MAY 2014 – JULY 2014, Vol. 4, No. 3; 2165-2174. E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org

Section B: Biological Sciences

CODEN (USA): JCBPAT Research Article

Growth, Moulting and Metamorphosis Inhibitory Activity of Calotropis Procera Extract against Poikilocerus Pictus Fabr

*Surjeet Singh Jat1, R.C. Saxena1 and Jiya Lal Solanki2

*1Pest Control & Ayurveda Drug Research Laboratory, Department of Zoology, S.S.L. Jain College, Vidisha (M.P.), India 2Department of Chemistry, Government College Badwah, Khargone, (M.P.), India

Received: 27 April 2014; Revised: 31 May 2014; Accepted: 06 June 2014

Abstract: The extract isolated from plant Calotropis procera, when applied topically on Poikilocerus pictus at three different doses out of 10 , certain could not immersed into the adult and remains half ecdysed which were found to be quite dose dependent as the dose increases from 10-30 µg, the number of half ecdysed adult also got increased. Moreover, ecdysed adult was inversely proportionate to the doses. The nymphal maturation period which was 15±1 days in controlled got decreased to 12±2 days at 30 µg extract treatment. When 5 different dose of extract were applied on the . First two concentrations caused 100% mortality of egg i.e. fertility was found to be almost scant as compared to the control group where it showed 94% egg hatching. Similarly mortality was found to be inversely proportional to the dosage the concentration caused delayed metamorphosis with several deformities. The effect of extract on fecundity and fertility was also witnessed to be decreased as against the control group. In the present study, IR spectrum exhibited characteristics absorption bands at 2075 cm-1 and 1636.13 cm-1 for un-saturation and mass spectrum. Finally, on the basis of interpretation of graphs, the structure of compound pentacyclic triterpinic acetate was elucidated. Keywords: Calotropis procera, Poikilocerus pictus, Phytoecdysone.

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INTRODUCTION

Phytoecdysone are mimics of hormones used by the (insects) and crustacean (crabs/lobsters) families in the moulting process known as ecdysis. Hoffmeister1 isolated a compound named “ecdysone” from the plant extract, that’s why they are called phytoecdysone and are quite abundant in plants. When insects eat the plants with these chemicals they may prematurely moult, lose weight or suffer other metabolic damage and die. Chemically, phytoecdysone are classified as triterpenoids, the group of compounds that includes triterpene saponins, phytosterols and phytoecdysteroids. Phytoecdysteroids are steroidal compounds produced by plants that also interfere with insect ecdysis.2 About 5–6% of plants species, including primitive groups such as ferns, contain measurable amounts of phytoecdysteroids.3 Some plants or fungi that produce phytoecdysteroids include Achyranthes bidentata4, Tinospora cordifolia5, Pfaffia paniculata6, Leuzea carthamoides7, Serratula coronata8, Cordyceps and Asparagus.9 Phytoecdysteroid are structural analogs of the insect moulting steroid hormone ecdysone occurring in plants. Plants comprise rich sources of ecdysteroids in high concentration and with broad structural diversity. Plants contain not only the steroidal compounds but also have another type of compounds known as Juvenile hormone analogue (JHA) which simply called “Juvabione”, the term coined by Stall10. More than 2000 plants species belonging to different family have been reported in India containing JH analogue, ecdysone and Anti-juvenile hormones. The pictus (Fabr.) of order and family Acrididae is a well-known pest of Calotropis plants. Exposures of sub-lethal dose of the insecticides greatly affect the growth and development of gonads of insects11-15. JHAs have already been developed as effective insect control agents16 and are currently receiving a significant amount of attention at every stage in the life cycle of insects regulating moulting, metamorphosis, development, reproduction and many of the physiological and biochemical processes associated with these compounds disrupting ecdysteroid biosynthesis, further metabolism or mode of action could make of effective insect control agents. Moreover, Morsy et al.17 have isolated some alkaloids, phytosteroids and resinous substances from Calotropis procera which when applied topically to the 3rd instar larvae which killed them before maturation. That’s why it was thought important to seek out phytoecdysone as antagonists from Calotropis procera plant for applying it on Poikilocerus pictus insects which were reared for the study of various aspects of their life cycle viz. growth, moulting and metamorphosis.

MATERIALS AND METHODS

The whole plant Calotropis procera R. Br. of family Asclepiadaceae was used as plant materials for the isolation of active principles to see the effect on Poikilocerus pictus. Grasshopper Poekilocerus pictus (Order Orthoptera and family Acrididae) is a well-known pest of Calotropis plants. The plant after proper identification and authentification were shade dried in the lab and powdered material was prepared of 40-60 mesh size. Soxhlet extraction method as per Harborne18 was followed for the extraction and isolation of plant materials and powdered material of 40-60 mesh size of the whole plant of Calotropis procera were loaded in the Soxhlet and petroleum ether, benzene, methanol and chloroform were used as solvent. Then, 6-8 cycles were run with different-2 solvents to get a good percentage yield (Table 1). The obtained semi solid crude extract were evaporated in the water bath to get solid extract. Then TLC and column chromatography was done for the further purification (Table 2). Purified fractions were sent to SAIF, CDRI Lucknow for the spectral analysis. Obtained purified extract was applied topically on the nymph of P. pictus to see the effect on growth, moulting and metamorphosis of Poikilocerus pictus.

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Table-1: Percentage yields of crude extract isolated by Soxhlet apparatus.

Plant Solvent Wt. of dried Volume of Weight of % yield name powder solvent extract Calotropis P. ether 50 gm 500 ml 4.12 8.24 procera Benzene 50 gm 500 ml 5.05 10.1 Chloroform 50 gm 500 ml 6.10 12.22

Table-2: TLC of extract of plant C. procera with Rf value & color characteristics.

S. No. Solvent Spots Rf Color characterization System Value Visual light UV light Iodine chamber

Plant Extract CHCL3: Spot 1 0.28 Yellow Green yellow Brown MeOH Spot 2 0.85 Light green Green Dark brown (97:3) Spot 3 0.92 Green Black-green Black

Authentic CHCL3: Similar to 0.81 light Green Dark green Brown marker value MeOH (97:3) Spot 2

RESULTS AND DISCUSSION

As the result mentioned in (Table 3) described the effect of extract of plant Calotropis procera on Poikilocerus pictus when applied topically at three different doses out of 10 insects taken in each group treated as well as controlled due to effect of plant extract many of them could not immersed into the adult and remains half ecdysed. In the treatment group the number of half ecdysed were found to be quite dose dependent as the dose increases from 10-30 µg, the number of half ecdysed adult also got increased. Regarding ecdysed adult, it was inversely proportionate to the doses. The nymphal maturation period which was 15±1 days in controlled got decreased to 12±2 days at 30 µg extract treatment. This clearly indicates that the extract is quite effective on insects for reducing the ecdysed adult as well as the nymphal maturation period. When standard extract of Juvavione (5 µg) was applied topically the maturation period of nymph got decreased one day more i.e. 11±2 (Graph 1).

Table-3: Effect of Calotropis procera extracts on Poikilocerus pictus when applied topically.

Treatment P. Pictus Number of half Number of Nymph maturation (n=10 each) ecdysed adult ecdysed adult period in days Control group 10 1 9 15±1 Not treated Calotropis procera 10 2 8 14±1 extract 10 µg Calotropis procera 10 4 6 13±1 extract 20 µg Calotropis procera 10 5 5 12±2 extract 30 µg Juvavione 5 µg 10 3 7 11±2

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Nymph Maturation Period in days

15

10

5

0 Nymph maturation period Control Treated in days Treated with C. Treated with C. procera with C. Treated procera 10µg procera with 20µg 30µg Juvavione 5 µg

Graph-1: Effect of C. procera extracts on Poikilocerus pictus during growth, moulting and metamorphosis.

100 90 80 70 60 50 Percentage of egg hatching 40 Percentage of mortality 30 20 10 0 Control Treated Treated Treated Treated Treated with C. with C. with C. with C. with C. procera procera procera procera procera 50 µg 40 µg 30 µg 20 µg 10 µg

Graph-2: Effect of different doses of Calotropis procera extract upon development stages of Poikilocerus pictus.

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Table-4: Effect of Calotropis procera on development of Poikilocerus pictus.

Food Total No. of nymph No. of nymph found died No. of adult emerged Control 9 1 8 Treated* 10µg 8 1* 7 Treated* 20µg 8 2 6 Treated* 30µg 9 4 5

*Leaves of cotton smeared with extract of Calotropis procera

Results mentioned in (Table 4) showed the effect of pure extract of Calotropis procera upon the developmental stages of Poikilocerus pictus when 5 different doses in µg were applied on the insect. First two concentrations caused 100% mortality of egg i.e. fertility was found to be almost scant as compared to the control group where it showed 94% egg hatching. Similarly mortality was found to be inversely proportional to the dosage the concentration caused delayed metamorphosis with several deformities. When, 10 µg concentrations of Calotropis procera extract were applied on developmental stages of Poikilocerus pictus. It was found that Poikilocerus pictus showed 60 % fertility and 40 % mortality was found (Graph 2). Results as shown in (Table 5) described that the effect on fecundity and fertility which was also witnessed to be decreased as against the control group. During fecundity and fertility of Poikilocerus pictus number of eggs laid by a female were 35 in control and 24, 18, 13 number of eggs were found when treated with 10, 20, 30µg concentration of C. procera extract. Out of these eggs 33 eggs were hatched in control group and 15, 9, 4, 2 eggs were hatched in treated group. Hence the percentage of egg hatching got reduced during treatments by the extract of C. procera which was compared with the standard group treated with of Juvavione (Graph 3).

Table-5: Effect of purified Calotropis procera extract upon development stages of Poikilocerus pictus.

Dose (µg) No. of eggs Percentage of Incubation Percent mortality treated egg hatching period in days Control 25 94 4-6 6 Treated with C. 25 - 4-5 100 procera 50* Treated with C. 25 8 4-5 92 procera 40 Treated with C. 25 24 4-6 76 procera 30 Treated with C. 25 48 4-6 52 procera 20 Treated with C. 25 60 4-6 40 procera 10 *First two concentrations caused 100 % mortality of the egg. Remaining concentration caused delayed metamorphosis with several deformities.

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100 90 80 70 60

50 No. of eggs laid by a female 40 No. of eggs hatched 30 Percentage of egg hatching 20 10 0 Control Treated Treated Treated Treated with C. with C. with C. with procera 10 procera 20 procera 30 Juvavione µg µg µg 5 µg

Graph-3: Fecundity and fertility of Poikilocerus pictus under influence of Calotropis procera.

Almost similar results have been observed by Bathori and Kalasz19 where they have isolated 20-hydroxy ecdystroid which showed loss in fecundity and fertility. Delaying of the sexual maturation of adult female and loss in egg laying and egg hatching after the Calotropis procera extract on grasshopper have also been reported by Elsayed and Al-Otaibi20. Similarly, Sawant et al.21 have reported the study of the newly hatched nymphs of Poecilocerus pictus (Fabr.) feeding on and found a strong correlation between length and weight, in addition to extended longevity and shortened nymphal periods. Very recently, Rufaie et al.22 have also noticed the effect of phytoecdysteroid (β-Ecdysone) on synchronization of maturation in silkworm Bombyx mori. In the present study, the compound present in the extract is invaluable resource for the determination of their growth, moulting and metamorphosis activity. Thus, it is worthwhile to identify new plant species containing high levels of phytoecdysone or novel phytoecdysone. During the study, the plant extract from the plant Calotropis procera was extracted in Soxhlet apparatus by applying different solvent viz. Petroleum ether, Benzene and chloroform and during Soxhletion maximum percentage yield was observed in chloroform (12.22 %) which was followed by 10.1% in benzene, 8.24 % in petroleum ether (Table 1). Moreover, three fractions Cp-1 to Cp-3 were obtained by column chromatography. However, Ghosh and Laddha23 reported extraction and monitoring of phytoecdysteroids through HPLC. In the present study, thin layer chromatography was also done by using CHCL3: MeOH (97:3) solvent system (Table 2). The obtained Rf value (0.92) were compared with authentic marker value of phytoecdysone (Juvavione). Similarly, Alam and Ali24 have isolated the extract from root of Calotropis procera by Soxhletion and different combination of chloroform and methanol (99:1, 98:2, 96:4, 95: 5, 9:1) was tried in column

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Growth … Jat et al. chromatography to obtain similar fractions. The purified fractions were sent to the SAIF, CDRI, Lucknow from where IR, GC, 1HNMR and 13CNMR and Mass spectrum of the compound was obtained. On the basis of spectral analysis, the structural elucidation was carried out and compound pentacyclic triterpinic acetate was obtained as colorless crystals from CHCL3: MeOH (97:3) eluants. It is responded positively to Liebermann-Burchardt test for triterpenes. Similarly, Mittal and Ali25 have reported terpenoid glycosides from the roots of Calotropis procera and investigated two new terpenyl constituents characterized as bisabolan-11, 14-diol-14-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside and 2- limonenyl-oxybenzoyl-1β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D glucurono pyranosyl-(1→2)-β-D-glucuronopyranoside along with the known compounds tricapryl glyceride and α- amyrin acetate have been isolated for the first time from the methanolic extract of the roots of Calotropis procera. Mittal and Ali25 have also reported aliphatic and phenolic glycosides from the roots of Calotropis procera. In the present study, IR spectrum exhibited characteristics absorption bands at 2075 cm-1 and 1636.13 cm-1 for un-saturation and mass spectrum displayed a molecular ion peak at m/z 466, 1 corresponding to triterpinic acetate C32H50O2 (Graph 4). The HNMR (Graph 5) value was compared with those of calotropenyl acetate and other similar triterpenes. The 13CNMR spectrum (Graph 6) of the present compound exhibited important carbon resonances of triterpenes26-28 On the basis of above discussion; the structure of compound pentacyclic triterpinic acetate is given below (Fig.1):

Graph-4: IR spectrum of fraction Cp-2 of Calotropis procera.

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Graph-5: 1HNMR spectrum of fraction Cp-2 of Calotropis procera.

Graph-6: 13CNMR spectrum of fraction Cp-2 of Calotropis procera.

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O

H

H

OAc H

Fig. 1 Pentacyclic Triterpinic Acetate

Shaker et al.29 have isolated three new secondary metabolites like phytoecdsyteroids from the plant Calotropis procera to protect itself from the host insect like Poikilocerus pictus. These secondary metabolites are 5-hydroxy-3, 7-dimethoxyflavone-4’-O-β-glucopyranoside, 2β, 19-epoxy- 3β, 14β- dihydroxy-19-methoxy-5α-card-20-enolide and β-anhydroepidigitoxigenin-3β-Oglucopyranoside, along with two known compounds, uzarigenine and β-anhydroepidigitoxigenin were isolated from Calotropis procera (Asclepiadaceae). The structure elucidation was accomplished mainly by nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric methods.

CONCLUSION

Finally, it can be concluded that the compound pentacyclic triterpinic acetate isolated from Calotropis procera was found to be effective against growth, moulting and metamorphosis of Poikilocerus pictus which is an agricultural pest harmful for the development of crops. REFERENCES 1. H. Hoffmeister. Ein. Neves. Hautungs Hormone Der Insekten. Angew. Chemic. 1966, 78: 269-272. 2. R. Lafont. Archives of Insect Biochemistry and Physiology, 1997, 35: 3–20. 3. L. Dinan. Russian Journal of Plant Physiology. 1998, 45: 296–305. 4. X.Y. Gao, D.W. Wang and F. M. Li. Yao Xue Xue Bao, 2000, 35(11): 868–870. 5. C.Q. Song and R.S. Xu. Chinese Chemical Letters. 1991, 2(1): 13–14. 6. D.Courtheyn, B.Le Bizec and G.Brambilla et al. Analytica Chemica Acta. 2002, 473: 71–82. 7. J. Pis, M. Budesinsky and K. Vokac et al. Phytochemistry, 1994, 37(3): 707–711. 8. M. Bathori, H. Kalasz and S.A. Csikkelne. Acta Pharm Hung, 1999, 69(2): 72–76. 9. L. Dinan, T. Savchenko and P. Whiting. Cell & Molecular Life Sci, 2001, 58(8): 1121–1132.

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* Corresponding author: Surjeet Singh Jat; Pest Control & Ayurveda Drug Research Laboratory, Department of Zoology, S.S.L. Jain College, Vidisha (M.P.), India

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