ISJ 13: 153-163, 2016 ISJ 13: 153-163, 2016 ISSN 1824-307X RESEARCH REPORT Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura E Edwin1, P Vasantha-Srinivasan1, S Senthil-Nathan1, A Thanigaivel1, A Ponsankar1, S Selin-Rani1, K Kalaivani2, WB Hunter3, V Duraipandiyan4, NA Al- Dhabi4 1Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi - 627 412, Tirunelveli, Tamil Nadu, India 2Post Graduate and Research Department of Zoology, Sri Parasakthi College for Women,Courtrallam-627 802, Tirunelveli, Tamil Nadu, India  3United States Department of Agriculture, ARS, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA 4Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P.O.Box.2455, Riyadh 11451, Kingdom of Saudi Arabia Accepted May 12, 2016 Abstract Andrographolide was isolated from ethanol extraction of Andrographis paniculata by column chromatography. Evaluation of larvicidal efficacy, enzymatic changes and cytotoxic activities against Spodoptera litura were conducted across a range of concentrations. The compound showed significant larvicidal activity between 5 - 25 ppm, post ingestion. Morphological deformities observed in larval-pupal stages. Enzymatic profiles were altered by reduction in acid phosphatase, ACP activity by 69.18 %, alkaline phosphatase, ALP activity 75.3 % and 74.9 % reduction in ATPase. Binding affinity to midgut epithelium cells suggests disintegration of cellular organelles observed was directly associated with ingestion of the compound. The results suggest that andrographolide has potential for development as a significant inhibitor of development against the pest Spodoptera litura. Key Words: biorational insecticides; HPLC; GC-Mass; toxicity; Noctuidae; histology   Introduction Chemical pesticides provide important protection of crops, animals, and humans from pests and pathogens, improper use in developing countries has resulted in insecticide resistance development, and various environmental pollution disputes (Matsumura, 1975). Chemicals including synthetic pesticides can sometimes form by- products which can be linked to unwanted side effects, in beneficial insects (Abudulai et al., 2001). Increasing development of resistance to synthetic insecticides, promotes investigations into the potential of biorational chemistries isolated from plants (Abudulai et al., 2001). Plants are potential producer of novel chemical compounds which cannot ___________________________________________________________________________ Corresponding author: S. Senthil-Nathan Division of Biopesticides and Environmental Toxicology Sri Paramakalyani Centre for Excellence in Environmental Sciences Manonmaniam Sundaranar University Alwarkurichi - 627 412, Tirunelveli, Tamil Nadu, India E-mail: senthil@msuniv.ac.in; senthilkalaidr@hotmail.com yet be synthesized. Estimations suggest that over 2000 plant species have the potential to identify and develop new chemistries to reduce bacteria, fungal, and insect/arthropod pests (Klocke, 1989). The complexity in chemical compounds in biorational products can also make development of resistance by insect pests is more difficult (Regnault-Roger et al., 2002). These environmental issues have driven agricultural researchers to search for better ecofriendly based pesticides (Wood and Granados, 1991). Andrographolide is a labdane diterpene lactone from Andrographis paniculata, an annual herbaceous plant in the family Acanthaceae, is native to India and Sri Lanka, which has been used for its therapeutic activity. The therapeutic value is for its impact on enzyme induction (Meenatchi- Sundaram et al., 2009). Unfortunately the limitation to wider direct use as a therapeutic agent is due to poor solubility in water (Xiaoa et al., 2013). Acid phosphatase (ACP, E.C.3.1.3.2) and Alkaline phosphatase (ALP, E.C.3.1.3.1) are 153   mailto:senthilkalaidr@hotmail.com enzymes, which supports to hydrolyse phosphomonoesters in acid or alkaline environments. Epithelim of intestine in animals is a common place for ALP which helps to provide phosphate ions for metabolic processes mainly in transphosphorylation reaction (Sakharov et al., 1989). During larval moulting stage the ACP and ALP activity decrease and increase after moulting (Miao, 2002;  Ajamhassani et al., 2012). Adenosine triphosphatases (ATPases) helps to transport glucose, amino acids, and other organic molecules. ATPases are present in the guts and nerve cells of the lepidopteron insects (Horie, 1958). Thus if a reduction in ATPase efficacy could be caused, there would be a defect in the gut physiology of the insect. Spodoptera litura (Lepidoptera: Noctuidae) is an economically important polyphagous pest in India, China and Japan (Wan et al., 2014), causing major loss to many vegetables and field crops. The pest is active throughout the year on a wide variety of crops. The larvae are especially serious pests during the seedling stage (Chandrasekaran et al., 2012). Synthetic pesticides or chemical insecticides used for controlling S. litura have started to fail as populations of S. litura start to develop chemical resistance (Sintim et al., 2009; Senthil-Nathan et al., 2013). A. paniculata extracts have been reported as an antifeedant and ovicidal activity against insects (Hermawan et al., 1993), but activity of andrographolide on S. litura has yet to be evaluated. Hence, the objectives of the present study includes, isolation and characterization of Andrographolide from Andrographis paniculata and evaluation of Andrographolide for its larvicidal efficacy, phosphatases and cytotoxic activities against Spodoptera litura. Materials and Methods Spodoptera litura culture S. litura larvae were collected from Ricinus communis plant in Nagercoil, Tamil Nadu, India was cultured and maintained according to Senthil- Nathan and Kalaivani (2005) and Senthil-Nathan et al. (2008a). Larvae were cultured in the laboratory on R. communis plant leaves. The R. communis plant leaves were collected from approximately 45 days old plant. For leaf assays and mass culturing, in-between young and mature leaves were used. Pre-pupae were provided with vermiculture soil as pupation sites. Adult moths emerged from the pupation site were transferred to the cages and fed on a 10 % sucrose solution. The fine cloths containing eggs were removed every 24hrs and eggs present were surface sterilized in situ by dipping in 10 % formaldehyde solution for approximately 3 mts, then washing with distilled water. The eggs in the fine cloths were moistened and kept in aerated containers for hatching. The culture and experiments were carried out at 27 (±3°C), 65 % relative humidity, with a 14:10 light: dark cycle. Isolation of plant compound andrographolide The ethanolic extracted plant material was analysed using chromatograph column chromatography with chloroform and methanol. Solutes were eluted with different gradients of 90:10, 80:20, 70:30, 60:40, 50:50 and 30:60 Chloroform: Methanol. Active fraction analyses by GC-MS revealed andrographolide as a major compound along with a few trace elements. Further purification by column chromatography and analyses by HPLC was compared with a purchased standard and chromatogram of andrographolide for confirmation. Insect bioassay Bioassays were performed with second, third, fourth and fifth instar larvae of S. litura using different concentrations. Methanol treated leaves was used as control. Five replication was performend with twenty larvae per concentration (n = 100). The fresh R. communis leaves (75 - 125 cm2) were sprayed with different concentrations of (5, 10, 15, 25 ppm) on both surfaces and allowed for dry. All the treatment instars larvae were starved for 4 h, then fed with treated leaves. The uneaten leaves were removed every day and provide fresh leaves. Replication was done five times (totally n = 50). Mortality was recorded every 24 h for all treatments. The percentage mortality was calculated using the formula (1) and corrections for mortality when necessary were done using Abbott’s (1925) formula (2): (1) Percentage of mortality = Number of dead larvae Number of larvae introduced ×100 (2) percentage of mortality = in T after t t t ×100Corrected (1 - n in C after t t t n ) The mean lethal concentration (LC50 and LC90) were calculated by subjecting mortality data to Probit analysis (Finney, 1971). From these results the developmental studies were performed. Developmental studies S. litura 3rd instar larvae were used for developmental studies. Individual larvae were reared in containers on the R. communis leaves treated with the lethal concentrations of active fraction as 3, 6, 9 and 12 ppm. Control leaves were treated with methanol. The containers of the treated and control groups were maintained at 27 (± 2 °C) in a 14L:10D photoperiod at 85 % relative humidity. Records were made daily to document deformities in the pupae and abnormalities in emerged adults. Enzymatic profile Preparation of enzyme extract Treated larvae of third, fourth and fifth instars were used for enzyme activities at different concentrations of 3, 6 and 9 ppm respectively. The 154   extraction procedure as in Applebaum (1964) and Applebaum et al. (1961). Anaesthetized larvae were dissected out and entire digestive tract was treated within ice-cold insect Ringer’s solution. The gut contents, malphigian tubules and adhering tissues were carefully removed. The separated gut regions was weighed (in mg) and homogenized in volume 300µl of ice-cold citrate-phosphate buffer (pH 6.8) with tissue grinder for 3 min at 4 C. The homogenate was suspended in ice-cold buffer bringing the volume up to 1ml with citrate buffer. Samples were centrifuged at 500xg, (Eppendorf 5415C table top centrifuge) for one min and the resultant supernatants were collected as the enzyme source. Histology The effects of the active fraction ingestion was studied on larvae of S. litura. Treated and control larval gut tissue was fixed in Bouin’s Solution overnight. The blocks were cooled about 27 °C for 3 h and cut into 1.5 µm slices with an ultra-cryo- microtome (Cryocut 1800, Leica, Germany). The slices were stained with Delafield’s haematoxylin and counter-stained with eosin, and mounted after drying. The sections were observed and photographed under Optika, Flow series HBO light microscope (model: B-600 TiFl-Italy). Statistical analysis Each experiment was replicated five times and the data’s obtained from mortality were expressed in mean of five replications and arcsine-square root transformation of percentages was used to normalise the data. The analysis of variance (ANOVA) was used to identify the difference in percentage of mortality and were fitted with linear regression using Minitab®17 for lethal concentration. Differences between the treatments were determined using the Tukey-Kramer HSD test (p ≤ 0.05). The highest mean difference detected by statistical testing are marked with letter “a” the next text lower “b” etc. (Snedecor and Cochran, 1989; SAS Institute, 2001). For enzyme activity linear regression technique of Microcal software (OriginPro 8) was used to graphs. The lethal concentrations (LC50 and LC90) were calculated using Probit analysis (Finney, 1971). Acid phosphatase (E.C.3.1.3.2) and alkaline phosphatase (E.C.3.1.3.1) Bessey et al. (1946) procedure was used to carried out the experiment The 100 uL substrates were incubated for about 30 min and 1 mL of alkali (1.0M pH 8.0) was added to stop the reaction. The p-nitrophenolate spectral absorbance was maximal at 310 nm. The molar absorbance of p- nitrophenolate at 400 nm is about double that of p- nitrophenyl phosphate at 310 nm. On converting the p-nitrophenolate into p-nitrophenol by acidification, the absorption maximum is shifted to about 320 nm with no detectable absorption at 400 nm. Estimation of adenosine triphosphatase Shiosaka et al. (1971) protocols was followed during the experiment. In the gut sodium and potassium dependent ATPase activities were assayed. Fiske and Subbarow (1925) methodology was adopted for quantitative assay of inorganic phosphorous liberated during experiment. In detail first the protein is precipitated with trichloroacetic acid, then the protein-free filtrate is treated with acid molybdate solution and the phosphoric acid formed is reduced by the addition of 1-amino-2-naphthol-4- sulfonic acid (ANSA) reagent to produce blue color. The intensity of the color is proportional to the amount of phosphorous present. Results Analysis of purified plant compound andrographolide The isolated plant compound from column chromatography analysed by HPLC revealed the purity at 88 % (Fig.1) when compared with the standard of andrographolide (Fig. 2). The analysed compound eluted at the Rt 3.01, which was detected at 224 nm compared with the standard chromatogram. The andrographolide standard eluted Fig. 1 HPLC chromatogram of standard andrographolide 155   Fig. 2 HPLC chromatogram of isolated compound andrographolide at the Rt of 2.92. The comparison analyses confirms the compound isolated was andrographolide. Mortality of S. litura against andrographolide The active compound andrographolide had pronounced larvicidal activity against S. litura. Mortality rates of larvae increased gradually with increasing concentrations (5, 10, 15 and 25 ppm). Second instar larvae had the highest mortality. The mortality percentage of andrographolide against S. litura is shown in (Fig. 3). The leaf disc treated with andrographolide was permitted to feed and the larvae were observed at 24 h. Mortality rate showed significant difference between second instar (F4,20 = 54.61, p ≤ 0.001), third instar (F4,20 = 56.62, p ≤ 0.001), fourth instar (F4,20 = 67.35, P≤0.001) and (F4,20 = 29.02, p ≤ 0.001) fifth instar larvae respectively. The greatest mortality rate was observed in all instar larvae at 25 ppm concentration. The results shown that the andrographolide had significant larvicidal activity. The lowest (5 ppm) concentration of andrographolide had the lowest mortality rate in all instars, but it caused several abnormalities in larval growth. Low concentrations were ample enough to give prominent LC50 and LC90 values for all the instars. Lethal concentration (LC50) for second instar larvae was observed at 8.31 and 28.86 ppm for LC90. In third instar larvae LC50 was observed at 9.59 and 30.78 ppm for LC90. For fourth and fifth instar larvae the LC50 and LC90 was observed at 10.38 ppm, 34.53 ppm and 11.33 ppm, 36.88 ppm respectively. Fig. 3 Percentage mortality of second, third, fourth and fifth instar of S. litura after treatment with andrographolide. Means (SEM±) followed by the same letters above bars indicate no significant difference (p ≤ 0.05) by using Probit analysis. 156   Fig. 4 Morphological effects of andrographolide on S. litura. (A,B,C) larval pupal intermediate (D,E) pupal deformities (F) control pupa (G) treated and unhealthy adult (died) (H) adult emerged with wrinkled wings, molting disorder (shrinking of larval body) (I) healthy adult. Behavioural effects of S. litura against andrographolide Effective dosage of andrographolide produced a typical colour change, with fluid ooze out from the body of the S. litura larvae. Larval-pupal intermediates due to growth-regulatory effects of andrographolide and immature adults were noticed in all the treated concentrations but was more prominent at the highest concentrations (Fig. 4). In some cases pre-pupae were affected often causing death, or formation of abnormal pupae were noticed at higher concentrations of 9 and 12 ppm. In lower dosage (3 and 6 ppm) durations of the development life cycle became extended, larval growth was retarded and colour change occurred. At the concentration of 12 ppm larvae appeared unhealthy, with severely damaged cuticles. The adult emergence showed lower longevity, deformed wings and egg produced by the adults were nonviable, immature. 157   Fig. 5 Enzyme activity (ACP) in (A) third, (B) fourth and (C) fifth instar larvae of S. litura after treatment with andrographolide. ACP activity Concentration used below the LC50 value showed reduction in enzymatic activity. Acid phosphatase activity was reduced in larvae treated even at lower concentrations 3, 6 and 9 ppm. The ACP inhibition was significantly different for third (F3,16 = 22.65; p ≤ 0.001), fourth (F3,16 = 42.01; p ≤ 0.001) and fifth (F3,16 = 49.99; p ≤ 0.001) instar larvae (Fig. 5). When compared with control the estimated highest reduction of 69.18 % was observed in third instar larvae treated with 9 ppm concentration. The reduction in acid phosphatase level reduces metabolism by minimal release of phosphorus. The reduction observed suggests that the cause is predominantly due to the physiological activity of andrographolide. ALP activity Activity of andrographolide against S. litura showed ALP activity reduction across all concentrations. It showed significant reduction at 9 ppm. Inhibition rate was protuberant in third instar larvae compared with fourth and fifth instars of S. litura (Fig. 6). The greatest reduction of 75.3 % was observed in third instars treated with 9 ppm concentration of andrographolide. ATPase Adenosine triphosphate was reduced in treated larvae compared with control larvae. The greatest reduction of 74.9 % occurred with treatment of 9 ppm andrographolide (Fig. 7). The reduction rate of third, fourth and fifth instar larvae are significantly 158   Fig. 6 Enzyme activity (ALP) in (A) third, (B) fourth and (C) fifth instar larvae of S. litura after treatment with andrographolide. different with each other (p ≤ 0.001). This reduction affects the physiology of the gut by inhibiting the transphosphorylation activity. This activity clearly implies the compound can produce significant influence on enzymatic profile. Histological study of S. litura Differences in the midgut region was observed in 3, 6 and 9 ppm treated larvae of S. litura. The histology of treated larvae showed damaged cellular components to the epithelial and columnar cells. The untreated larvae had well distinguished epithelial and columnar cells, peritrophic membrane, brush border membrane and microvilli (Fig. 8A). Larvae treated at 3 ppm showed some disturbed cells, epithelial cells were enlarged, getting to rupture and partially disintegrated gut lumen was observed (Fig. 8B). In 6 ppm treatment, larvae showed damaged gut lumen and cytoplasmic regions of oozing cell material into the alimentary tract that mixed within the food column. Columnar cells enlarged, brush boarder membrane was damaged with no clear identification of epithelial layer (Fig. 8C). Larvae at 9 ppm concentration showed damage to cell organelles such as brush boarder membrane being reduced, columnar cells becoming thin, enlarged spacing of cells, mixing with other cell organelles and post disruption of membranes (Fig. 8D). At higher concentration of 12 ppm, the epithelial cells were broken and fully collapsed. There was no distinct columnar cells, epithelial layer, cytoplasmic disappeared and other cellular components appeared to have disintegrated completely (Fig. 8E). The space between the cytoplasm appeared larger and very distinct. Thus at each increasing concentration the damage of cells became more significant and obvious. 159   Fig. 7 Percentage reductions of enzyme activity (ATP) in (A) third, (B) fourth and (C) fifth instar larvae of S. litura after treatment with andrographolide Discussion Mortality rate was highest in second instar larvae compared with all other instars, possibly because of their earlier developmental stage. Hexane and chloroform extract of A. paniculata showed 100 % mortality at 1000 ppm treated mosquitoes, Anopheles subpictus (Elango et al., 2011). Young instars were more susceptible than older instars, as insects mature the mortality rate was reduced. This observation may be due to the increased physiological power of a maturing larvae which has greater capacity to excrete or breakdown ingested toxins. Earlier instars were observed to be more susceptible to andrographolide exposure, when compared with later instars. Morphological changes in the lifecycle of the insect was due to inhibitory effects of andrographolide. Similar results were reported from ethanolic leaf extract of Tribulus terrestris, family (Zygophyllaceae), on S. litura (Gunasekaran and Chellaiah, 1985). The darkish black colour pupae was observed and unable to emerge into adults due to ingested andrographolide. The deformed adults showed, darker wings and unable to fly for a longer distance. Similar results were reported by Narendran et al. (1999), of deformities in head size, length of the body, remains of old cuticle, and darkened colouration on wings when treated with bark extracts of Cassia fistula and leaf extract of Murraya koenigii, L. family Rutaceae, at 1000 ppm. Martinez and van Emden. (2001) also showed that azadiractin induced a wide range of abnormalities such as larval, pupal and adult abnormalities and small sized adults of the lepidopteran, Spodoptera littoralis. The enzyme activity was reduced due to ingestion of treated diet, which reduced gut enzymes such as ACP, ALP and ATPase. Reports suggest that these enzymes are the most sensitive to pesticide exposure (Wu and Lam, 1997; Diamantino et al., 2001;  de Almeida et al., 2014; Ottaviani, 2014). Decreased ACP levels from treatments suggests a decreased release of phosphorous needed metabolism (energy), which caused decline rate of transport of metabolites of that further reduced the overall rate of metabolism (Senthil-Nathan and Kalaivani, 2005, 2006; Senthil- Nathan et al. 2005a, b). 160   Fig. 8 Histological section of the midgut region of fourth instar larvae of S. litura (A) control, (B) Treated concentrations of 3 ppm, (C) 6 ppm, (D) 9 ppm and (E) 12 ppm of andrographolide. CC- Columnar cells EL- Epithelial layer GL- Gut lumen BBM- Brush border membrane 161   Adenosine triphosphate (ATP) is considered the energy currency of cells, particularly in the epithelial cells of intestine which aid transport and metabolites reabsorption, nutrients and secondary transport of ions (Lechleitner and Phillips, 1988; Fogg et al., 1991; Zibaee et al., 2010). As ingested food cannot be digested or oxidised, the metabolism stops, thus ATPase levels are reduced. The digestion and absorption of nutrients occurs in midgut (Selin-Rani et al., 2016). Histological studies revealed that andrographolide treatment disrupted cell and organelle integrity in larval midguts of S. litura. Destruction of epithelial cells would reduce nutrition absorption of ingested food. The midgut cytoplasm were severely damaged at the highest concentration ingested by larvae. Corruption of the epithelial layer due to andrographolide ingestion led to oozing of cytoplasmic components which mixed with food in the digestive tract. Cell destruction leads to the reduction rate of digestion and food intake. Similar observations were reported by Senthil-Nathan et al. (2008a, b) on S. exigua which ingested styryl- pyrone in an artificial diet, and for S. litura treated with 100 ppm of Melia extract. Melia is a genus of flowering trees in the mahogany family, Meliaceae (Schmidt et al., 1997). The apoptotic histopathological changes support reports that andrographolide has strong effect on cell physiology, and midgut structure of S. litura larvae. The results that the active compound bound to specific receptors on the midgut may have led to blockage of the digestive tract. Furthermore, the subsequently destructions of the transmembrane potential and the osmotic lysis of cells lining the midgut would have contributed to the death of the larvae (Aronson and Shai, 2001). The present study suggest that andrographolide derived from A. paniculata had acute toxicity to S. litura and also produced morphological changes in their lifecycles. The enzyme activity was also reduced due to ingestion of andrographolide through their diet. Similarly, andrographolide treatment strongly disrupted the midgut cells. Overall, our results gain evidence to develop novel and safer natural pesticides against the polyphagous pest S. litura. 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