Drugs and chemicals
Analytical grade solvents (ethanol and petroleum ether), Cyclophosphamide (Alkem Laboratories Ltd. Mumbai), Levamisole (Khandelwal pharma. Ltd. Mumbai), Quercetin (Modern Industries, Sinner), Colloidal carbon (camel India Pvt. Ltd.), Nylon fibres (Local market, Nashik, India). Pasteurella multocida of bovine origin vaccine (Government Veterinary Hospital, Nashik).
Plant procurement and authentication
A. ficulneus L. herb was collected and procured in the month of July and leaves were dried in the shade. The A. ficulneus L. was authenticated by an herbarium section of a botanical survey of India, Maharashtra, Pune. No. BSI/WRC/Cert./2014.
Experimental animals
For the evaluation of immunomodulatory effect, adult healthy Swiss albino mice (Mus musculus, having 10–12 weeks old, 18–22 g body weight were used in this study, n = 6 in each group of models) either sex was used and housed at 25 ± 5 °C in a ventilated animal house under 12/12 h light/dark cycle. Animals were purchased from LACSMI Bio Farms (Laboratory Animal Centre for Safe Medical Innovations), # 12, “Rachana Blossom”, Jagdishnagar, Aundh, Pune-411 007, Maharashtra CPCSEA Registration Number: 1277/PO/RcBt/S/09. The all mice were maintained under standard conditions in an animal house approved as per the guidelines of CPCSEA, Department of Animal Welfare, and Government of India. All mice were fed with a pelleted diet and distilled water ad libitum. The experimental animal protocol was approved by the Institutional Animal Ethical Committee (IAEC) (Protocol No. 884/PO/Re/S/05/CPCSEA).
Preparation of leaves extracts
The dried leaves coarse powder (500 g) was extracted by using Soxhlet apparatus by petroleum ether (40–60 °C) to remove resinous and fatty material for 12 h. Then defatted marc was subjected through 95% ethanol for 24 h to obtain the ethanolic extract. For aqueous extract dried leaves, coarse powder macerated with chloroform and distilled powder (1:9) for 7 days. The aqueous extract of A. ficulneus L. (AEAF) and ethanolic extracts A. ficulneus L. (EEAF) were evaporated up to 30 °C temperature to yield a yellowish-brown powder. Both extracts were sealed in an airtight polythene or container and stored at 4 °C for further experimental work [8, 9].
Preliminary phytochemical investigations
Preliminary phytochemical analysis was carried out to check and identify the effective constituents of leaves extracts A. ficulneus L. investigated for as per the standard procedure reported practical pharmacognosy books [9, 10].
Total flavonoid content determination
For estimation of total flavonoid content (TFC), aluminium chloride method was used and quercetin is used as a standard. The TFC was determined from the calibration curve of quercetin and expressed as mg of QE/gm of dry weight. Method was repeated 3 times for precision and values were expressed in mean ± STD deviation in terms flavonoid content, quercetin equivalent per g of dry weight [11, 12].
Total phenol determination
Folin-Ciocalteu’s assay was used to determine total phenol content using gallic acid as a standard. Absorbance of sample was measured with the blank at 750 nm using a spectrophotometer. Method was repeated three times for precision and values were expressed in mean ± standard deviation in terms of phenol content, gallic acid equivalent, GAE per g of dry weight [13].
Thin layer chromatography
Active constituents (flavonoids) of A. ficulneus L. leaves extracts were detected by the method thin layer chromatography using silica gel 60 TLC plates. These phytochemicals were identified and compared with their retention time obtained, which is compared with the standard (quercetin) retention time for flavonoids. The volume of the spots applied on the chromatographic plates was 5 μl and TLC were performed by ethyl acetate:water:formic acid:glacial acetic acid (100:26:11:11) mobile phase for both extracts. The presence of flavonoid was detected by the formation of yellowish-brown colour on TLC plate by exposure of polyethylene glycol reagent on TLC plate. The chromatograms were evaluated without chemical treatment, under UV 254 and UV 365 nm light. Rf value was calculated by the formula, distance travelled by the sample and distance travelled by the solvent [14, 15].
HPTLC instrumentation and method
Chromatographic analysis carried out on plate size 10 × 10 cm HPTLC plates silica gel 60 F254 purchased from E. MERCK KGaA instrument (Germany). Samples of standards and extracts were applied as band length 6.0 mm wide and 8.0 mm apart by Camag Linomat 5 sample applicator. The application rate of sample on plate was 150 nl/s. Analysis was done at 380 nm in absorbance remission mode by win CATS Chromatography software [16].
Preparation of standard
A stock solution of quercetin (100 μg/ml) was prepared by dissolving 10 mg of accurately weighed quercetin in 100 ml ethanol. For calibration 5–20 μl standard solution were applied to HPTLC plate in the range 500–2000 ng per band.
Preparation of sample solution
Extraction of leaves of A. ficulneus L. were carried out by solubility of the marker constituents, ethanolic leaves extract prepared by weighing 50 g of dried powdered of leaves of A. ficulneus L. and extraction done by maceration method for 24 h. Solution was filtered, concentrated, evaporated through distillation and used for further HPTLC analysis.
Method validation
The analytical method was validated for specificity, linearity, limit of detection (LOD) and limit of quantization (LOQ) according to the International Council for Harmonization (ICH, 2005) guidelines. The specificity calculated by comparing Rf values and ultraviolet–visible (UV) spectra of peaks of components in sample and standard chromatogram.
Acute toxicity study
The oral acute toxicity study for both the extracts were carried out as per the OECD 420 guidelines in female mice at a single dose of 2000 mg/kg. As per the guidelines, mice were observed for 48 h for behavioural changes of toxicity and animals kept for 14 days under observation for any toxicological signs and lethal outcome [17].
Evaluation of immunomodulatory effect
The effect of both extracts (AEAF and EEAF) on immunomodulation the specific and nonspecific immune response was evaluated in mice using in vivo experimental animal models.
Specific immune response: humoral antibody response
Antigen: sheep red blood cells
In the specific immune responses, sheep red blood cells (SRBCs) are used as an antigen. Fresh blood was collected in freshly prepared sterile Alsevere’s solution in 1:1 proportion. Three times sheep blood was washed by centrifugation method at 3000–5000 rpm for 15 min. And after the final wash, red blood cells were suspended in normal saline and makeup final concentration of SRBCs up to 1 × 109 cells and used for immunization and challenge. One millilitre of the 2% suspension contained approximately 109 red blood cells.
Evaluation of humoral antibody response
For humoral immune response, mice grouped into total eight groups consisting of six mice (n = 6) in each group. Group 1 received (negative control) Cyclophosphamide (Cyp.100 mg/kg, p.o.), single-dose on 9th and 16th day; group 2 received (vehicle control ) distilled water (10 ml/kg, p.o.), for 21 days; group 3 (test extract 1) received AEAF (200 mg/kg p.o.) for 21 days; group 4 (test extract 2) received EEAF (200 mg/kg p.o.) for 21 days; group 5 (standard) received Levamisole (LMS 50 mg/kg, p.o.) for 21 days; group 6 received AEAF (200 mg/kg p.o.) for 21 days and a single dose of Cyp.100 mg/kg, p.o. each on 9th and 16th day; group 7 received EEAF (200 mg/kg p.o.) for 21 days and Cyp.100 mg/kg, p.o. single dose each on 9th and 16th day; group 8 received LMS (50 mg/kg, p.o.) for 21 days and Cyp.100 mg/kg, p.o. single dose each on 9th and 16th day. Mice from all groups were immunized by 0.1 ml intraperitoneal of suspension 1 × 109 cells SRBCs/ml on the 7th and 14th day considered for immunization. Blood samples were collected from all groups of mice by retro-orbital plexus on 14th day of study considered for primary antibody response and also blood samples withdrawn on the 21st day of study considered for secondary antibody response, after immunization. Blood was centrifuged and serum was isolated for determination of antibody levels by the haemagglutination technique by 96-wells U-shaped microtiter plates. Two-fold dilutions (0.025 ml) of serum samples were performed in the 96-wells microtiter plates with normal saline. In each well, 0.025 ml of 1% (V/V) SRBCs were added. The plates kept for incubation for 1 h at room temperature and then evaluated for haemagglutination. The highest dilution set out haemagglutination was considered for antibody titre; titre was expressed in a graded manner and the minimum dilution (1/2) being ranked as 1. The mean ranks of all groups were compared for statistical analysis [3, 18].
Cell-mediated immune response: delayed type hypersensitivity reaction
Cell-mediated immune response was measured using delayed type hypersensitivity (DTH) reaction by foot paw oedema method. Mice were divided into eight groups same as humoral antibody (HA) titre. On the 21st day of the study, all animals from each group were immunized with 0.1 ml of 1% SRBCs suspension in the sub plantar route of the right-hand paw. After 24 h, animals in all groups were assessed for foot paw oedema reaction, i.e. on the 22nd day. The oedematous thickness of the right footpad injected with SRBCs was determined by the vernier calliper method. The hypersensitivity reaction was expressed as the difference in the thickness (mm) between the right and left footpad injected with normal saline [3, 18, 19].
Non-specific immune response
Neutrophil adhesion test
Nonspecific immune response was assessed by neutrophil adhesion method. Mice are divided into four groups of six animals in each group. Group 1 control received distilled water, 10 ml/kg, p.o. for 14 days; group 2 and group 3 (test 1 and test 2 respectively) received AEAF 200 mg/kg and EEAF 200 mg/kg p.o. for 14 days. Group 4 standard received Levamisole 50 mg/kg for 14 days. On the 14th day of the study, blood samples were collected in pre-treated EDTA vials from all groups of mice by retro-orbital plexus and analyzed for differential leukocyte count (DLC) and total leucocyte count (TLC). After initial count analysis, all blood samples were incubated in nylon fibres 80 mg nylon fibres/ml for 15 min at 37 °C and again blood samples of incubated in nylon fibres were again analyzed for DLC and TLC respectively to give a neutrophil index and adhesion of blood specimens.
The percent of neutrophil adhesion was calculated by given formula:
$$ NeutrophilIndex= TLC\times \% Netrophils $$
$$ NeutrophilAdhesion= NIu- NIt\div NIu\times 100 $$
Were,
NIu: Neutrophil Index of blood samples
NIt: Neutrophil Index of blood samples incubated with nylon fibres [20].
Carbon clearance test
Mice were divided into four groups, the same as described in neutrophil adhesion tests. Carbon ink suspension and normal saline used in a 1:8 proportion and used for test in a dose of 10 μl/g body weight of mice. Carbon ink solution was injected by tail vein route to each mouse 48 h after the 14th days of treatment, i.e. on the 14th day. Then, 25 μl blood samples were collected by retro-orbital plexus at 0 and 15 min after injection carbon ink solution and blood was mixed with 2 ml of 0.1% sodium carbonate solution. The optical density (OD) of the samples was measured spectrophotometrically at 660 nm. The phagocytic index (K) was calculated by equation: K = log OD1 − log OD2/15, where OD1 (0 min) and OD2 (15 min) are the optical densities respectively [21].
Mice lethality test
Mice were divided into five groups of 6 mice in all groups. Control group 1 received no drug, no vaccination; group 2 vehicle/positive control, received distilled water, 10 ml/kg, p.o. and vaccination; groups 3 and 4 received AEAF 200 mg/kg and EEAF 200 mg/kg, p.o. for 21 days; and group 5 standard Levamisole received 50 mg/kg for 21 days. On the 7th and 17th day of an experiment, all groups were immunized through the intraperitoneal route with haemorrhagic septicaemia vaccine (0.2 ml/mouse s.c.), except control group. On the 21st days of an experiment, the mice were challenged with 0.2 ml of 25 ml LD50 dose of Pasteurella multocida (bovine origin) containing 107 cells per ml subcutaneously and were examined for about 72 h and percentage mortality is calculated as [22],
$$ \mathrm{Percent}\ \mathrm{mortality}=\left(\mathrm{Number}\ \mathrm{of}\ \mathrm{animal}'\mathrm{s}\ \mathrm{dead}\right)/\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{animal}\mathrm{s}\times 100. $$
Method of anaesthesia
In present work, all animals (Except mice lethality test animals) were anaesthetized by pharmacological method with Pentobarbital sodium 25 mg/kg i.p. (Mfg by Alcami corp., Charleston, USA.) for post operational studies.
Statistical analysis
All the investigational data were expressed as mean ± SEM. The parameters such as humoral immune response and cellular immune response were analysed by one-way ANOVA followed by Tukey-Kramar multiple comparison test. Whereas neutrophil adhesion test, carbon clearance and mice lethality test were analysed by one-way ANOVA by Dunnett’s multiple comparison test using GraphPad Prism trial version 5.01 (GraphPad Software, Inc); p < 0.05 was considered statistically significant for comparison.