Isolation of chemical constituents from Spilanthes calva DC: Toxicity, anthelmintic efficacy and in silico studies

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Original article

Isolation of chemical constituents from Spilanthes calva DC: Toxicity, anthelmintic efficacy and in silico studies P. Jayaraj a,∗ , B. Mathew b , C. Mani c , R. Govindarajan d a

Department of Pharmacy, Periyar Maniammai University, Vallam 613403, Tamilnadu, India Division of Drug Design and Medicinal Chemistry Research Lab, Department of Pharmaceutical chemistry, Grace College of Pharmacy, Palakkad 678004, Kerala, India c Department of Chemistry, Srimad Andavan Arts and Science College, Tiruchirappalli, Tamilnadu, India d Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow, India b

a r t i c l e

i n f o

Article history: Received 16 March 2014 Accepted 29 April 2014 Keywords: Spilanthes In silico ␤-Tubulin Pheretima posthuma Helminthic Paralysis time ArgusLab

a b s t r a c t Aqueous and ethanol extracts of this Spilanthes calva DC is widely used in folk medicine in South India for treating various parasitic diseases. In vitro anthelmintic activities of crude aqueous and alcoholic extract of aerial parts of the plant was investigated to provide experimental evidence for its use in folk medicine. Investigations of in vitro anthelmintic efficacy were evaluated separately on adult Pheretima posthuma and Ascaridia galli compared with Albendazole. Ethanol extract showed more anthelmintic activity than aqueous extract. Six compounds were isolated from ethanol extract compounds 3, 4 and 6 showed significant anthelmintic activity with LC50 values of 12, 11, 9.9 and 11.47, 10.56, 8.35 respectively against both the worms. The oral LD50 of the aqueous and ethanol extracts estimated in mice is greater than 5000 mg/kg. Molecular docking studies were carried out for compound 6 by using ArgusLab 4.0.1. and Molegrow 2012.2.5.0 generated the enzyme binding interaction which suggested the lactone ring attained a non-coplanar conformation with benzisoxazole can contribute two significant hydrogen bonding interaction with Tyr 50 and Gln 134. This could be attributed to the slight structural resemblance with Albendazole and hydrogen bonding, ␲-␲ and non-polar interactions towards the inhibitor-binding cavity of the ␤-tubulin enzyme. Our study shows ingredients in S. calva DC ethanol extract contains an effective anthelmintic composition that could potentially developed as a promising plant origin anthelmintic. © 2014 Published by Elsevier Masson SAS.

1. Introduction Spilanthes calva DC (Asteraceae) is a therapeutically important annual or short-lived perennial herb with yellow and non-fragrant flowers. It is commonly distributed throughout India as a weedy herb and it is used extensively in traditional medicine for various ailments including pain, toothache, throat complaints, dry cough and for treating various parasitic diseases and tuberculosis [1–4]. Previously analgesic and antioxidant potential of the methanol extract of the leaves of the plant was reported [5]. Literature review shows no chemical constituents were isolated from this plant but in this genus a wide range of phytoconstituents have been isolated which includes spilanthol, undeca-2E, 7Z, 9E-trienoic acid isobutylamide,

∗ Corresponding author. Tel.: +91 9600 152 250; fax: +91 4362 264 600. E-mail addresses: [email protected], [email protected] (P. Jayaraj).

undeca-2E-en-8, 10-diynoic acid isobutylamide and ␣,␤-amyrin and sitosterol [6,7]. Helminths infection is one of the most prevalent diseases in humans and animals, and it is a big public health issue as well. It is estimated that one-fourth of the world population is infected. It is interesting to note helminths differ from many other parasites because these organisms multiply inside the definitive host for the development which in turn develop anthelmintic resistance with unique biochemical and genetic mechanisms and they have ability to evade host immune defenses for reasons that are not fully understood [8]. The major impediment to the use of synthetic anthelmintics in humans and livestock production is the growing rate of parasite resistance [9]. Forages or plants that are rich in condensed tannins (proan thocyanidins) were found to improve performance of parasitized sheep [10]. This raised the discovery of new therapeutic anthelmintic candidates from natural source with low toxic issues for man and animals [11]. Alkaloids isolated from plants exhibit in vivo and in vitro anthelmintic activity [12] and nitrogen containing bicyclic

http://dx.doi.org/10.1016/j.bionut.2014.04.002 2210-5239/© 2014 Published by Elsevier Masson SAS.

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heteronucleus such as benzimidazole and benzoxazole were endowed with potent action against helminthiasis infections ability to bind with microtubules of the parasite thus altering its function and structure [13,14]. Some of the anthelmintic candidates showed activity by binding selectively to ␤-Tubulin of nematodes, cestodes and fluke, a protein subunit of microtubule and thereby disrupting microtubule structure and function [15]. Microtubules are very dynamic, ubiquitous cellular organelles serving a variety of vital functions including mitosis, motility and transport, in all eukaryotes. Many of these structures exist in a dynamic equilibrium in which assembly and disassembly of the soluble subunits are balanced. In such systems, the drug-tubulin interaction results in a shift of this equilibrium with a net loss of microtubules and accumulation of free tubulin. In view of the crucial roles that microtubules play in many cellular processes their drug-induced destruction eventually leads to the death of the organism and alkaloids destroys the parasites by interfering with microtubules [16]. 2. Materials and methods 2.1. Extraction and active compound isolation 2.1.1. Plant material The plant S. calva was collected from Thirunelveli, Tamilnadu, India in the month of June 2010 and authenticated by Dr. P. Jayaraman, Director of Plant Anatomy Research Centre, Tambaram, Chennai. A voucher specimen of S. calva herbarium number PARC/2010/200 was deposited in the library of Plant Anatomy Research Centre, Chennai, Tamilnadu, India. 2.1.2. Drugs and chemicals Bendex Suspension (Albendazole) was purchased from Cipla India Ltd, Saline water (Claris Lifesciences Ltd., Ahmadabad), analytical gradechemicals, including various organic solvents from merk and loba chemicals, India, were used for the extraction and to study the phytochemical constituents. Ethanol AR (Ganis Scientific & Surgicals) and DMSO (S.D Fine chemicals, Mumbai) were used in this study. 2.1.3. Preparation of SCA extract Aerial parts of S. calva was dried at room temperature and then powdered (500 g) and the aqueous extract was obtained by boiling with distilled water, then filtered and freeze-dried. The powder (yield: 12.5% w/w) was stored at 4 ◦ C until used. 2.1.4. Preparation of SCE extract Aerial parts of S. calva was dried at room temperature and then powdered (500 g) and extracted three times in a reflux condenser for 4 h with 3 L of 95% ethanol by sonication at room temperature (25 ± 2 ◦ C). The solutions were combined, filtered, concentrated under reduced pressure and lyophilized into powders. The final yield was 8.65% (w/w). 2.1.5. Phytochemical studies The stock solution was prepared from each of the SCA and SCE extracts were dissolved in 10 mL of its own mother solvents. The obtained stock solutions were subjected to preliminary phytochemical screening was subjected to phytochemical screening tests [17]. 2.1.6. Extraction, fractionation, and isolation procedure The air-dried powdered aerial parts of S. calva DC (3 kg) were extracted with 95% ethanol (5 L × 3) at room temperature for 4 days. The solvents were evaporated under reduced pressure and the combined extracts were concentrated to obtained crude residue

ethanol extract (260 g). The crude extract was acidified to pH 4 with dilute HCl after removing neutral material with n-Hexane, basifying the solution to pH 9 with aqueous ammonia and extracting again with CHCl3 afforded Fraction A (220 g). With attention to isolate the alkaloids, the CHCl3 fraction (160 g) was chromatographed on silica gel (40–63 ␮m, 9 × 73 cm), eluting with a gradient solvent system of on a silica gel (120 g, 200–300 mesh) column, eluting with a gradient solvent system of MeOH: CHCl3 with total combined volume of 200 mL in a v/v ratio of 0: 100, 1: 99, 2: 98, 3: 97, 4: 96, 5: 95, 7: 93, 10: 90, 15: 85, 20: 80, 30: 70, 40: 60, 60: 40, 80: 20 and 90: 10 to obtain fractions F1-F15. Fraction F3, up on crystallization with n-BuOH: CH3 COOH: H2 O (4: 1: 5) yielded compound 1 (90 mg). Its mother liquor was purified by column chromatography eluting with a gradient solvent system of MeOH: CHCl3 to obtain five sub-fractions F3.1-F3.5. The separation of these sub-fractions F3.3 to F3.5 using preparative TLC plates (mobile phase; CHCl3 -ethyl acetate-HCOOH (5: 4: 1) yielded compound 2 (75 mg). Fractions 4, 5 and 6 were combined (F50) and then separated by the column chromatography with a gradient solvent system of ethylacetate: CHCl3 with 50 mL, v/v ratio of 0: 100, 20: 80, 40: 60, 50: 50, 60: 40 and 80: 20 to obtain sub-fractions F50.1-F50.6. Further separation of sub-fraction F50.2 on preparative TLC plates mobile phase; n-BuOH-HOAc-H2O (4: 1: 5) gave compound 3 (59 mg). Fractions 7, 8 and 9 (F51) were combined and separated by the column chromatography with a gradient solvent system of ethylacetate: CHCl3 with 50 mL, v/v ratio of 20: 80, 40: 60, 50: 50, 60: 40 and 80: 20 to obtain subfractions F50.1-F50.6, crystallization of the sub-fraction F51.3from CHCl3: MeOH (1: 1) and on preparative TLC plates mobile phase CHCl3: EtOAc: HCOOH (5: 4: 1) furnished compound 4 (66 mg). Fraction F10 and F11 was crystallized from the MeOH: dil.NH4OH (70: 30) to give compound 5 (54 mg) and fraction F12 and F13 was crystalized from acetic acid: CHCl3 (1: 9) to give compound 6 (64 mg). All the 4 alkaloids isolated are minor alkaloids from this plant. 3. Pharmacological studies 3.1. Animals Albino mice of either sex (Swiss albino and C57/BL 6 J strains) weighing 28 ± 1 g, bred in the animal house of Novel Nutrients Pvt Ltd Banglore, India were used in the study. The animals were maintained at 25 ± 1 ◦ C, with 12/12 h light/dark cycle and 55 ± 10% relative humidity. The animals were fed with normal pellet diet containing 74% carbohydrate, 21% protein and 4% fat, purchased from Pranav Agro Industries Ltd., Maharashtra. All efforts were made to minimize animal suffering and to reduce the number of animal used. All the drugs were administered intraperitoneally (i.p.) 30 min prior to the tests. This study got clearance from Institutional Animal Ethics Committee letter number: NNLabEthics 20010/145. 3.2. Acute toxicity study (LD50 ) The acute toxicity of the SCA and SCE extracts was estimated by per oral route. The investigation was carried out with a minimum number of experimental animals. The extracts were given at doses of 10, 100 and 1000 mg/kg in the first phase and 1600, 3000 and 5000 mg/kg in the second phase. This method estimates the dose of the extract that will kill 50% of the population by a given route. Mice were kept under constant observation for the following 14 days, their weights were noted and at the end of the study a macroscopic tissue evaluation was done [18].

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3.3. Anthelmintic efficacy assay 3.3.1. Used organisms Adult earthworms Pheretima posthuma and Round worms Ascaridia galli were used in the study. Earthworms were collected from moist soil from Trichy, Tamilnadu, India and washed with normal saline to remove all the adhering debris and worms of 5–7 cm in length and 0.1–0.2 cm in diameter were selected for the study. Adult round worms were collected from the intestines of freshly slaughtered fowls (chicken) 4–6 cm in length at Trichy, Tamilnadu, India and preserved in normal saline. Both worms were authenticated at Dr. M. Johnson Department of Zoology Srimad Andavan Arts and Science College, Tiruchirappalli, Tamilnadu, India. They were used immediately in the study after collection. 3.3.2. Anthelmintic activity in vitro assay The labelled SCA extract, SCE extract and compounds 3, 4 and 6 were dissolved in distilled water and DMSO at concentrations of 3.13, 6.25, 12.5, 25, 50/mL and 1, 2, 4, 8, 16/mL for extracts and compounds respectively. Six worms, approximately of equal size, each of P. posthuma and A. galli, were placed in petri dishes and each petri dish had 25 mL of test solution of the extracts. For reference standards, Albendazole (20 mg/mL each) were used as positive controls, and distilled water was used as the negative control. The experiments were run in triplicate and before starting the experiments, standard drugs and test solutions were freshly prepared. Time for paralysis and death of worms were noted after determining worms neither moved when shaken vigorously nor when dipped in warm water of 50 ◦ C [19]. 4. In silico studies for compound 6 4.1. Toxicity prediction Toxicity prediction tool is an effective in the drug discovery process because many of the newly synthesized potential candidates had failed in clinical trial evaluation due to the pharmacokinetics and toxicity issues. Many computational predictions are nowadays available to overcome such scenario in the drug discovery process. Toxicity prediction of the newly designed scaffold was retrieved from a web-based application for Organic Chemistry Portal (http://www.organic-chemistry.org/prog) [20]. 4.2. Molecular docking study The crystal structure of ␤-tubulin (PDB entry: 1OJ0) was retrieved from the Protein Data Bank (http://www.rcsb.org) [21]. The enzyme structure is refined by removing the inhibitor and water molecules. Hydrogen atoms were added to correct the tautomeric and ionization states of amino acid residues. The active sites of the enzyme (PDB entry:1OJ0) were identified by using Q-Site Finder: an energy-based method for the prediction of protein-ligand binding sites [22]. The predicted sites comprised of Ile 4, 16, 24, His 6, Phe 20, 167, 200, Tyr 50, Gln 134, Leu 135, 250, Thr 136, 232, 237, 238, Ser 165, 166, 234, Glu 198, Met 233, Val 236 and Cys 239 were the interacting residues. These predicted amino acid residues were selected and saved as the binding site for the docking study for compound 6. The remaining protocol of docking studies was described in our previous study [23]. 4.3. Toxicity prediction for compound 6 Preclinical evaluation of toxicity of the compound 6 was done by computational tool. Mutagenic, tumerogenic, irritant and reproductive effective are the toxicity issues predicted by OSIRIS, an

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Table 1 Qualitative phytochemical parameters of S. calva DC. Phytochemicals

Alkaloids Glycosides Flavonoids Phenols Steroids Saponins Terpenoid Tannins Carbohydrates

Aerial parts of the plant Ethanol extract

Aqueous extract

+ + + + + + + + +

+ + + + – + + – +

NH N N O

OH

HO OH Fig. 1. 2-[4–(benzylamino)-7 H-cyclopenta methyl)tetrahydrofuran-3,4-diol (1).

–[d]pyramidin-7-yl]-5-(hydroxy-

entirely in-house developed drug discovery informatics systems [24].

5. Results 5.1. Phytochemical analysis The phyto chemical investigation of SCA extract and SCE extract showed the presence of alkaloids, glycosides, flavonoids, phenols, steroids, saponins, terpenoid, tannins and carbohydrates (Table 1).

5.2. Characterization of isolated compounds 5.2.1. [4-(benzylamino)-7H-cyclopenta[d]pyrimidin-7-yl]-5(hydroxymethyl) tetrahydrofuran-3,4-diol (compound 1) Yellow amorphous powder (Fig. 1): M.P. 144.38 ◦ C; 3600, 3328, 1215, 1180, 1130, 1115, 1080, 1060, 1025 cm−1 ; 1 HNMR(DMSO-d6 , 400 MHz), ␦ppm : 3.6 (1H,d, 5 -CH2 OH), 3.9(2H, d, 5 –H), 4.1(1H,dd, 3 -H), 4.6(1H,dd, 4 -H), 4.7(2H,d, 2b-H), 5.2(1H, s, 3 -OH), 5.4(1H,s, 4 -OH), 5.5(1H,dd, 8-H), 7.2(1H,dd, 4 -H), 7.3(1H,dd, 2 -H) and 8.4(1H,s, 6-H); 13 CNMR (DMSO-d6) ␦ppm : 44.1(2b-C), 56.1(9-C), 62.4(5 -C), 71.4(4 -C), 74.3(3 -C), 88.8(2 -C), 120.4(3-C), 127.8(2 C), 128.9(3 -C), 149.5(1 -C), 140.7(8-C), 153.1(6-C), 155.4(2-C); ESI-TOF (m/z): 355.15, 338, 328, 295.

5.2.2. 2E)-3-(4-ethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en1-one (compound 2) Yellow powder; (Fig. 2); M.P. 242.33; FT-IR (KBr) vmax /cm−1 : 3610(OH), 1645(C O). 1 H NMR (CDCl3 , 400 MHz, ␦ ppm): 0.91(s, CH3 ), 1.42(s, OCH3 ), 4.06(m, CH2 ), 4.60(s,OH), 6.91-6.97(s, CH CH ), 7.42-8.02(m, Ar-H). 13 C NMR (DMSO6 , 100 MHz, ␦ ppm): 14.72(CH3 ), 55.46(OCH3 ), 119.46, 127.66(CO CH CH), 130.67-163.24(ArC), 188.749(C O). ESI-TOF (m/z): 355.15, 251.12, 161.23, 147.22.

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CH3

O

H3C

CH3

O N

HN

O

O CH3

O

Fig. 5. 4-Methyl-1-propyl-1 H pyrrollo [2,3-c]pyridine-5,7(4 H 6H)-dione (5). Fig. 2. (2 E)-3–(4 ethoxy phenyl) -1-(4-methoxyphenyl) prop -2-en-1-one (2).

O H3C

HO

N

CH3 O O

O

N H HO

Fig. 6. 3,4-dihydro [1,4]oxazino [4,3,b] [1,2]benxoxazol-1 (10b H)-one (6) amorphous powder.

O

Fig. 3. 8-hydroxy-1,1-dimethyl-3-oxo-1,3,4,5-tetrahydro-2,4 carboxylic acid (3).

benzoxipine-5-

H3C H3C

N

O

O

O

CH3 Fig. 4. 7-(dimethylamino)-4-methyl-2-H-chrome-en-2-one. (4).

5.2.3. 8-hydroxy-1,1-dimethyl-3-oxo-1,3,4,5-tetrahydro-2,4benzoxazepine-5-carboxylic acid (compound 3) Colourless solid; (Fig. 3); M.P. 174.38; FT-IR (KBr) vmax /cm−1 : 3460(OH), 1695(C O). 1 H NMR (CDCl3 , 400 MHz, ␦ ppm): 1.38(s, CH3 ), 5.12(d, CH), 6.51(s, Ar-OH), 7.31-7.42(m, Ar-H), 7.52(d, N-H), 12.72(s, COOH). 13 C NMR (DMSO6 , 100 MHz, ␦ ppm): 28.08(CH3 ), 57.50(C5 ), 78.27(C1 ), 127.61-137.34(ArC), 155.75(COOH), 172.15(C O). ESI-TOF (m/z): 355.15, 251.12, 161.23, 147.22. ESI-TOF (m/z): 251.15, 225, 150, 107.

5.2.4. 7-(diethylamino)-4-methyl-2H-chromen-2-one (compound 4) Yellowish amorphous powder; (Fig. 4); M.P. 325.2; FT-IR (KBr) vmax /cm−1 : 1725(C O), 1340(C N). 1 H NMR (CDCl3 , 400 MHz, ␦ ppm): 1.38(m, CH3 ), 3.41(m, CH2 ), 5.91(s, C3 ), 6.48-7.36(m, Ar-H). 13 C NMR (DMSO6 , 100 MHz, ␦ ppm): 12.47, 18.37(CH3 ), 44.70(CH2 ), 109.01-125.57(ArC), 156.0(C3 ), 162.02(C O). ESI-TOF (m/z): 231.12, 202, 190.

5.2.5. 4-methyl-1-propyl-1H-pyrrolo[2,3-c]pyridine-5,7(4H,6H)-dione (compound 5) Colourless solid; (Fig. 5); M.P. 279.5; FT-IR (KBr) vmax /cm−1 : 3364(NH), 1689(C O), 1340(C N). 1 H NMR (CDCl3 , 400 MHz, ␦ ppm): 0.87(m, CH3 ), 1.71(t, CH2 ), 1.81(s, HC C O), 4.16(d, C4 ,), 8.23(d, C2 , C3 ), 11.12(s, NH). 13 C NMR (DMSO6 , 100 MHz, ␦ ppm): 10.41, 23.41(CH3 , CH2 ), 28.36(N CH2 ), 76.00(C2 ), 109.12(C3 ), 142.33(C1 ), 154.65(C O). ESI-TOF (m/z): 206.15. 5.2.6. 3,4-dihydro[1,4]oxazino[4,3-b][1,2]benzoxazol-1(10bH)-one (compound 6) Yellow solid; (Fig. 6); mp 242.30 C; IR (vmax/cm-1 ): 2925(CHstr), 1735(C O), 1611(C C); 1H NMR (500 MHz, DMSO6, ␦): 1.4 (t, 2H, H-3), 4.5 (t, 2H, H-4), 1.5 (s, 1H, H-7), 7.6 (d, 1H, H-10), 7.66(dd, 1H, H-11), 7.4(dd, 1H, H-12), 8.10(d, 1H, H-13). 13CNMR (125 MHz, DMSO6,␦): 60.1(CH2, C3), 62.3(CH2, C4), 110.0(CH, ArCH10), 119.9(C,C8), 123.1(CH, ArCH13), 125.0(CH, ArCH12), 130.4(CH, ArCH11), 133(150.3(CH, C7), (160.1(C O, C6), 164.3(=C O, C9). m/z = 191.18(C10H9NO3); 147, 119, 70, 45 (Fig. 6). 5.2.7. Acute toxicity studies SCA extract and SCE extract until 5000 mg/kg, per oral, did not produce mortality, nor macroscopic tissue injury or weight loss during the observation period for14 days. Lethal effects were not observed at any of the administered doses, the oral LD50 of the ethanol extract estimated in mice is > 5000 mg/kg (Table 2). 5.2.8. Anthelmintic efficacy All results were shown in Table 3 and expressed as a mean ± SEM of six worms in each group. All the data are expressed as mean ± SEM. The values obtained were compared with control group using one-way ANOVA and by Dunnett’s test. LC50 values of SCA and SCE extracts and compounds 3, 4, 6 are shown in Table 4.

Table 2 Effect of SCE extract and SAE extract on the determination of acute lethal dose 50 (LD50 ) per os. 1st part of investigation

2nd part of investigation

Doses (mg/kg)

Mortality

Doses (mg/kg)

100 500 750

0/3 0/3 0/3

1250 2500 5000

Animals showing mortality in each group 1

2

3

5

Total

0/1 0/1 0/1 LD50 > 5000

0/2 0/2 0/2

0/3 0/3 0/3

0/5 0/5 0/5

0/11 0/11 0/11

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Table 3 Results of anthelmintic activity of aqueous (SCAE), ethanol (SCEE) extracts and of S. calva DC and compounds 3, 4, 6. Sample/Groups

Control (normal saline) Albendazole Albendazole Albendazole Albendazole Albendazole SCAE SCAE SCAE SCAE SCAE SCEE SCEE SCEE SCEE SCEE Comp. 3 Comp. 3 Comp. 3 Comp. 3 Comp. 3 Comp. 4 Comp. 4 Comp. 4 Comp. 4 Comp. 4 Comp. 6 Comp. 6 Comp. 6 Comp. 6 Comp. 6

Conc. mg/mL

– 1 2 4 8 16 3.13 6.25 12.5 25 50 3.13 6.25 12.5 25 50 1 2 4 8 16 1 2 4 8 16 1 2 4 8 16

Pheretima posthuma (Earthworm)

Ascaridia galli (Roundworm)

Paralytic time (min)

Death time (min)

Paralytic time (min)

Death time (min)

– 148 ± 0.56 75 ± 0.93 38 ± 0.57 20 ± 0.45 10 ± 0.20 224 ± 0.42 123 ± 0.56 62 ± 0.45 32 ± 0.74 17 ± 0.42 170 ± 0.45 87 ± 0.46 45 ± 0.46 23 ± 0.15 12 ± 0.36 320 ± 0.52 162 ± 0.42 82 ± 0.56 42 ± 0.42 22 ± 0.78 253 ± 0.42 128 ± 0.75 65 ± 0.24 34 ± 0.83 18 ± 0. 67 152 ± 0.76 78 ± 0.86 40 ± 0. 86 21 ± 0.63 11 ± 0.74

– 222 ± 0.45 112 ± 0.54 56 ± 0.56 29 ± 0.20 16 ± 0.45 345 ± 0.46 175 ± 0.76 90 ± 0.65 47 ± 0.64 24 ± 0.56 280 ± 0.64 142 ± 0.56 72 ± 0.48 37 ± 0.18 19 ± 0.32 381 ± 0.86 192 ± 0.48 98 ± 0. 48 57 ± 0.68 30 ± 0. 86 354 ± 0.86 180 ± 0. 86 92 ± 0.74 48 ± 0.87 25 ± 0. 86 224 ± 0.82 114 ± 0.92 58 ± 0.74 30 ± 0. 86 17 ± 0.92

– 79 ± 0.90 40 ± 0.20 20 ± 0.24 10 ± 0.64 6 ± 0.86 167 ± 0.98 85 ± 0.86 43 ± 0.76 21 ± 0.89 12 ± 0.58 110 ± 0.62 56 ± 0.86 29 ± 0.43 15 ± 0.98 8 ± 0.56 220 ± 0.54 112 ± 0.86 57 ± 0. 64 30 ± 0. 86 16 ± 0.54 180 ± 0.54 92 ± 0.64 48 ± 0.86 26 ± 0.84 14 ± 0.82 82 ± 0.93 42 ± 0.74 22 ± 0.72 12 ± 0.75 7 ± 0.74

– 162 ± 0.20 82 ± 0.65 42 ± 0.72 22 ± 0.74 10 ± 0.56 337 ± 0.34 170 ± 0.74 85 ± 0.42 43 ± 0.93 21 ± 0.86 202 ± 0.64 102 ± 034. 52 ± 0.94 27 ± 0.92 15 ± 0.54 341 ± 0.38 172 ± 0.97 88 ± 0. 86 45 ± 0.96 24 ± 0.88 261 ± 0.64 134 ± 0.45 68 ± 0.54 35 ± 0.98 19 ± 0.56 166 ± 0.68 85 ± 0.82 45 ± 0.83 24 ± 0.82 13 ± 0.82

Each value represents mean ± SEM (n = 6). P < 0.001 significantly different compared with reference compound, Albendazole, Student’s t test.

5.2.9. Molecular docking study Molecular docking study of compound 6 with the enzyme ␤tubulin was performed to understand the drug-receptor interaction at the molecular level. On drug discovery perspective, ␤-tubulin can be considered as the effective target of anthelmintic candidates [25,26]. The molecular docking study emphasized that the bioactive 3, 4-dihydro oxazino [4,3-b][1,2]benzoxazol-1(10bH)one occupied in the active site of the enzyme. The docking pose was established by Molegrow 2012.2.5.0 version which can visualize hydrogen bonding, electrostatic and hydrophobic interaction of the ligand molecule. It has been noted that three significant hydrogen-bonding binding appeared during the docking calculation between the isolate and the inhibitor-binding cavity of the ␤-tubulin (Fig. 7). In order to establish the detailed structure activity relationship, it is logical to divide the structure of the compound 6 into two fragments namely:

The docking suggested that the lactone ring attained a noncoplanar conformation with benzisoxazole that can contribute two significant hydrogen bonding interaction with Tyr 50 &Gln 134. The nitrogen atom of the benzisoxazole showed another hydrogen bonding interaction with Thr 237. A crucial finding was noted that a significant ␲-␲ interaction of phenyl system of Phe 200 and aromatic nucleus of benzisoxazole. The molecular docking study also revealed a non-polar interaction between methylene unit of lactone system and methyl group of Leu 250 also contribute an effective interaction of 3, 4-dihydro [1,4]oxazino[4,3-b][1,2]benzoxazol1(10bH)-one towards the catalytic site of the enzyme. The significant activity of compound 6 could be attributed to the slight structural resemblance with standard drug. Both Albendazole

• bicyclic 3-hydro benzisoxaole; • ethyl lactone.

Table 4 LC50 values of anthelmintic activity of aqueous (SCAE),ethanol extracts SCEE of S. calva DC and compounds 3, 4, 6. Sample/Groups

Pheretima posthuma (Earthworm) LC50

Ascaridia galli (Roundworm) LC50

Albendazole SCAE SCEE Comp. 3 Comp. 4 Comp. 6

9.77 36.02 33.62 12.00 11.61 9.90

5.35 35.33 29.22 11.47 10.56 8.35

Fig. 7. Molecular interaction of the compound 6 towards the active site of ␤-tubulin.

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Fig. 8. Toxicity prediction of compound 6.

and compound 6 have benzene ring fused with five membered heterocyclic ring which contains two heteroatoms but differs in one heteroatom and side attachment. The assay was performed by using adult earthworms and round worms in vitro because these have high resemblance, both anatomically and physiologically, with the intestinal roundworm parasite Ascaris lumbricoides of Humans. 5.2.10. Toxicity prediction for compound 6 The green and red signal indicates the safe and risk of molecule towards that particular system. In the current study, compound 6 showed a high risk in the reproductive system. It is highlighted that a fragment of (C6 H5 )ON from the molecule can produce this toxicity issues. The details of the toxicity analysis are shown in (Fig. 8). 6. Discussion The S. calva aqueous, ethanol extract and three isolated compounds were screened for in vitro anthelmintic activity against P. posthuma and A. galli, owing to its anatomy and physiology resemblance with the intestinal roundworm parasites of human beings for preliminary evaluation of anthelmintic activity [27]. It is evident from Table 1 extracts and isolated compounds showed significant anthelmintic activity (P < 0.001) in a dose dependent manner in tested worms. The ethanol extract was more effective in causing death of worms at all concentrations than aqueous extract at 99.99% significant level, and also effective (P < 0.001) in causing paralysis and death. The data reported here indicates that compound 3, 4 and 6 are potentially active anthelmintic compounds. The results indicate the active constituents were mainly contained in ethanol extract. Based on the results we isolated and characterized 6 phytochemicals from ethanol extract. Among that 8-hydroxy-1,1dimethyl-3-oxo-1,3,4,5-tetrahydro-2,4 benzoxipine-5-carboxylic acid (compound 3), 7-(dimethylamino)-4-methyl-2-H-chromeen-2-one (compound 4) and 3,4-dihydro [1,4]oxazino [4,3,b] [1,2]benxoxazol-1 (10b H)-one (compound 6) showed significant anthelmintic activity. Compound 6 showed strong anthelmintic efficacy against tested worms with LC 50 values of 9.90 and 8.35 against the tested worms. The results obtained for the compounds were very promising and have provided scientific evidence for the development of this plant as an alternative anthelmintic agent. In addition, compounds 3, 4 and 6 showed a much higher activity when compared with the ethanol extract. Our results indicate these alkaloids are responsible for anthelmintic activity of SCE extract. From this study it may be presumed that in vitro, anthelmintic activity shows the paralyzed worms are easily expelled from the host gut by peristaltic movements [28]. Apparently, the active principles of the ethanol extract in particular, is effective against the tested worms the precise mode of its action needs to be investigated further for development of potential herbal anthelmintic agent. More active 3,4-dihydro [1,4]oxazino [4,3,b] [1,2]benxoxazol-1 (10b H)-one (compound 6) molecular docking studies were carried out and it showed minimum binding energy and increased affinity with the protein and it was found that hydrogen bond formation with amino acid residues of active pocket may be responsible for

the anthelmintic activity as referred to Albendazole. A preclinical evaluation of the compound 6 was done by suitable computational tool and suggested the toxicity issue associate with reproductive system. By suitable molecular modification this problem can be overcome in future. Molecular docking study gave clear insight of the structural recognition of the molecule (compound 6) towards the inhibitor-binding cavity of ␤-tubulin. The structural similarity of the molecule with the standard drug (Albendazole) also helped to prove its anthelmintic property. The appearance of a non coplanar conformation of lactone ring is key feature of the compound 6 at its molecular interaction level with receptor site. However, based on the results it is not appropriate to arrive at the conclusion of structure activity aspects of the moiety and further evaluation is necessary for their clinical use. The computational study gave a new insight of compound 6. 7. Conclusion In conclusion, aerial parts of S. calva ethanol extract was found to be effective when tested against the P. posthuma and A. galli. The present study also revealed that among the isolated compounds – 3,4-dihydro [1,4]oxazino [4,3,b] [1,2]benxoxazol-1 (10b H)-one (compound 6) showed strong activity against tested worms. This compound can be chosen as lead compound for the development of new drugs for the treatment of anthelmintic infection in animals and humans; however, more investigations including preclinical pharmacological evaluations before clinical trials, evaluation of toxicity and their mechanism of action must be done. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement Authors are thankful to the management of Novel Nutrients Pvt Ltd Banglore, India and this research was supported by Novel Nutrients Pvt Ltd Grant Scheme 2010 (NN 101). References [1] Jayaraj P, Govindarajan R, Pushpangadan P. The genus spilanthes ethnopharmacology, phytochemistry, and pharmacological properties: a review. Adv Pharm Sci 2013, http://dx.doi.org/10.1155/2013/510298 [Article ID 510298]. [2] Ignacimuthu S, Ayyanar M, Sankarasivaraman K. Ethnobotanical study of medicinal plants used by Paliyar tribals in Theni district of Tamil Nadu, India. Fitoterapia 2008;79(7–8):562–8. [3] Patil HM, Bhaskar VV. Medicinal knowledge system of tribals of Nandurbar District, Maharashtra. Indian. Indian J Tradit Know 2006;5:327–30. [4] Ganesan S, Suresh N, L. Kesaven L. Ethnomedicinal survey of lower Plani hills of Tamilnadu. Indian Journal of Traditional Knowledge 2004;3:299–304. [5] Sekendar Ali MD, Razibul Habib MD, Rafikul Islam MD, Saiful Islam MD, Mominur Rahman MD, Hasan N. Analgesic and antioxidant activities of the methanolic extract of Spilanthes calva (d.c) leaves in male rats. Global J Pharmacol 2012;6:12–8. [6] Krishnaswamy NR, Prasanna S, Seshandri TR, Vedantham TNC. ␣&␤-Amyrin esters and sitosterol glucoside from Spilanthes acmella. Phtytochemistry 1975;14:1666–7. [7] Ramsewak RS, Erickson AJ, Nair MG. Bioactive N-isobutylamides from the flower buds of Spilanthes acmella. Phytochemistry 1999;51:729–32. [8] Lemke TL, Williams DA, Roche VF, Zito SW. Foye’s principles of medicinal chemistry. New Delhi: Wolters Kluter; 2013. p. 24–60. [9] Nweze NE, Ogidi A, Ngongeh LA. Anthelmintic potential of three plants used in Nigerian ethnoveterinary medicine. Pharm Biol 2013;51:311–5. [10] Niezen JH, Robertson HA, Waghorn GC, Charleston WA. Production, faecal egg counts and worm burdens of ewe lambs which grazed six contrasting forages. Vet Parasitol 1998;80:15–27. [11] Guarrera MP. Traditional anthelmintic, antiparasitic and repellent uses of plants in central Italy. J Ethnopharmacol 1999;68:183–92. [12] Wang GX, Zhou Z, Jiang DX, Han J, Wang JF, Zhao LW, et al. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim)

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Please cite this article in press as: Jayaraj P, et al. Isolation of chemical constituents from Spilanthes calva DC: Toxicity, anthelmintic efficacy and in silico studies. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.04.002