Isolation and in vitro antiamoebic activity of iridoids isolated from Kigelia pinnata

.


Introduction
Amoebiasis is an ubiquitous disease and fifty million cases of amoebic dysentery and liver abscess are reported each year around the world. 1 It is responsible for 100,000 deaths per year worldwide and is the second leading cause of death due to parasitic disease. 2 Nitroimidazole drugs such as metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] are presently the most effective antiamoebic medications (Figure 1).The drug is known to have common side effects; 3 It is mutagenic in bacteria and high doses in rodents can cause carcinoma. 4As a result of this and also taking into account the possibility of the development of resistant strains of the Entamoeba against metronidazole, a search for new effective amoebicidal agents is required in order to provide a new dimension in the therapy of amoebiasis.Medicinal plants are considered as an important source of potentially useful structures for the development of new chemotherapeutic agents.The first step towards this goal is the biological and phytochemical screening of plants extracts from traditional preparations used in popular medicine.][7][8][9][10][11] They require more thorough investigation in order to validate their activity and that may uncover new leads for amoebicidal drugs.
Kigelia pinnata (K.pinnata) is an African tree which is widely grown in tropics.All parts of the tree are used for a variety of purposes in all the regions where it grows. 12Its leaves and bark was used for treating diarrhea and dysentery. 13The fruits are used as dressing for ulcers, purgative and to increase the flow of milk in lactating women. 14Different fractions of the stem bark and fruits of K. pinnata had shown growth inhibitory effect on four melanoma cell lines and found potential antineoplastic agent. 15The methanolic extract of the plant was found to be a potent antibacterial and antifungal agent. 16In West Africa, the root is used as a vermifuge and as a treatment for haemorrhoids and rheumatism. 17Steriods, iridiods and coumarins have been isolated from the root bark 18 and flavonoids and iridoids from the fruit and leaves. 19ichloromethane extracts from the root and stem bark of K. pinnata containg naphthoquinones 20 showed anti-trypanosomal 21 and anti-microbial activity. 22Verminoside was isolated from root bark and showed promising anti-inflammatory activity. 23Govindachari and his colleages isolated kiglin and 6-methoxymellein together with two known compounds, stigmasterol and lapachol from the roots of this plant. 24They also isolated kigelin, β-sitosterol, 3-dimethylkigelin and ferulic acid from its bark. 25Dorothy et al. isolated five minor constituents from root bark of K. pinnata, which consisted of two known napthaquinones and three new aromatic monoterpenes. 26wo non-quinonoid aldehydes, norviburtinal and pinnatal were obtained from the root bark by Joshi et al. 27 Literature survey showed that no antiamoebic screening has been reported on this plant.
The present paper reports isolation and in vitro antiamoebic activity of three iridoids as well as extracts from stem bark of K. pinnata.Biologically monitored fractionation of the butanol extract from stem bark of K. pinnata led us to the isolation of three known iridoids; specioside, verminoside and minecoside.Specioside, verminoside and minecoside were isolated earlier from root bark of K. pinnata 18 and further characterized by El-Naggar et al 28 and Sticher et al 29 All the compounds were identified by comparing their UV, IR and 1 H NMR data with the literature values. 18,28,29In vitro antiamoebic activity of extracts and isolated compounds were carried out against HK-9 strain of E. histolytica by using micro dilution method.
Powered dried stem bark was defatted with hexane and exhaustively extracted from cold methanol.The extract was concentrated under vacuum and the residue was dissolved in water.The aqueous layer was extracted with butanol and ethyl acetate successively.The butanol extract was digested with chloroform and the residue was subjected to column chromatography which leads to the isolation of three major constituents.Three known iridoids, specioside (I), verminoside (II) and minecoside (III) (Figure 2) were isolated from biological monitored fractionation of the butanol extract.The structures of these compounds I, II and III were confirmed by comparing their spectroscopic data (UV, IR and 1 H NMR) with the literature values. 18,28,29xenic culture of HK-9 strains of E. histolytica was maintained in diamond's TYIS-33 medium. 30Heat inactivated bovine serum was used to give a more favorable growth.Metronidazole was used as the reference drug in all the biological experiments.All the plant extracts and compounds including metronidazole were dissolved in dimethylsulfoxide (DMSO).The maximum concentration of DMSO in the test did not exceed 0.1% at which level no inhibition of amoebal growth occurred. 31,32IC 50 (50% inhibition concentration) values of plant extracts and the isolated compound have been determined by using the microdilution method. 33he antiamoebic effect was compared with the most widely used antiamoebic medication metronidazole.The percentage inhibition values obtained was transformed into probit values and regression analysis was undertaken in order to find the best fitting straight line through the point obtained.At least three experiments were performed for each compound or plant extract tested.The in vitro antiamoebic screening of fractions of plant extract and compound isolated against HK-9 strain of E. histolytica are summarized in Table 1 and 2. The Metronidazole had a 50% inhibitory concentration (IC 50 ) of 0.33 mg/mL in our experiment which is close to the previously reported IC 50 of 0.32 mg/mL obtained against E. histolytica. 34When all the fractions of plant extract are tested against E. histolytica it is found that the butanol fractions of K. pinnata possessed good antiamoebic activity.The major iridoids constituents were present in the butanol extract of K. pinnata.These iridoids were highly active against amoeba and account for the high ARKAT potency of the butanol extract.Verminoside was found highly active (IC 50 0.19 µg/mL as compared to metronidazole IC 50 0.33 µg/mL) than the standard drug while specioside (IC 50 0.39 µg/mL) was as active as metronidazole.

Conclusions
The natural products have potential in the search for new and selective agents for the treatment of important tropical diseases caused by protozoa.In this study, K. pinnata was studied for its antiamoebic evaluation which is being used in traditional systems of medicine against dysentery and diarrhea.Three known iridoids specioside, verminoside and minecoside were isolated and screened in vitro against HK-9 strain of E. histolytica.Verminoside was found to be more active than the standard drug while specioside showed activity comparable to metronidazole.It is hoped that these studies will stimulate further efforts towards the development of new and urgently needed medications for the treatment of amoebiasis.Extraction and Isolation of Iridoids (I-III) from K. pinnata.The plant sample was cut into small pieces and air-dried in the shade for several days.A 2 kg air dried stem bark was defatted with hexane and percolated with methanol at room temperature and then extracted exhaustively with cold methanol.The methanol extract was concentrated under reduced pressure to yield 300 g residue.The residue was dissolved in 500 mL water and the aqueous layer was extracted with 6 x 100 mL 2-butanol and ethyl acetate.The extracts were concentrated under vacuo to yield 6.63g and 4.80g, respectively.The butanol fractions residue was digested with chloroform, filtered and subjected to column chromatography.It was eluted with dichloromethane, chloroform with increasing proportion of acetone.After elution with acetone, proportions of methanol were gradually increased as the eluting solvent until methanol was used.Each elute was examined on TLC using the solvent system chloroform-methanol-water (65:20:15) and fractions with the similar Rf values were combined.Many minor components were detected in elutes of chloroform-acetone.Large amounts of another three compounds were detected in acetonemethanol (9:1) elutes and these have been shown to be iridoids (I-III).In vitro testing against E. histolytica.Activity against E. histolytica (strain HK-9) in vitro was assessed using a microtiter plate method. 33DMSO (40 µL) 31,32 was added to sample of plant extracts (~10 mg) or compounds (~1 mg) followed by enough culture medium to obtain concentrations of 10 or 1 mg/mL.Samples were dissolved by mild sonication in a sonicator bath for a few minutes and then further diluted with medium to concentrations of 1.0 or 0.1 mg/mL.Two-fold serial dilutions were made in the wells of 96-well microtiter plate (Nunc) in 170 µL of medium.Each test included metronidazole as a standard amoebicidal drug, control wells (culture medium plus amoebae) was prepared from a confluent culture by pouring off the medium, adding 2 mL of medium and chilling the culture on ice to detach the organisms from the side of the flask.The number of amoeba per mL was estimated with a haemocytometer and trypan blue ARKAT exclusion was used to confirm viability.Fresh culture medium was added to dilute the suspension to 10 5 organism/mL, and 170 µL of this suspension was added to the test and control wells in the plate so that the wells were completely filled (total volume, 340 µL).An inoculum of 1.7 x 10 4 organisms/well was chosen so that confluent, but not excessive growth took place in the control wells.The plate was sealed with expanded polystyrene.Secured with tape, placed in a modular incubating chamber (flow laboratories, High Wycombe, UK), and gassed for 10 min with nitrogen before incubation at 37 °C for 72 h.Assessment of antiamoebic activity.After incubation, the growth of amoebae in the plate was checked with a low power microscope.The culture medium was removed by inverting the plate and shaking gently.The plate was then immediately washed once in sodium chloride solution (0.9 %) at 37 °C.This procedure was completed quickly, and the plate was not allowed to cool in order to prevent the detachment of amoebae.The plate was allowed to dry at room temperature, and the amoebae were fixed with methanol, when dry, stained with (0.5 %) aqueous eosin for 15 min.The stained plate was washed once with tap water and then twice with distilled water and allowed to dry.A 200 µL portion of 0.1 N sodium hydroxide solution was added to each well to dissolve the protein and release the dye.The optical density of the resulting solution in each well was determined at 490 nm with a microplate reader (Labsystem Multiskane Bichromatic, UK).The % inhibition of amoebal growth was calculated from the optical densities of the control and test wells and plotted against the logarithm of the dose of the drug tested.Linear regression analysis was used to determine the best-fitting straight line from which the IC 50 value was determined.

Table 1 .
In vitro antiamoebic activity of crude extracts from Kigelia pinnata stem bark against E. histolytica

Table 2 .
In vitro antiamoebic activity of isolated iridoids from Kigelia pinnata against E. histolytica The stem barks of Kigelia pinnata were collected from New Delhi, India.The plant was identified by a botanist at the Department of Botany, Chaudhary Charan Singh University, INDIA and specimens are deposited in the herbarium of the Chaudhary Charan Singh University.
Procedures.UV/Vis spectra were recorded in methanol on a Shimadzu UV-1601 PC UV/Visible spectrophotometer, IR spectra in KBr on a Perkin-Elmer model 1620 FT-IR spectrometer and 1 H NMR spectra on Bruker spectrospin DPX-300 MHz in CD 3 OD.Column