Chapter 3 Materials & Methods
3.MATERIALS AND METHODS
3.1.Study Plan
The plants were selected on the basis of their traditional medicinal importance, these were Annona squamosa, Murraya koenigii, Withania coagulans and Withania somnifera chemotype WS-101R. The immunomodulatory activities in the extracts of these plants were investigated by studying their effect on humoral and cellular immune responses of mice. Once the immunomodulatory activity was detected in the crude extract, efforts were further made to carry out bioactivity guided fractionation and isolation of pure molecule/s possessing immunomodulatory action.
The identified extract/s fraction/s and pure molecule/s were further investigated for their immunoprophylactic and chemotherapy adjunct efficacy against an experimental infection of human lymphatic filarial parasite, Brugia malayi in rodent host, Mastomys coucha.
3.2.Laboratory Chemicals
Medium RPMI-1640 (Sigma, USA) with or without phenol red was used in all cell culture experiments. The medium was fortified with 1% antibiotic-antimycotic cocktail (Sigma, USA) and 10% Foetal bovine serum (GIBCO, USA). Minimum essential medium (MEM; Sigma, USA) was used for in vitro cyto-toxicity studies. Concanavalin A (Con A), Lipopolysaccharide (LPS), HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], Brefeldin-A, Phosphate buffer saline (PBS) tablets, DCF-DA (2'-7'-dichlorofluorescein diacetate), Griess reagent, Dimethyl Sulfoxide (DMSO), TMB (3, 3', 5, 5'-tetramethylbenzidine), Cocktail O, and Alamar blue or Resazurin and Ortho-phenyl-diamine (OPD) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Carboxy methyl cellulose/ Gum acacia was purchased from HiMedia, Mumbai, India. Picroliv was obtained from Medicinal and Process Chemistry Division of CDRI, Lucknow, India. The standard antifilarial drugs Ivermectin and DEC were purchased from (Sigma, USA). The fluorochrome [Fluorescein isothiocyanate (FITC) or Phycoerythrin (PE)] conjugated monoclonal antibodies to T and B cell surface antigens (CD) as well as to various cytokines were purchased from Becton and Dickinson (BD, San Diego, CA, USA). The fixing and permeabilization solutions (Leucoperm A and B) were purchased from Serotec, UK while the remaining chemicals used were of analytical grade available locally. [3H]-thymidine (3H-Tdr, specific activity 18Ci/m mole) was procured from Bhabha Atomic Research Centre (BARC), Mumbai, Maharashtra, India and the glass fibre filters (GF/C grade) from Whatman, England, UK for cell proliferation assay. Yeast extract tablets (Magrita Laboratories Pvt. Ltd., Lucknow, India) and Dog bix (Tetragon Chemie Pvt. Ltd., Bangalore, India) were purchased locally.
3.3.Animal Models
3.3.1.BALB/C MOUSE
BALB/c albino mouse (derived its name ‘B’ from Halsey J. Bagg, and ‘ALB’ from albino, however the letter ‘/c’ was added by George D. Snell to denote its white/ albino colour). It is an inbred, immunocompetent host and an animal model of choice for studies in many disciplines including immunological assays. BALB/c is very small and active animal having an average weight of 20 g and represents a good breeding performance with long reproductive life-span. The females have gestation period of 20-21 days with an average litter size of 4 babies (Potter, 1985; Hansen et al., 1973).
3.3.2.Mastomys coucha
Mastomys coucha (GRA: Giessen strain) (family-Muridae) is a multi-mammate, prolific breeder, straw coloured rodent with red eyes, originally brought from Giessen, Germany in 1974 through the courtesy of Late Prof. G. Lammler (Institute for Parasitology, Giessen University) and since then is being maintained in the Animal House of CDRI. The female may have her young at any time of the year and if conditions favored, may do so regularly at intervals of 33 days. The litter usually consists of 8-14 babies and the gestation period is around 21 days. It is highly susceptible to B. malayi infection. This animal model is being in wide use by scientific community for experimental research on chemotherapy and immunology of filariasis (Petranyi et al., 1975; Singh et al., 2009).
3.3.3.Meriones unguiculatus
Another rodent, commonly known as Mongolian jird/ gerbil (Meriones unguiculatus, family - Gerbillinae) with agouti or mixed brown body colour, has proven to be an excellent permissive rodent model for the study of lymphatic filariasis using B. pahangi or B. malayi. The animal is being routinely bred at National Laboratory Animal Centre at CDRI. It has small litter size and is used for the propagation of B. malayi parasites (McCall et al., 1973) at our Institute.
All the animals were housed under standard conditions of temperature (23 ± 1 °C), relative humidity (55 ± 10 %), and 12/12 h light/dark cycles at CDRI Animal House and fed with standard pellet diet (initially developed for Syrian hamsters (Schuster et al., 1973), substituted with proteins and fats) and water ad libitum. The studies were carried out in accordance with the current guidelines for the care of laboratory animals. Animal handling and experimental protocols including the number of animals employed in this thesis work were duly approved by our Institutional Animal Ethics Committee (IAEC) which assigns IAEC approval numbers to each study.
3.4.Collection of Plant Material
The leaves of Murraya koenigii (MK) and fruits of Withania coagulans (WC) were collected in the month of March, 2008 while twigs of Annona squamosa (AS) were collected in March, 2009 by Botany Division of CDRI from Lucknow, India, and identified by Dr. A. K. Mangal, Central Council for Research in Ayurveda and Siddha. Voucher specimens of the above three plants (4763 for MK, 4554 for WC and 4738 for AS) have been preserved in the investigators Laboratory. The plant material was dried and supplied to Medicinal and Process Chemistry Division of CDRI where it was extracted and fractionated based on its bioactivity.
The fourth plant Withania somnifera (WS) was chemotype WS-101R. A set of discrete chemotypes of Ashwagandha (Withania somnifera) were developed at Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP, Lucknow, India) as a consequence of large scale phytochemical screening of accessions of the plant collected from across almost all major wild habitats of the country followed by genetic improvement of the selected lines in the identified core collection (Chaurasiya et al., 2009). Of these, three chemotype varieties were found to possess immunostimulant activity in preliminary testing and one of these varieties i.e. WS 101R was selected for detailed investigations. This chemotype was raised at the experimental farm of CIMAP at Lucknow, India, following standard agronomic practices. Roots of the 5 months old plants were harvested for the preparation of aqueous-ethanol extract.
3.4.1.Extraction and Fractionation of Annona squamosa
The extraction and fractionation of Annona squamosa was carried out by Phytochemists in the Medicinal and Process Chemistry division of CSIR-CDRI, Lucknow. In brief, the plant material (6.0 Kg, wet weight) was shade dried, finely powdered, placed in percolator with ethanol (18 L) and allowed to stand at room temperature (RT) for forty eight hours. The percolate was collected. This process of extraction was repeated for four times, till the plant material was extracted exhaustively. The total percolate was collected, filtered and concentrated under vacuum using rota-vapor at 40-45 °C which yielded 520 g (8.66%) ethanolic extract. The ethanolic extract was evaluated for its immunomodulatory activities (as described below) at various log doses in BALB/c mice. Based on the bioactivity profile, the ethanolic extract was further fractionated by the chemists to yield four fractions viz. Hexane, chloroform, n-butanol and aqueous fractions. The ethanolic extract (500g) was triturated with n-hexane (250 ml x 15) and the hexane soluble fraction (F1) was then concentrated (100 g) under reduced pressure at 40 °C. Residue obtained after triturating with hexane was further triturated with chloroform (250 ml x 15) and chloroform soluble fraction (F2) was concentrated (95 g) under reduced pressure. Residue obtained after triturating with chloroform was then suspended in distilled water (2 Litres) and then fractionated with n-butanol saturated with water (500 ml X 10). n-butanol soluble fraction (F3) was concentrated (136 g) under vacuum at 50 °C. Water soluble fraction (F4) was also concentrated (168 g) under vacuum at 45-50 °C. These fractions were again subjected to bioactivity evaluation (immunomodulation) which demonstrated the localization of best immunomodulatory activity in the chloroform fraction. The active chloroform fraction was further followed for purification by chromatography to obtain 12 major pure molecules which were characterized by spectral analysis using NMR, 13C and Mass. In brief, the chloroform fraction (80.0 g) was subjected to repeated column chromatography over silica gel (60-120 mesh) and eluted with gradient solvents using method as described earlier (Yadav et al., 2011). F2 fraction thus yielded twelve known pure compounds which were identified by comparing their spectroscopic data with those previously reported in literature.
Fraction F2 was eluted with a gradient of solvents hexane-ethyl acetate (95:05) to ethyl acetate–methanol (95:05), fifty five fractions were collected (500 ml each) and their composition was monitored by TLC, those fractions showing similar TLC profiles were grouped into nine major fractions (SF1–SF9). Successive flash chromatography of fraction SF1 using hexane-EtOAc as binary mixture of increasing polarity yielded compound 1 (200 mg), as light brown powder. Flash column chromatography over neutral alumina of SF2 using hexane-EtOAc as binary mixture of increasing polarity yielded compound 2 (21 mg). Again CC (column chromatography) of fraction SF3 using hexane-EtOAc as binary mixture yielded compound 3 (6.0 mg). Compound 4 (180 mg) was obtained as yellow amorphous powder from flash chromatography of fraction SF4 using hexane-EtOAc eluate. Flash chromatography of fraction SF5 by elution with hexane-EtOAc as binary mixture of increasing polarity yielded compound 5 (19 mg), which was again purified by loading over neutral alumina using chloroform, and hexane (90:10) as eluent. Successive flash chromatography of fraction SF6 using hexane-EtOAc as binary mixture of increasing polarity yielded compound 6 (80 mg). Again flash chromatography of fraction SF7 using hexane, EtOAc as binary mixture of increasing polarity, yielded mixture of two compounds so these were again purified on neutral alumina using chloroform as eluent, and this yielded compound 7 (23 mg) and chloroform, and methanol (95:5) as eluent yielded compound 8 (24 mg). Compound 9 (200 mg) was crystallized in ethanol, from the residue of fraction SF7. Fraction SF8 was subjected to dianion HP20 resin then eluted with methanol–water (40:60) to methanol: water (70:30) yielded two compounds 10 (73 mg) and 11 (152 mg). Fraction SF9 was purified over HP-20 resin then reverse chromatographed using gradient of water: methanol (8:2) resulted in compound 12 (2.0 g).
Optical rotations were measured on a Perkin-Elmer model 241 digital polari-meter. UV spectra were obtained on a Perkin-Elmer λ-15 UV spectrophotometer. IR spectra were recorded on a Perkin-Elmer RX-1 spectrophotometer using KBr pellets. 1 H and 13 C NMR spectra were recorded on a Bruker DRX 300MHz NMR spectrometer. ESMS on an Advantage Max LCQ Thermo-Finnigan mass spectrometer and FABMS were carried out on a JEOL SX 102/DA-6000 mass spectrometer. CC was performed using silica gel (230–400 mesh). TLC was carried out on pre-coated silica gel plates 60 F254 or RP-18 F254 plates (Merck). Spots were visualized by UV light or by spraying with H2SO4–MeOH or Dragondorff's reagent. All the compounds were known and already reported in the literature. However, of these 12, only five were supplied for detection of immunomodulatory activity based on their availability with the Phytochemists. Therefore, immunomodulatory activity of only five available compounds was undertaken.
3.4.2.Extraction of MK and WC
The air-dried and powdered MK leaves (500 g) and WC fruits (500 g) were extracted exhaustively with 95% ethanol (4 x 1L) at RT. Each extraction was carried out for 16 h. The combined percolates were filtered and solvent was evaporated under vacuum using rota-vapour at 45 °C which afforded ethanolic extract (55 g, 11% for MK and 60 g, 12% for WC). Since the crude extracts derived from MK and WC did not demonstrate profound immunomodulatory properties, the crude extracts were neither fractionated nor investigated for immunoprophylactic or chemotherapy adjunct studies.
3.4.3.Extraction of WS-101R chemotype of WS and isolation of pure withanolideS
The WS-101R chemotype extract was prepared by extraction of the liquid nitrogen powdered fresh root tissue in two volumes (g/ml) of aqueous-ethanol (75:25 v/v) for 24 h with occasional shaking followed by filtration through two layers of muslin cloth and re-filtration of the filtrate through a filter paper. The filtrate was saved and residue was re-extracted two more times as above. The three filtrates were pooled, concentrated in a flask evaporator at 50 °C followed by complete dryness in a freeze dryer. The lyophilized extract was weighed and analyzed by TLC and HPLC and evaluated for immunomodulatory efficacy in BALB/c mice. The yield of extract on fresh weight basis was 4.28% and 16.0% on dry weight basis. Since the crude extract possessed promising immunostimulatory efficacy, it was further fractionated and several pure compounds were isolated (Chaurasiya et al., 2009). However, of these, only three viz. Withanone, Withanolide A, and Withaferin A were supplied in sufficient quantity based on their availability with the Phytochemists. Therefore, immunomodulatory activity of only these three available compounds was undertaken. Since Withaferin A possessed the best immunostimulatory activity and its yield was also higher than other withanolides, it was further investigated for immunoprophylactic and chemotherapy adjunct efficacy against filarial infection of B. malayi. The compounds were isolated by Silica-gel column chromatography and crystallization cycles, essentially as described earlier (Chaurasiya et al., 2011). This whole extraction exercise was carried out at CSIR-CIMAP, Lucknow, India.
3.5.Host-Parasite system
Brugia malayi, a sub-periodic strain of human lymphatic filarial parasite was used in the present study to evaluate the immunoprophylactic and chemotherapy adjunct activities of the extracts, fractions or compounds demonstrating promising immunostimulatory activity. The infection can be successfully transmitted to various vertebrate hosts including variety of rodents, cats and monkeys. The infection is experimentally transmitted through black eyed susceptible strain of Aedes aegypti mosquitoes developed by McDonald (Liverpool School of Tropical Medicine and Hygiene, U.K.) as described below:
3.5.1.Experimental maintenance of B. malayi
3.5.1.1. Rearing and breeding of mosquito vector (Aedes aegypti)
Rearing and breeding of mosquito vector (Figure: 3.1) was carried out in the temperature (271 C) and humidity (755%) controlled insectarium. For maintaining the life cycle, stored mosquitoes eggs were transferred to wide enamel bowls containing stored tap water. The eggs hatched into first stage larvae (L1) within 24-72 h. Yeast extract tablets and dog biscuits (Dog bix) were made into fine powder separately in a mixer grinder. The two were mixed in 3:1 ratio and used as a feed for growing mosquito larvae. After 5-6 days, the hatched larvae transform into pupae, the non-feeding stage which were separated from the larvae with the help of locally fabricated wide mouth glass pipette and transferred to small crystallizing glass dishes containing water to be kept inside the nylon netted mosquito cages fabricated locally. Young mosquitoes emerge from the pupae within 24 to 48 h. Small Petri dishes containing cotton soaked in 3% glucose solution covered with a moist filter paper disc of the size of Petri dish were kept inside each mosquito cage for feeding and a small beaker containing water was kept for females to lay eggs.
3.5.1.2. Transmission of infection to vector
Within 4-5 days of their emergence from pupae, young mosquitoes were fed on B. malayi infected donor mastomys (microfilaria density 100-200 Mf/10 l of tail blood) between 12.00 and 12.45 h, the time when maximum number of Mf appear into peripheral blood circulation. The donor animal was kept in small cylindrical wire netting cage with lid which can be closed. Each lid carries a small hole through which the tail of animal was kept out. The cage carrying donor was kept inside the mosquito cage. Before blood meal the mosquitoes were starved for approximately 3-4 h by removing glucose. Four to five day old mosquitoes were allowed to feed on donor mastomys for 20-30 minutes (Figure: 3.2). After completion of blood meal donor was taken out and left in the original animal cage. Within 9 1 day of blood meal, ingested Mf develop into infective larvae (L3) inside the mosquito after 2 successive molts. On day 8, 4-5 female infected mosquitoes were dissected to have an idea of the development of larvae and to assess the exact date of mosquito dissection for collection of B. malayi L3.
3.5.1.3. Collection of mosquito eggs
Engorged female mosquito generally starts laying eggs on the surface of water or on wet filter paper discs after 72 h of blood feeding. The eggs laid on water were picked up with fine brush along with the filter discs and washed gently with fresh water in a filter paper cone. Filters carrying eggs were left to dry and stored in desiccators to be used within 6 months.
3.5.1.4. Collection of infective larvae (L3)
L3 of B. malayi were recovered from laboratory bred vector mosquitoes (Aedes aegypti) fed on donor mastomys 9±1 days earlier. The mosquitoes were paralyzed by vigorous shaking of the mosquito cage. The dead mosquitoes, their broken wings and appendages were removed. The mosquitoes were crushed gently in sterile Ringer's solution and poured on to the funnel of Baerman's apparatus containing two layers of fine meshed muslin cloth. After 40 min, all the larvae wriggle to the lower end of rubber tubing just above the clamp which was slightly loosened to collect a small volume of Ringer containing most of the L3. After 5 min, small amount of Ringer was recollected to ensure complete larval recovery. Larvae were collected in glass cavity blocks and washed repeatedly under a dissecting microscope. Fully mature, actively motile L3 were counted and distributed in separate cavity blocks to infect healthy naïve rodent species (Figure: 3.2).
3.5.1.5. Experimental transmission of infection to rodent host
Six weeks old male mastomys weighing between 30 and 35 g were inoculated with 100 L3 subcutaneously in the back region (Petranyi et al., 1975) while eight week old male gerbils (Meriones unguiculatus) received 150 L3 intra-peritoneally (McCall et al., 1973) (Figure: 3.2).
3.5.1.6. Assessment of Parasitemia
10 µl blood was drawn by pricking the tail vein of mastomys between 12:00 and 12:45 h (time of peak microfilaraemia) first on day 90 after L3 inoculation and thereafter at monthly interval. The blood was spread into a thick smear on a clean glass slide with a needle, air dried for 24 h, dehemoglobinized by overlaying smear with tap water for 5 min and air dried. Dehemoglobinized smear was stained with 0.15% Leishmann stain for 20 min, washed with tap water, air dried and examined under the compound microscope (Nikon, Japan) for assessment of Mf density. Mf were counted from one end of the blood smear to the other end with the help of a Hand Tally counter. Based on the Mf densities in the blood, mastomys (80-150 Mf/10 µl tail blood) were selected as donors for infecting mosquito by allowing them to feed on their blood thus maintaining the cyclical transmission of B. malayi from animal to animal. Gerbils infected intraperitoneally were used for recovery of adult parasites and Mf for in vitro antifilarial evaluation and antigen preparation to investigate specific immune response of the host.