42.88±4.41e 61.33±2.98e 73.55±4.93f 417.83 1445.05 29.55*
7 18.22±3.97de 26.44±4.93d 38.66±2.98d 46.88±4.54e 1012.38 2495.13 27.41
8 8.22±4.62bc 14.22±5.29bc 20.44±0.99c 28.44±3.47d 1648.17 3229.83 29.14*
9 00±00a 10.22±0.49b 14.22±5.29bc 22.44±4.33cd 1609.25 2755.03 61.03*
10 00±00a 00±00a 6.22±5.69ab 14.22±5.29bc 1583.93 2327.70 77.49*
11 00±00a 8.22±4.62b 14.22±5.29bc 20.44±0.99cd 1656.66 2799.79 70.32*
12 00±00a 00±00a 00±00a 00±00a - - -

Effective concentrations (LC50 and LC90) of fractions were evaluated against H. armigera showed that fraction 5 had least LC50 and LC90 values of 198.23 and 1094.23 ppm, respectively followed by fractions 6 and 4, which recorded the LC50values of 417.83 and 480.49 ppm, respectively and the LC90values of 1445 and 1343 ppm, respectively. Chi-square values were statistically significant for all the fractions except fraction 7.

Growth inhibitory activity

Fractions 4, 5 and 6 completely prevented the adult emergence of H. armigera at 1000 ppm concentration. Also 500 ppm concentration of fraction 5 showed no adult emergence. Fraction 6 at a concentration of 500 ppm recorded 26.66% adult emergence while concentrations 250 and 125 ppm of fraction 5 showed 31.0 and 36.66% adult emergence, respectively. Less than 50% adult emergence was observed in fractions 7, 8, 9 and 10 at 1000 ppm concentrations and fractions 7, 9 and 10 at 500 ppm concentrations. Irrespective of the concentrations tested, fractions 1, 2, 11 and 12 showed maximum adult emergence (Table 3).

Table 3  Percentage of adult emergence of Helicoverpa armigera treated with different fractions isolated from chloroform extract of Clerodendrum phlomidis. 

Fractions Concentrations (ppm)
125 250 500 1000
1 91.55±4.75de 88.61±0.62f 83.33±6.44f 75.47±6.76c
2 100±00e 100±00g 89.05±1.03f 84.16±5.85c
3 83.61±5.32d 74.28±1.59e 69.33±5.96e 44.00±5.47b
4 58.21±9.07bc 50.66±10.90 bcd 46.00±5.47cd 00±00a
5 36.66±7.45a 31.00±8.21a 00±00a 00±00a
6 52.85±6.38b 46.00±5.47b 26.66±3.72b 00±00a
7 52.50±5.59b 47.14±6.38bc 40.00±9.12c 38.66±2.98b
8 64.50±3.09c 59.72±3.80d 51.42±3.19d 45.71±6.38b
9 59.11±1.98bc 56.94±3.10cd 47.77±3.04cd 42.5±8.50b
10 57.11±4.41bc 51.11±2.48bcd 43.77±4.12cd 38.33±6.84b
11 89.77±0.49de 88.83±0.88f 85.83±4.54f 82.14±6.56c
12 100±00e 97.77±4.96fg 89.77±0.49f 83.77±5.18c
Control 100±00e 100±00g 100±00g 100±00d

Values represent Mean ± SD of five replicates; Similar alphabets in a column do not differ significantly using Tukey's test (P≤0.05).

Ovicidal activity

Maximum ovicidal activity was noticed in fraction 5 at 1000 and 500 ppm concentrations which recorded 69.25 and 61.76%, respectively followed by fractions 4 and 6 against H. armigera. All the fractions had notable amount of ovicidal activity in a dose dependent manner. Among the fractions, fraction 9 showed poor ovicidal activity (Table 4).

Table 4  Ovicidal activity (%) of different fractions isolated from chloroform extract of Clerodendrum phlomidis against Helicoverpa armigera. 

Fractions Concentrations (ppm)
125 250 500 1000
1 15.87±3.17bcd 24.41±2.07def 36.12±3.59efg 43.56±3.21cde
2 12.64±4.41abc 21.19±3.00bcde 30.68±5.86bcde 36.01±5.67bc
3 19.02±5.62cde 28.80±3.53ef 32.95±5.50def 42.56±3.42cd
4 25.41±4.96ef 31.90±3.43f 42.45±5.12g 52.05±5.80e
5 31.90±3.43f 41.45±3.55g 61.76±3.33h 69.25±3.04f
6 22.25±3.76de 28.63±3.90ef 40.40±4.03fg 49.05±4.03de
7 19.14±2.79cde 23.47±6.51cde 34.01±4.40defg 45.61±5.64de
8 8.43±4.43ab 13.81±2.80ab 21.19±4.78ab 30.85±2.08ab
9 7.38±2.64a 11.76±4.63a 18.14±3.26a 24.47±2.80a
10 13.87±4.82abc 18.09±2.88abcd 23.42±2.92abc 35.07±1.75bc
11 15.98±0.70bcd 20.19±2.12bcd 31.90±3.43cdef 43.56±3.21cde
12 9.54±2.26ab 15.87±4.89abc 25.41±4.96abcd 36.12±3.59bc

Values represent Mean ± SD of five replicates; Similar alphabets in a column do not differ significantly using Tukey's test (P≤0.05).

Oviposition deterrent activity

Oviposition deterrent activity of different fractions against H. armigera is presented in Table 5. Fraction 5 at all the tested concentrations exhibited 100% oviposition deterrent activity followed by fraction 4 at 1000 and 500 ppm concentrations and fraction 6 at 1000 ppm. Fractions 1 to 8 at all the concentrations revealed notable amount of oviposition deterrent activity in a dose dependent manner. Fractions 9-12 showed poor oviposition deterrent activity compared to other fractions. In control, oviposition deterrent activity was observed as 11.23%.

Table 5  Oviposition deterrent activity of chloroform fractions of Clerodendrum phlomoides against Helicoverpa armigera. 

Fractions Concentrations (ppm)
125 250 500 1000
1 55.26±4.13fg 67.25±3.60d 74.11±4.38cd 88.24±1.40ef
2 51.43±4.61ef 69.57±5.95de 85.21±3.29e 89.33±1.57ef
3 64.09±3.35hi 79.67±3.56f 93.40±1.20f 96.18±1.64gh
4 85.22±1.78j 92.58±1.13g 100.0±00f 100.0±00h
5 100.0±00k 100.0±00g 100.0±00f 100.0±00h
6 71.40±3.38i 78.04±2.39ef 96.44±1.26f 100.0±00h
7 67.42±3.51hi 71.65±3.68def 78.57±4.84de 91.80±2.03fg
8 62.62±5.11gh 72.77±3.42def 79.48±3.74de 85.21±0.50e
9 38.81±1.59cd 43.16±6.38b 54.15±4.03b 63.04±6.33bc
10 33.37±4.74bc 38.34±4.32b 47.23±5.38b 56.88±4.75b
11 44.24±5.30de 51.78±4.48c 69.15±4.09c 74.48±1.77d
12 29.21±3.84b 37.48±2.44b 51.28±2.20b 65.88±3.60c
Control 11.23±3.95a

Values represent Mean ± SD of five replicates; Similar alphabets in a column do not differ significantly using Tukey's test (P≤0.05).

Isolation and characterization of the compound

The fraction eluted with the solvent hexane: ethyl acetate (1:1) gave a compound as pale yellow crystal crystallized from methanol (melting point 210°C, Lit. mp 210 - 212°C). The yield was 850 mg. It gave positive ferric reaction by giving green colour with alcoholic ferric chloride for phenol. It also answered Shinoda test for flavonoid by giving reddish pink colour with mg/HCl. TLC over silica gel G with Chloroform: Ethyl acetate (9:1) as the developing system gave a single spot, pale yellow turning dark yellow, on exposure to ammonia (Rf = 0.35). The purity of the compound was (98.2%) checked using HPLC. Spectral analyses were performed using UV, IR, 1HNMR, 13CNMR and EI-MS.

The 1HNMR spectrum showed the absence of substituent at C-3, H-3 appearing as singlet at δ = 6.56. The lone aromatic proton H-8 appeared as singlet at δ = 6.54. The A2B2 system in ring B was shown by two sets of ortho coupled protons at δ 7.85 and 7.03 (J=9.0 Hz) corresponding to H-2' , H-6' and H-3' and H-5'. 4'-OMe appeared as three proton singlet at δ - 3.94 and 6-OMe appeared at 3.90. 7-OH appeared as a broad at singlet δ 9.39 and the chelated 5-OH appeared downfield at δ 12.96.

The 13CNMR also confirmed the structure of the compound as pectolinaringenin in the present investigation. The flavonoid carbonyl appeared slightly downfield at δ = 182.33. This suggested the unsubstituted C-3 with 5-OH. The single peak at δ 93.09 was assigned to C-8 and the absence of slightly downfield peak around δ 97.0 found for C-6 in 5,7-oxygenated flavones showed that C-6 was substituted. C-6 OMe appeared at δ 131.08. C-5 attached to chelated OH appeared at δ 152.72. The δc values in the ring B corresponded to pectolinaringenin.

The EI-MS gave M+, the molecular ion at m/z 314 corresponded to the molecular formula C17H14O6. The peak at m/z 296 corresponded to [M-H2O]+. The above data confirmed the structure as pectolinaringenin (Fig 1). The molecular formula is C17H14O6.

Figure 1  Pectolinaringenin (5,7-dihydroxy- 4¢,6- dimethoxy-flavone). 

Bioeffficacy of pectolinaringenin

Antifeedant activity

Pectolinaringenin exhibited 74.68% antifeedant activity against H. armigera at 100 ppm concentration followed by 50 ppm, which was 66.89%. The LC50 and LC90 values were 33.95 and 140.44 ppm, respectively. Significant chi-square value was shown in Table 6.

Table 6  Percent antifeedant, larvicidal, adult emergence, ovicidal and oviposition deterrent activities of pectolinaringenin against H. armigera. 

Fractions Concentrations (ppm)
125 250 500 1000
1 55.26±4.13fg 67.25±3.60d 74.11±4.38cd 88.24±1.40ef
2 51.43±4.61ef 69.57±5.95de 85.21±3.29e 89.33±1.57ef
3 64.09±3.35hi 79.67±3.56f 93.40±1.20f 96.18±1.64gh
4 85.22±1.78j 92.58±1.13g 100.0±00f 100.0±00h
5 100.0±00k 100.0±00g 100.0±00f 100.0±00h
6 71.40±3.38i 78.04±2.39ef 96.44±1.26f 100.0±00h
7 67.42±3.51hi 71.65±3.68def 78.57±4.84de 91.80±2.03fg
8 62.62±5.11gh 72.77±3.42def 79.48±3.74de 85.21±0.50e
9 38.81±1.59cd 43.16±6.38b 54.15±4.03b 63.04±6.33bc
10 33.37±4.74bc 38.34±4.32b 47.23±5.38b 56.88±4.75b
11 44.24±5.30de 51.78±4.48c 69.15±4.09c 74.48±1.77d
12 29.21±3.84b 37.48±2.44b 51.28±2.20b 65.88±3.60c
Control 11.23±3.95a

Values represent Mean ± SD of five replicates; Similar alphabets in a column do not differ significantly using Tukey's test (P≤0.05)

* Chi-square values are significant at (P <0.05)

Larvicidal activity

Maximum larvicidal activity of 81.11% was observed in pectolinaringenin againstH. armigera at 100 ppm concentration followed by 50 ppm concentration, which was 68.22%. The LC50 and LC90 values were 27.31 and 122.72 ppm, respectively. The Chi-square value was significant (Table 6).

Growth inhibitory activity

Pectolinaringenin at 100 ppm concentration completely prevented the adult emergence of H. armigera; 50 ppm resulted 35% adult emergence. In the case of control, the adult emergence was observed as 95.55% (Table 6).

Ovicidal activity

Pectolinaringenin at 100 ppm exhibited 67.95% ovicidal activity againstH. armigera followed by 50 ppm concentration, which was 56.67% (Table 6).

Oviposition deterrent activity

The oviposition deterrent activity was 100% at 100 and 50 ppm concentrations against H. armigera. More than 84.8% oviposition deterrent activity was observed at 25 and 12.5% concentrations (Table 6).

DISCUSSION

Antifeedant activity

Fractions eluted with hexane: ethyl acetate (1:1) of chloroform extract ofC. phlomidis recorded maximum feeding protection againstH. armigera at 1000 ppm concentration. The result was in agreement with Baskar et al. (2010) who reported that hexane extract and effective fraction 8 from Couroupita guianensis exhibited more than 80% antifeedant activity againstH. armigera.

Pectolinaringenin at 100 ppm concentration showed maximum antifeedant activity of 74.68% followed by 50 ppm with the LC50 and LC90 values of 33.95 and 140.44 ppm, respectively. This findings agreed with the report ofThoison et al.(2004) who isolated several triterpenes and flavonoids fromNothofagus dombeyi and N. pumilio using bioassay-guided fractionation and observed that pectolinarigenin and 12-hydroxyoleanolic lactone from N. dombeyi and dihydrooroxylin A from N. pumilio exhibited feeding deterrent toCtenopsteustis obliquana larvae. Kumari et al. (2003) studied the antifeedant activity of six neo-clerodane diterpenoides isolated fromClerodendrum species against E. vittellaand S. litura; they observed that all the compounds were exhibited more than 65% antifeedant activity at 10 µg/cm2. Akhtar and Isman (2004) evaluated antifeedant effects of digitoxin, cymarin, xanthotoxin, toosendanin, thymol and trans-anethole against Trichoplusia ni, Pseudaletia unipuncta, Plutella xylostella andEpilachna varivestis and they found that xanthotoxin was most effective deterrent for T. ni, whereas thymol acted as good antifeedant against P. unipuncta, P. xylostella andE. varivestis.

Pectilonaringenin isolated from C. phlomidis exhibited strong antifeedant activity against H. armigera at 100 ppm concentration with least LC50 and LC90 values. The present findings agreed with the reports of Singh et al. (2011), who evaluated eight essential oils and some compounds againstChilo partellus for their antifeedancy. They observed that thymol and 1,8-Cineole exhibited least LC50 values of 141.8 and 148.2 ppm, respectively. Similarly, Morimoto et al. (2000; 2003) reported the antifeedant activity of rotenone, flavone, and 5-hydroxy-3,6,7,8,40-pentamethoxyflavone and recorded the LC50values of 59.1 and 24.6 l and 42.8 mg/cm2, respectively againstS. litura.

Proportionately little quantity of pectolinaringenin detected by HPLC was isolated from chloroform extract of C. phlomidis showed high antifeedant activity against H. armigera in the present study. This finding corroborated the earlier findings of Morimoto and Komai (2006) who isolated four flavonoids namely, 5-hydroxy-3,6,7,8,4'-pentamethoxyflavone, 5-hydroxy-3,6,7,8-tetramethoxyflavone, 5,6-dihydroxy-3,7-dimethoxy flavone, and 4,4',6'-trihydroxy-2'-methoxychalcone from cudweed Gnaphalium affine and were screened against S. litura for feeding deterrent activity. Except 4,4',6'-trihydroxy-2'-methoxychalcone, all the other flavonoids showed strong antifeedant activity and were detected in small quantity by HPLC.

Positions of different groups in the structure decide the activity of the compound. In this study the presence of hydroxyl group at 5th and 7th position in A-ring and 4-pyran ring (a carbonyl in C-ring) contributed towards strong antifeedant activity. Methyoxyl group at C-4' in the B-ring contributed moderate antifeedant activity (Fig. 1). Feeding deterrency of pectolinaringenin in the present study agreed with the findings of Ohmura et al. (2000) who reported that the presence of hydroxyl group at 7th position in the A-ring and carbonyl group at C-4 in the pyran ring in naringenin contributed strong antifeedant activity ofCoptotermes formosanus than the C-4' in the B-ring. Similarly, pinocembrin isolated from Flourensia oolepis having hydroxyl groups at 7th position in A-ring and carbonyl at 4th position in pyran ring C were responsible for the highest antifeedant activity against Epilachna paenulata (Georgina et al. 2009). Pectolinaringenin isolated from Clerodendron siphonenthus byPal et al. (1989) showed feeding inhibition against the rice weevil, Sitophilus oryzae and fromC. phlomidis by Muthu et al. (2013) showed ovicidal and oviposition deterrent activities against E. vittella, which also confirmed the present findings.

Larvicidal activity

Fraction 5 isolated from hexane: ethyl acetate system from chloroform extract ofC. phlomidis exhibited the least LC50 value of 198.23 ppm against H. armigera larvae in the present study. This result coincided with the findings of Adeloma and Eloff (2011) who reported that acetone: chloroform fractions of Cassia alata exhibited the LC50 values of 0.505 mg/mL against the larvae of Haemonchus controtus.Recently, Anshul et al (2013) reported that methanol extract containing isomeric flavonoids had potent larval mortality against H. armigera.

Chloroform extract fractions 5, 6 and 4 from C. phlomidis showed potent larvicidal activity against H. armigera in the present investigation. This result correlated with the earlier findings of Calle et al. (1990), who studied the insecticidal activity of four fractions of Ageratum conyzoides petroleum extract, eluted with petroleum and increasing amount of chloroform. They reported that fraction 4 showed potent insecticidal activity of 80% against 3rd and 4th instar Lepidopteran larvae, Cynthia carye. They hypothesised that the activity might be due to the presence of flavonoidal compounds. Also, some flavonoids and flavonoid-containing extracts possessed anthelmintic and nematicidal activities which induced embryonic and larval lethality (Ayers et al. 2008; Kim et al. 2009).

Fraction 5 at 1000 ppm and pectolinaringenin at 100 ppm exhibited potent larvicidal activity with least LC50 values of 198.23 and 27.31ppm, respectively against H. armigera in this investigation. Similarly, Muthu et al. (2012a) reported that fraction 5 exhibited LC50 5.02 and 61.63 ppm and pectolinaringenin LC50 0.62 and 2.87 ppm against C. quinquefasciatus and A. aegypti, respectively. At lower concentration (12.5 ppm), the compound exhibited more than 38% larval mortality against H. armigera. It was in accordance with the earlier findings of Kumari et al. (2003)who reported that six neo-clerodane diterpenoides isolated fromClerodendrum species at 10 µg/cm2 exhibited 20 and 44% larval mortality against E. vittella and S. litura, respectively. There are many reports on various flavonoids isolated from plants which also supported the present findings: quercetin and kaempferol isolated from Ricinus communis showed insecticidal activity against C. chinensis (Upasani et al. 2003), bioassay with quercetin and rutin againstH. armigera drastically reduced the larval survival were reported by Jadhav et al. (2012) which evidenced the larvicidal activity of flavonoids.

Growth inhibitory activities

Fractions and the compound either completely reduced the adult mergence or larval-pupal intermediate or pupicidal activities were observed in this study. This agreed with the earlier observation of Muthu et al. (2010) who reported very low adult emergence was observed in hexane and ethyl acetate extracts of Atalantia monophylla against E. vittella; Baskar et al. (2009) observed complete prevention of adult emergence of H. armigera at higher concentration. Further, the fractions from the chloroform extract ofCaesalpinia bonduc reduced the emergence of S. litura (Baskar et al. 2012a).

In the present finding, pectolinaringenin completely prevented the adult emergence at 100 ppm concentration and low amount of adult emergence was observed in the lower concentrations against H. armigera. This was in agreement with the earlier findings of Muthu et al. (2012b) who found that chloroform extract of C. phlomidis completely prevented the adult emergence of E. vittella. Further, Isomeric flavonoid from methanol extract ofA. annua exhibited promising growth inhibitory activities against H. armigera (Anshul et al. 2013).

Ovicidal activity

Maximum ovicidal activity of 69.25% was recorded in fraction 5 from chloroform extract of C. phlomidis at 1000 ppm concentration againstH. armigera. This agreed with the findings of Raja et al. (2005) who studied the ethyl acetate derived fractions and their compounds from Hyptis suaveolensshowed more than 60% ovicidal activity against S. litura and H. armigera. Ovicidal activity of different fractions from the hexane extract of Atalantia monophyllaagainst S. litura and H. armigera was studied by Baskar et al. (2012b) andBaskar and Ignacimuthu (2012b); they reported that fraction 9 at 1000 ppm exhibited 75.61 and 72.21%, respectively.Jeyasankar et al. (2013) reported that Syzygium lineare derived fraction 3 and compound 3-(3-Hydroxy-hexyl)-detrahydro-pyron-4-one 3 showed ovicidal activity of 66.74 and 69.50% against S. litura.

Higher concentration of fraction 5 and 100 ppm of the compound reduced the egg hatchability in this study and the ovicidal activity was in dose dependent. Results of this investigation narrowly correlated with the results of Muthu et al. (2013) who observed maximum ovicidal activity of 69.25% at 1000 ppm of fraction 5 and 67.95% activity in pectolinaringenin at 100 ppm against E. vittella. Earlier,Malarvannan et al. (2009) observed higher concentration of plant derived extracts either completely or maximum percentage prevention of egg hatchability of H. armigera. Ovicidal activity may due to interference of the plant derived substances with the normal embryonic development of the eggs by suppressing hormonal and biochemical process, incomplete blastokinensis and abnormal breakage of extra embryonic membrane in the embryo (Enslee and Riddifold 1977; Fagoonee and Lauge 1981). Garlic volatile was studied against the eggs of S. litura, Dysdercus koenigii, E. vittellaand H. armigera by Gurusubramanian and Krishna (1996) who observed that the chemicals present in the volatiles of garlic could diffuse into the eggs, thereby affecting the important physiological and biochemical processes associated with embryonic development.

Oviposition deterrent activity

Oviposition deterrency may be caused due to the adverse effects of secondary plant chemicals on ovarian development, fecundity and fertility of adults. In the present investigation, the fractions 4-6 and pectolinaringenin exhibited 100% oviposition deterrent activity against H. armigera. The observations of Muthu et al. (2013)confirmed the present findings. They reported that fractions 3-6 from chloroform extract of C. phlomidis completely prevented the oviposition ofE. vittella at 1000 and 500 ppm concentrations and pectolinaringenin also showed 100% oviposition deterrent activity at 100 ppm concentration. Similarly, Singh et al. (2011) observed maximum oviposition deterrent activity in thymol against Chilo partellus both in choice and no-choice methods.

The presence of flavonoids in active fraction 5 and pectolinaringenin exhibited maximum oviposition deterrent activity against H. armigera. The present finding agreed with the findings of Hashem et al. (2013) who studied the frass extract of S. littoralis fed with castor leaf against female adults of S. littoralis and A. ipsilon for oviposition deterrent activity; they observed that the presence of low amount of phenolics/ flavonoids effectively prevented the egg laying.

CONCLUSION

To the best of our knowledge, this is the first report on the fractions and pectolinaringenin against H. armigera for antifeedant, larvicidal, growth regulation, ovicidal and oviposition deterrent activities. The semi-purified extracts and an isolated compound from C. phlomidis exhibited various biological activities and could be effectively used to develop a new pesticidal formulation to control economically important agricultural pests and disease causing vector mosquitoes.

ACKNOWLEDGEMENT

This project was supported by King Saud University, Deanship Scientific Research, College of Science, Research Center

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Received: May 03, 2014; Accepted: August 04, 2014

*Author for correspondence: suribaskar@hotmail.com

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