In vitro and in vivo immunomodulatory activities of iridoids fraction from Barleria prionitis Linn

Journal of Ethnopharmacology 141 (2012) 424–431

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In vitro and in vivo immunomodulatory activities of iridoids fraction from Barleria prionitis Linn B.V. Ghule ∗ , P.G. Yeole Institute of Pharmaceutical Education and Research, Wardha 442 001, Maharashtra State, India

a r t i c l e

i n f o

Article history: Received 10 September 2011 Received in revised form 8 December 2011 Accepted 3 March 2012 Available online 13 March 2012 Keywords: Barleria prionitis Iridoids fraction Immunostimulant activity HPTLC standardization

a b s t r a c t Ethnopharmacological relevance: Barleria prionitis Linn. (Family: Acanthaceae), one of the important Ayurvedic medicinal plant in India, has long been used to treat variety of ailments including swellings, gout, arthritic and rheumatic disorders, nervine and skin diseases, and also acts as immunorestorative. Aim of the study: The present study was aimed to explore in vitro and in vivo immunomodulatory activities of the iridoids fraction i.e. n-butanol fraction of methanol extract from Barleria prionitis aerial parts (IFBp). Materials and methods: IFBp was studied for in vitro [nitroblue tetrazolium (NBT) test and neutrophils candidacidal assay] and in vivo immunomodulatory activity on cellular and humoral immune responses to the antigenic challenge by sheep red blood cells (SRBCs) and by neutrophil adhesion test, phagocytic activity and cyclophosphamide-induced myelosuppression. The study comprised the preliminary phytochemical screening, HPTLC standardization and maximum tolerable dose determination of IFBp. Results: IFBp (50, 100 and 200 ␮g/ml) significantly (P ≤ 0.01) increased the intracellular killing activity of stimulated neutrophils assayed by in vitro NBT reduction test and neutrophils candidacidal assay. Pretreatment of IFBp (100 and 200 mg/kg; p.o.) evoked a significant increase in percent neutrophils and neutrophils adhesion to nylon fibres. Oral administration of IFBp augmented the humoral immune response to SRBCs, evidenced by increase in antibody titres and dose dependently potentiated the delayed-type hypersensitivity reaction induced by SRBCs in mice. IFBp potentiated significantly (P ≤ 0.01) the macrophage phagocytic activity and ameliorated the red blood cells, total white blood cells and platelets count and haemoglobin concentration, and also restored the myelosuppressive effects induced by cyclophosphamide. The content (% w/w; mean ± SD, n = 3) of main iridoids i.e. shanzhiside methyl ester and barlerin was found to be 21.55 ± 2.40 and 10.03 ± 1.69 in IFBp of BP, respectively. Conclusion: The present investigation reveals that IFBp is a potent immunostimulant, stimulating both the specific and non-specific immune mechanisms. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Modulation of immune responses to alleviate disease has been of interest for many years and the concept of ‘Rasayana’ in Ayurveda is based on related principles (Charak Samhita, 1949). Many medicinal plants classified as “Rasayana” in Ayurveda are believed to be useful in strengthening the immune system of an individual (Patwardhan et al., 1991). The concept of immunomodulation

Abbreviations: BP, Barleria prionitis; IFBp, iridoids fraction i.e. n-butanol fraction of methanol extract from Barleria prionitis aerial parts; HPTLC, high performance thin layer chromatography; NBT, nitroblue tetrazolium; SRBCs, sheep red blood cells; HBS, Hank’s balanced salt; NaCl, sodium chloride; RBCs, red blood cells; PBS, phosphate buffer saline; MTD, maximum tolerable dose; TLC, total leukocyte cells; DLC, differential leukocyte cells; HA, haemagglutinating antibody; DTH, delayedtype hypersensitivity; RES, reticulo-endothelial system; WBCs, white blood cells; CMI, cell-mediated immunity. ∗ Corresponding author. Tel.: +91 7152 240284; fax: +91 7152 241684. E-mail address: [email protected] (B.V. Ghule). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.03.005

relates to a non-specific activation of the immune system. It implies primarily a non-antigen dependent stimulation of the function and efficiency of macrophages, granulocytes, complement, natural killer cells, lymphocytes and also the production of various effector molecules by activated cells (para-immunity). Being non-specific, it is expected to give protection against different pathogens including bacteria, fungi, viruses etc. and constitutes an alternative or adjunct to conventional chemotherapy (Wagner, 1984). Barleria prionitis Linn. (BP) (Family: Acanthaceae) is found throughout the tropical regions of India, Sri Lanka and South Africa. BP is an annual shrub, locally known as “Vajradanti” in India and “Katukaradu” in Sri Lanka, and is widely used in folk medicines. The herb’s extract is prescribed for massage in toothache, swellings, arthritis and gout. In folk medicine, BP is widely used to treat nervine disorders, boils and glandular swellings, leprosy and other skin diseases, rheumatic affections, internal abscesses, chronic sinusitis etc. (Nadkarni, 1954; Chopra et al., 1966; Asolkar et al., 1992; The Aurvedic Pharmacopoeia of India, 1999; Khare, 2004).

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In line with these traditional medicinal uses, BP has been reported to possess anti-respiratory syncytial virus (Chen et al., 1998), antiarthritic, anti-inflammatory and hepatoprotective activities (Sing et al., 2003, 2005), antihepatotoxic, antistress and immunorestorative (Suri et al., 2003), glutathione S-transferase, acetylcholinesterase inhibitory and antibacterial (Kosmulalage et al., 2007) activities. Previous work on the iridoid constituents (monoterpene lactone glucosides) of BP have led to the isolation and identification of shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester (barlerin), 6,8-O,O-diacetyl shanzhiside methyl ester (acetylbarlerin) (Taneja and Tiwari, 1975; Damtoft et al., 1982; Byrne et al., 1987; Fathalla et al., 2009) and 6-O-trans-p-coumaryl8-O-acetyl shanzhiside methyl ester and its cis isomer (Chen et al., 1998). The present study was therefore undertaken to explore in vitro [nitroblue tetrazolium (NBT) test and neutrophils candidacidal assay] and in vivo immunomodulatory activities of the iridoids fraction i.e. n-butanol fraction of methanol extract from Barleria prionitis aerial parts (IFBp) on cellular and humoral immune responses to the antigenic challenge by sheep RBCs and by neutrophil adhesion test, phagocytic activity and cyclophosphamide-induced myelosuppression.

thimble of Soxhlet apparatus in a porcelin dish and ensuring that no residue remains after evaporating the solvent. Methanol extract was dried at 50 ◦ C under vaccum, the extractive value (% w/w) was found to be 16.82 of dry weight of plant material. Previous works on BP have led to the isolation and identification of mainly iridoid constituents (Taneja and Tiwari, 1975; Damtoft et al., 1982; Byrne et al., 1987; Chen et al., 1998). Wagner and Bladt (1996) suggested that iridoids can be enriched into n-butanol fraction from the parent extract. Therefore methanol extract (100 g) was suspended in 200 ml of 20% v/v methanol in distilled water and fractionated successively, in a separating funnel, with different volumes of nbutanol (50, 50, 50, 40, 40, 40, 35, 35, 30, 20, 20 ml) until complete fractionation. The n-butanol fractions were combined and vaccum evaporated to dryness to obtain iridoids fraction (IFBp). The extractive value of IFBp was found to be 42.83% w/w of the methanol extract from BP.

2. Materials and methods

2.5. Compositional analysis of IFBp by HPTLC method

2.1. Materials

A Camag HPTLC system (Muttenz, Switzerland) including a Linomat V sample applicator, a Camag Twin-Trough TLC chamber, Camag TLC scanner III and Wincats integration software was used. Aluminium-backed HPTLC plates (10 cm × 20 cm) with 200 nm thickness of silica gel 60 F254 (Merck, Darmstadt, Germany), prewashed with methanol, were used. Shanzhiside methyl ester and barlerin being main iridoids present in BP were used as biomarkers for the standardization of methanol extract and IFBp from BP by HPTLC method. Methanol solvent was used to prepare stock solutions of the samples and the standard markers. From stock solutions of methanol extract (3 mg/ml) and IFBp (2 mg/ml) of BP, different concentrations (5, 10 and 15 ␮l) were spotted in the form of bands of width 6 mm by means of a Linomat V sample applicator to the plate. Similarly from stock solutions of shanzhiside methyl ester and barlerin (100 ␮g/ml), different volumes i.e. 2, 4, 6, 8 and 10 ␮l were spotted on the TLC plates to obtain concentration of 200, 400, 600, 800 and 1000 ng per spot. A constant application rate of 1 ␮l/15 s was employed and the chromatogram was developed upto 80 mm under chamber saturation (20 min) conditions with chloroform-methanol (80:20, v/v) as the mobile phase in a Camag twin-trough TLC chamber. Subsequent to the development, TLC plates were dried in a current of air with the help of air dryer. Densitometric scanning was performed on Camag TLC scanner III in the absorbance mode at 240 nm. The source of radiation utilized was a Deuterium lamp. The data of peak area plotted against the corresponding concentrations were treated by linear regression analysis.

A Camag HPTLC (high performance thin layer chromatography) system (Muttenz, Switzerland) including a Linomat V sample applicator was used. HPTLC precoated Silica gel 60 F254 plates (20 cm × 20 cm) (Merck, Darmstadt, Germany) were used. Double beam UV visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan), vaccum evaporator (Bio-Technics IndiaTM ) and digital plethysmometer were used. Shanzhiside methyl ester and barlerin, isolated and identified in our laboratory, were used as biomarkers. Hanks balanced salt (HBS) solution, Sabouraud’s 2% dextrose broth and Candida albicans (ATCC 10231) fungal culture were purchased from HiMedia, Mumbai, India. All other chemicals like dimethylsulphoxide, sodium chloride, trypan blue, eosin, sodium deoxycholate, methylene blue, nitroblue tetrazolium (NBT), glucose, sodium citrate, citric acid, sodium carboxymethyl cellulose, gelatin, sodium carbonate etc. were purchased from Loba Chemie (Mumbai, India) and HiMedia (Mumbai, India). All the organic solvents and chemicals were of analytical grade and used as obtained. 2.2. Plant material At the flowering stage, aerial parts of Barleria prionitis Linn. (BP) (Family: Acanthaceae) were collected from Wardha district, Maharashtra State, India during the month of November–December and authenticated at Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. A voucher specimen (number 9188) is deposited in the Institute of Pharmaceutical Education and Research, Wardha for the future reference. Fresh aerial parts were cleaned, shade dried and coarse to finely powdered by grinder and then sieved through mesh sieve # 20 and stored in air tight container until further use. 2.3. Extraction of plant material and preparation of iridoids fraction (IFBp) The shade dried and coarse-finely powdered aerial parts (1 kg) of BP were extracted successively with petroleum ether (60–80 ◦ C) and methanol by Soxhlet extraction method. The completion of extraction was ensured by taking a few ml of extractant from the

2.4. Preliminary phytochemical screening IFBp was subjected to preliminary phytochemical screening (Harborne, 1984; Trease and Evans, 2008) for the detection of various phytoconstituents.

2.6. In vitro immunomodulatory activity 2.6.1. Preparation of neutrophils Neutrophils were isolated from venous blood of healthy volunteers. The heparinized blood (5 ml containing 100 units of heparin) was added to 1 ml of 4.5% dextran B in physiological saline. The mixture was gently shaken and allowed to stand for 60 min at room temperature to sediment erythrocytes. Neutrophils were isolated by Ficoll–Hypaque density gradient centrifugation according to Ferrante and Thong (1980). After removal of the residual erythrocytes by hypotonic lysis, the neutrophils were washed with HBS solution. The cells were suspended at a final concentration of 5 × 106 neutrophils/ml in HBS solution for NBT reduction test and

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107 neutrophils/ml for in vitro candidacidal assay (Lehrer and Cline, 1969; Basaran et al., 1997). The viability of neutrophils was tested by trypan blue exclusion and was greater than 92%. 2.6.2. Pretreatment of neutrophils (activation) with IFBp Neutrophils (5 × 106 cells/ml) were treated with IFBp (50, 100 and 200 ␮g/ml in 5.0% v/v dimethysulphoxide in 0.15 M phosphate buffer saline (PBS). Equal volumes (1 ml) of both neutrophils and IFBP were added into plastic tubes and incubated at 37 ◦ C for 4 h (Hay and Westwood, 2002). 2.6.3. Nitroblue tetrazolium (NBT) test Methods described by Miller et al. (1991) and Park et al. (1968) were followed. Briefly 0.1 ml of 0.1% NBT solution (prepared in equal volumes of 0.9% NaCl and 0.15 M PBS) was placed into each of 4 wells in a plastic microtitre plate. First well was served as control (added with unstimulated neutrophils) and 2nd, 3rd and 4th wells were added with 0.1 ml (5 × 106 cells/ml) of stimulated neutrophils (pretreated with IFBp). Contents of each well were mixed carefully with a plastic pipette and immediately microtitre plates were placed in a tray of moist atmosphere (containing cotton wet gauze to provide humidity) and incubated at 37 ◦ C for 10 min. Incubation was further continued at room temperature for 15 min and contents of each well were thoroughly mixed with a plastic pipette and placed one drop on a microscope slide (precleaned with 95% alcohol and dried) so as to prepare smear. The smears were air dried (note: at least 3 slide smears were separately prepared from each well). One slide from each well was selected and flooded with freshly prepared Safaranin stain. Staining was allowed for 1 min and an equal volume of Sorenson’s buffer (0.067 M) was added to each slide and waited for 10 min. Smears were examined under the microscope with oil immersion objective (100×), and 100 neutrophils were counted, recording both total neutrophils and numbers which contain deposits of black formazan (reduced NBT dye) i.e. cells containing black material larger than the granules normally appearing in neutrophils. 2.6.4. Neutrophils candidacidal assay 2.6.4.1. Preparation of Candida albicans cells suspensions. Test organism was grown (inoculated) in 50 ml of Saubouraud’s 2% dextrose broth for 72 h at 33 ◦ C. Under this condition, the Candida cells grew only in yeast phase (stationary phase). The Candida organisms were washed twice in HBS solution. The yeast viability was checked while counting the organism in hemacytometer. For this purpose 0.5 ml of 0.3% w/v trypan blue and 0.1% eosin (3:1) were added to 0.5 ml of the washed culture, mixed well and the hemacytometer chamber was filled with the mixture. The yeast cell looked as single spherical cells or attached doublets, the filamentous forms were absent and above 95% of Candida cells were viable. Candida cells concentration was adjusted to 107 cells/ml for use in neutrophils Candidacidal assay (Lehrer and Cline, 1969; Schmid and Brune, 1974). 2.6.4.2. Serum preparation. Fresh serum was obtained from normal group AB donor. Blood was withdrawn in a disposable syringe and transferred in a sterile centrifuge tube. It was allowed to clot for 1 h and centrifuged. The supernatant layer (faint yellow) was collected and stored in refrigerator at freeze temperature (4–7 ◦ C) until use. 2.6.4.3. In vitro neutrophils candidacidal assay. The methods described by Lehrer and Cline (1969) and Schmid and Brune (1974) were followed. Equal volumes (0.25 ml) of AB serum, pretreated neutrophils suspension (107 cells/ml) with IFBp and HBS solution were added to sterile plastic tubes. A third tube containing all components except neutrophils served as a control. The tubes were incubated for 10 min at 37 ◦ C. A 0.25 ml of Candida albicans (107

yeast phage cells/ml) were added, and the tubes were rotated (30 RPM) at 37 ◦ C for 60 min (note: after 15 min, a drop was taken for direct examination and preparation of stained smears to confirm the number of added organisms ingested by neutrophils). At 60 min, 0.25 ml of 2.5% sodium deoxycholate (pH: 8.7) was added to each tube (note: at this concentration, deoxycholate causes immediate lysis of the blood cells without damaging Candida cells). Methylene blue (0.01% in distilled water) was then added to achieve a final volume of 4–5 ml. The Candida cells suspension were centrifuged at 1100 × g for 15 min and resuspended in about 0.5 ml of the residual supernate fluid (note: thereafter, the tubes were kept in an ice water bath until they could be examined microscopically). At last, 300 Candida cells from each tube were examined to determine the percentage stained. To derive the Candidacidal activity due to the action of neutrophils, the percentage of stained yeast cells in the control tubes, usually 2.5% was substracted from that in the experimental tubes. Viable Candida cells, which were unstained, clearly differ from the non-viable organism which acquired a uniform, intense blue cytoplasm stain. 2.7. In vivo immunomodulatory activity 2.7.1. Preparation of iridoids fraction (IFBp) for oral administration Aqueous suspension (1% w/v) of IFBp was prepared in 1% w/v sodium carboxymethyl cellulose in distilled water. 2.7.2. Antigenic material The sheep red blood cells (SRBCs) were used as an antigenic material. The sheep blood (withdrawn from external Jugular vein of Sheep) was obtained from local Slaughterhouses in Wardha, collected in Alsever’s solution (prepared by adding glucose 2.05 g, sodium chloride 0.42 g, sodium citrate 0.80 g and citric acid 0.55 g in sterile water and volume was adjusted to 100 ml) and stored at 4–7 ◦ C in refrigerator. During the experimentation, adequate amount of stock solution of SRBCs was taken and washed three times with pyrogen-free normal saline by centrifugation at 3000 × g for 10 min on each occasion. The settled SRBCs were then suspended in normal saline. Each mouse received 0.5 × 109 cells in volume of 0.2 ml i.p. for sensitization and challenge at required time schedule. This cell count has been reported to induce optimum immune response in normal and immune suppressed conditions (Ghule et al., 2006). 2.7.3. Experimental animals All the experimental procedures used in present study were in accordance with Institutional guidelines for animal research (CPCSEA, 2003). Experimental protocols were approved (IPER/IAEC/2007-2008/11) by the Institutional Animal Ethics Committee of Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha. Albino mice were obtained from healthy animal colony maintained at the Institute’s Department of Pharmacology. Albino mice (male, 3–4 weeks old) were randomly distributed in groups as per experimental protocols (n = 6). They were kept in air-conditioned and pathogen-free isolators with temperature of 23 ± 2 ◦ C and humidity of 55.6 ± 10% on a regulated 12-h light and dark cycle. They were given standard laboratory chow diet and tap water ad libitum. Blood samples were collected through retro-orbital bleeding at specified time points under chloroform anesthesia. 2.7.4. Maximum tolerable dose (MTD) determination The OECD method was used to determine MTD in animals (OECD, 1996). IFBp orally administered in graded doses (100–4000 mg/kg of animal body weight) and animals were monitored for change in weight, general behavior and mortality at 0.5, 2,

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6 and 12 h intervals after IFBp administration and upto 7 days. The IFBp was found to be well tolerated up to the dose of 4000 mg/kg of animal body weight. 2.7.5. Neutrophil adhesion test The method originally described by Wilkinson (1978) was employed. Mice of Group I was served as control and received 1% w/v sodium carboxymethyl cellulose solution, whereas mice of Groups II and III, were pretreated with IFBp in divided doses. On 14th day of IFBp treatment, blood samples were collected (before antigenic challenge, SRBCs) by puncturing retro-orbital plexus by chloroform anesthesia into heparinized vials and analyzed for total leukocyte cell (TLC) and differential leukocyte cell (DLC) counts. After initial counts, blood samples were incubated with 80 mg/ml of nylon fibres for 15 min at 37 ◦ C. The incubated blood samples were again analyzed for TLC and DLC. The product of TLC and percent neutrophil gives neutrophil index of blood sample. The percent neutrophil adhesion was calculated as shown below Neutrophil adhesion (%) =

NIu − NIt × 100 NIu

Where NIu is the Neutrophil index of untreated blood samples and NIt is the Neutrophil index of treated blood samples. 2.7.6. Haemagglutinating antibody (HA) titre A microtechnique employing 96 wells microtitre plates was used (Hudson and Hay, 1980). The method used was similar to that described previously by Puri et al. (1993). On 14th and 21st day of the drugs treatment, each mouse was immunized with 0.2 ml of 0.5 × 109 SRBCs/mouse by i.p. route, including mice of control group. On 21st and 27th day of the treatment, primary and secondary antibody titres were determined by titrating serum dilutions with SRBCs (0.025 × 109 cells). Equal volumes of individual serum samples of each group were pooled and two-fold serial dilution of pooled serum samples made in 25 ␮l of 1% v/v suspension of SRBCs in saline. After mixing thoroughly, microtitre plates were incubated at 37 ◦ C for 1 h and examined visually for agglutination. Positive haemagglutination reaction was visualized as a mesh formation at the bottom whereas, negative haemagglutination reaction indicated button formation. HA titre was expressed in terms of maximum dilution which gave positive haemagglutination reaction and the lowest dilution of antibody (serum) was ranked as one. 2.7.7. Delayed-type hypersensitivity (DTH) response Treatment schedule, animals used, antigenic material used was similar to that described in the HA titer measurement. The method described by Lagrange et al. (1974) and modified by Doherty (1981) was used and modified. Albino mice of different groups were immunized (sensitized by antigenic challenge) intraperitonealy with 0.2 ml of SRBCs (0.5 × 109 SRBCs/mice) on 14th and 21st day of the drug treatment, as previously for the HA titre measurement. On 27th day of IFBp treatment, after measuring the normal foot pad volume of each mice on digital Plethysmometer, 108 SRBCs in volume of 25 ␮l was injected into left hind foot pad. The degree of swelling was measured at 24 (28th day) and 48 h (29th day) after antigenic (SRBCs, 108 cells) challenge. The foot pad reaction was expressed as the difference in thickness between one foot injected with SRBCs and other injected with 25 ␮l normal saline. 2.7.8. Macrophage phagocytosis by carbon clearance test For assay of the phagocytic activity of the reticulo-endothelial system (RES) in vivo, a carbon clearance test was used (Biozzi et al., 1953; Hudson and Hay, 1980). On day 30 after completion of IFBp treatment, the treated mice received an intravenous (tail vein) injection of carbon suspension, (Indian Ink, Camel) dilution with

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1:3 in a 1.5% solution of sterile gelatin in physiological saline, in a dose of 0.1 ml/10 g animal body weight (Ghule et al., 2006). Two minutes and 10 min following injection with carbon suspension, 0.05 ml of blood sample was collected from each mouse by retroorbital bleeding. Blood samples were directly added into 4 ml of 0.1% sodium carbonate solution to lyse the RBCs. Absorbance of the sample was measured at 675 nm using UV visible spectrophotometer. Rate of carbon clearance, termed as phagocytes index (granulopectic test) was calculated by the formula Log(OD2 ) − Log(OD10 ) T2 − T1 Whereas, OD2 is the log absorbance of blood at 2 min and OD10 is the log absorbance of blood at 10 min; T2 is the last time point of blood collection; T1 is the time point of blood collection. Rate of carbon clearance of treated group animals was compared with the control group animals. 2.7.9. Cyclophosphamide-induced immunosuppression This method used was as described by Ziauddin et al. (1996). Albino mice were divided into four groups, each group containing five mice. The control group received 1% w/v sodium carboxymethyl cellulose in distilled water. Group II was administered with only Cyclophosphamide at the dose of 30 mg/kg intraperitonealy, groups III–IV mice received IFBp (100–200 mg/kg; p.o.) for 10 days. On day 11, blood samples were collected from the retro-orbital plexus of individual animals and analyzed for hematological and serological parameters. 2.7.10. Statistical analysis All data are presented as mean ± S.D. Differences between groups were analyzed by using the one-way analysis of variance (ANOVA) with Dunnett’s t-test. A value of P ≤ 0.05 was considered statistically significant using GraphPad InStat 3 statistical analytical software. 3. Results and discussion Many plant products used in traditional medicine have been reported to have immunomodulating activities. While some of these stimulate both humoral and cell-mediated immunity (CMI), others activate only the cellular components of the immune system, i.e. phagocytic function without affecting the humoral immunity (Atal et al., 1986). Agents that activate host defense mechanisms in the presence of an impaired immune responsiveness can provide supportive therapy to conventional chemotherapy (Wagner, 1984). The pharmacological activity of a plant extract is largely dependant upon its composition, nature, and the structure of constituent(s) present in the extract. Plants of genus Barleria are rich sources of iridoids i.e. shanzhiside methyl ester and barlerin (Taneja and Tiwari, 1975; Damtoft et al., 1982; Byrne et al., 1987; Fathalla et al., 2009). Preliminary phytochemical screening of IFBp revealed the abundance of iridoids. Therefore, IFBp was standardized for the total content of main constituents before appraisal of its immunomodulatory activities. The developed HPTLC method is specific to be able to identify shanzhiside methyl ester and barlerin in methanol extract and IFBp from BP. Under the current chromatographic conditions, shanzhiside methyl ester and barlerin are satisfactorily separated. The representative HPTLC profile of these agents is shown in Fig. 1 at 240 nm. The HPTLC chromatograms of standard shanzhiside methyl ester and barlerin solutions showed the absorption peaks with retention factor values of 0.30 and 0.48, respectively. Similar peaks were observed in the HPTLC chromatograms of methanol extract and IFBP at similar retention factors (Fig. 1) indicating the presence of shanzhiside

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Fig. 1. Representative HPTLC chromatograms of all the tracks including (a) shanzhiside methyl ester (200, 400, 600, 800 and 1000 ng/spot; Rf = 0.30), (b) barlerin (200, 400, 600, 800 and 1000 ng/spot; Rf = 0.48), (c) methanol extract (15, 30, 45 ␮g/spot) and (d) IFBp (10, 20, 30 ␮g/spot) from Barleria prionitis at 240 nm; mobile phase: chloroform–methanol (80:20, v/v).

methyl ester and barlerin. The calibration curves for the biomarkers were linear over the range of 200–1000 ng/ml with regression equations (correlation coefficient) for shanzhiside methyl ester and barlerin as y = 3.9175x + 482.9 (r2 = 0.9956) and y = 4.2297x + 301.33 (r2 = 0.9932), respectively where y is the response as peak area and x is the concentration. Based on calibration curves, the content (% w/w; mean ± SD, n = 3) of shanzhiside methyl ester and barlerin was found to be 4.91 ± 0.21 and 4.69 ± 0.15 in methanol extract, and 21.55 ± 2.40 and 10.03 ± 1.69 in IFBp from BP, respectively. The NBT dye reduction test gives information about the phagocytic and intracellular killing functions of neutrophils which are necessary for normal microbiocidal activity. The dye is taken into neutrophils by phagocytosis and then stimulation of the hexose

monophosphate-shunt pathway (HMP) of glucose oxidation and concomitant changes in oxidative metabolism lead to the reduction of the dye (Akbay et al., 2002). IFBp, at the concentrations of 50, 100 and 200 ␮g/ml, significantly increased the intracellular killing activity of stimulated neutrophils assayed by in vitro NBT reduction test. It should be stated that IFBp might contain some constituents responsible for intracellular killing more than degranulation (Table 1). In the in vitro neutrophils candidacidal assay, Candida albicans are the foreign particles present in the assay medium and are actively engulfed by PMN cells. The average number of Candida cells ingested and associated with each PMN cells indicates the intensity of phagocytic activity of these cells. The results of the neutrophils

Table 1 Effect of IFBp on NBT-positive neutrophils (% and range) and percent Candida albicans cells killed in 60 min by stimulated normal neutrophils. Group(s)

Treatment (␮g/ml)

Mean (and range) NBT-positive neutrophils per cmm NBT-positive neutrophils (%)

I II III IV

Control IFBp 50 IFBp 100 IFBp 200

25.33 48.00 57.33 64.00

± ± ± ±

1.52 5.56a 2.08a 4.58a

Total Candida albicans cells counted (300)

NBT-positive neutrophils (range)

Viable cells

24–27 43–54 55–59 59–68

276.00 258.00 251.66 248.33

± ± ± ±

1.00 3.46a 4.35a 7.57a

Candidacidal activity (%)

Non-viable cells 26.00 42.00 49.00 51.66

± ± ± ±

3.00 3.46a 4.35a 7.57a

IFBp: iridoids fraction (50, 100 and 200 ␮g/ml) of methanol extract from Barleria prionitis. Tabulated values are mean ± S.D. a P ≤ 0.01, very significant when compared with blank control group. Statistical analysis was by ANOVA followed by Dunnett’s t-test (n = 3).

7.99 13.99 16.33 17.22

± ± ± ±

0.33 1.15a 1.45a 2.52a

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Table 2 Effect of IFBp on percent neutrophils and percent neutrophils adhesion. Group(s)

I II III

Treatment (mg/kg; p.o.) Control IFBp 100 IFBp 200

Neutrophil index [A × B]

TLC (103 /mm3 ) [A]

Neutrophils, % [B]

UB

FTB

UB

FTB

UB

FTB

4.98 ± 0.059 6.33 ± 0.062a 7.16 ± 0.042a

4.64 ± 0.047 5.66 ± 0.040a 6.27 ± 0.040a

42.00 ± 2.366 60.33 ± 2.160a 64.16 ± 2.858a

38.16 ± 1.472 51.16 ± 2.317a 53.83 ± 3.061a

209.34 ± 14.24 382.02 ± 17.36a 459.53 ± 23.19a

177.15 ± 8.591 289.71 ± 15.220a 337.63 ± 21.374a

Neutrophil adhesion (%) 15.28 ± 1.803 24.17 ± 0.792a 26.55 ± 1.142a

TLC: total leukocyte count; UB: untreated blood; FTB: fibre treated blood; IFBp 100 and 200: iridoids fraction (100 and 200 mg/kg) of methanol extract from Barleria prionitis. Tabulated values are mean ± S.D. a P ≤ 0.01, very significant when compared with the control group. Statistical analysis was by ANOVA followed by Dunnett’s t-test (n = 6). Table 3 Effect of IFBp on primary and secondary antibody titres and on foot pad reaction i.e. delayed-type hypersensitivity (DTH) in antigenically challenged mice. Group(s)

Treatment (mg/kg; p.o.)

I II III

Control IFBp 100 IFBp 200

Mean heamagglutinating antibody (HA) titre

Foot pad thickness i.e. mean % edema at

Primary HA titre

Secondary HA titre

24 h

48 h

6.83 ± 0.752 13.16 ± 1.472a 14.66 ± 1.211a

8.33 ± 0.816 16.50 ± 1.225a 17.83 ± 1.722a

24.59 ± 2.018 38.34 ± 4.163a 41.22 ± 3.153a

19.22 ± 1.421 33.22 ± 1.979a 35.47 ± 4.436a

IFBp 100 and 200: iridoids fraction (100 and 200 mg/kg) of methanol extract from Barleria prionitis. Tabulated values are mean ± S.D. a P ≤ 0.01, very significant when compared with the control group. Statistical analysis was by ANOVA followed by Dunnett’s t-test (n = 6).

Candidacidal activity after 4 h activation with IFBp are shown in Table 1. IFBp (50, 100 and 200 ␮g/ml) stimulated neutrophils to increase Candidacidal activity at a level of significance of P ≤ 0.01. The neutrophil, an end cell unable to divide and with limited capacity for protein synthesis is, nevertheless, capable of a wide range of responses, in particular chemotaxis, phagocytosis, exocytosis and both intracellular and extracellular killing. Margination of neutrophils from the blood stream requires a firm adhesion, which is mediated through the interactions of the b2 integrins present on the neutrophils (Smith et al., 1989; Srikumar et al., 2005). In the present study, pretreatment of IFBp (100-200 mg/kg; p.o.) evoked a significant (P ≤ 0.01) increase in the in vitro neutrophil adhesion to nylon fibres, which correlates the increase in percent neutrophils (Table 2). The HA titre was used to assess humoral immune response. The augmentation of the humoral immune response to SRBCS by drugs is evidenced by increase in the antibody titres in the blood of mice. Antibody molecules, a product of B lymphocytes and plasma cells, are central to humoral immune response; IgG and IgM are the major immunoglobulins which are involved in the complement activation, opsonization, neutralization of toxins, etc. (Benacerraf, 1978). When the effect of IFBp on primary and secondary humoral immune response to SRBCs was examined, it was found that oral administration of IFBp (100–200 mg/kg; p.o.) markedly augmented the antibody response to SRBCs in mice. The result indicated a significant stimulating effect of IFBp on the ability of mice to produce antibodies against a T cell dependent antigen (Table 3). In the early hypersensitivity reaction, antigen-antibody formed immune complexes (IC) are known to induce local inflammation with increased vascular permeability, edema and infiltration of PMN leukocytes. It is believed that the observed increase in the arthus reaction could be due to enhanced formation of IC consequent upon the elevated levels of antibody of precipitin type (namely IgM) (Sell, 1980). The cell-mediated immune response of

IFBp, assessed by foot pad reaction i.e. DTH reaction, is shown in Table 3. Oral administration of IFBp (100–200 mg/kg; p.o.) for 29 days produced a significant, dose-related increase in DTH reactivity in mice. Increase in DTH reaction in mice in response to cell dependent antigen revealed the stimulatory effect of IFBp on T cells. Phagocytosis is the process by which certain body cells, collectively known as phagocytes, ingest and removes microorganisms, malignant cells, inorganic particles and tissue debris (Miller et al., 1991). Macrophages play an important role in nonspecific and specific immune responses. The phagocytic activity of RES was measured by the rate of removal of gelatin-stabilized carbon particles from the blood circulation (Biozzi et al., 1953; Hudson and Hay, 1980). The result reveals that IFBp improve humoral immunity probably through the reproduction and differentiation of B-cells into antibody-secreting plasma cells (Table 3). Macrophages can process and present antigen to B-cells. Oral administration of IFBp (100–200 mg/kg; p.o.) for 30 days and 30 min prior to carbon injection exhibited a dose-related increase in the clearance rate of carbon by the cells of the RES (Table 4). The result indicates the stimulatory effect of IFBp on the cells of the mononuclear phagocytic system. Immunomodulatory study of IFBp was also performed using the Cyclophosphamide-induced myelosuppression animal model. The suppression of bone marrow activity reflecting myelosuppression by cyclophosphamide is considerable and is accompanied by a lowering of the hemoglobin concentration, RBCs, platelets and total WBCs counts. In differential WBCs count, a relative lowering of lymphocyte percentages and an increase in neutrophils was evident (Ziauddin et al., 1996). Oral administration of IFBp (100–200 mg/kg) significantly ameliorated the RBCs, total WBCs and platelets count, and haemoglobin concentration and also restored the myelosuppressive effects induced by cyclophosphamide (30 mg/kg; i.p.). IFBp was found to increase the total WBCs count, which was lowered by

Table 4 Effect of IFBp on carbon clearance (phagocytic index, %). Group(s)

I II III

Treatment (mg/kg; p.o.)

Control IFBp 100 IFBp 200

Legends are same as that of Table 3.

Carbon clearance at 2 min

10 min

0.7136 ± 0.026 0.8431 ± 0.042a 0.8911 ± 0.034a

0.5973 ± 0.033 0.3615 ± 0.028a 0.3155 ± 0.028a

Difference in absorbance

Phagocytic index (%)

0.1163 ± 0.047 0.4816 ± 0.045a 0.5756 ± 0.060a

0.0166 ± 0.006 0.0687 ± 0.006a 0.0822 ± 0.008a

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Table 5 Effect of IFBp on blood components of Cyclophosphamide-induced immunosuppression in mice for 10 days. Group(s)

Treatment (mg/kg; p.o.)

Blood components Hemoglobin concentration in g %

I II III IV

Control Cyclophosphamide 30 IFBp 100 IFBp 200

14.83 10.67 14.72 15.58

± ± ± ±

0.144x 0.178a 0.301a,x 0.354a,x

RBCs count in % million/cmm 6.12 3.47 5.65 6.08

± ± ± ±

0.0148x 0.298a 0.197a,x 0.206a,x

WBCs count in thousand/cmm 12.16 7.21 11.66 12.26

± ± ± ±

0.440x 0.256a 0.265a,x 0.308a,x

Platelets count in thousand/cmm 568.16 335.83 512.00 567.33

± ± ± ±

19.43x 13.45a 25.65a,x 15.09a,x

Control vs groups II–IV: a P < 0.01, very significant. Cyclophosphamide vs groups I, III and IV: x P ≤ 0.01, very significant. Remaining legends are same as that of Table 3.

cyclophosphamide, a cytotoxic drug, indicating that the IFBp can stimulate the bone marrow activity (Table 5). 4. Conclusions In conclusion, the data corroborate both the folk use of BP and utilization of BP, as plant drug with immunorestorative activity. This effect must be associated with the interaction between IFBp (i.e. mainly shanzhiside methyl ester and barlerin iridoids) and the immune system. The present investigation suggests that IFBp may stimulate both cellular and humoral immune responses. The results also indicate that IFBp is a potent immunostimulant, stimulating both the specific and non-specific immune mechanisms. Therefore the use of IFBp as an immunomodulator for developing and improving protective immunity even in normal individuals may be possible. Acknowledgements We owe our thanks to the authority of Botany Department, Nagpur University, Nagpur for the authentication of plant specimen. The facilities provided by the Institute of Pharmaceutical Education and Research, Wardha during this course of study are gratefully acknowledged. References Akbay, P., Calis, I., Undeger, U., Basaran, N., Basaran, A.A., 2002. In vitro immunomodulatory activity of verbascoside from Nepeta ucrainica Linn. Phytotherapy Research 16, 593–595. Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Second Supplement to Glossary of Indian Medicinal Plants with Active Principles. Part I (A–K) (1965–1981). National Institute of Science and Communication and Information Resources (CSIR), New Delhi. Atal, C.K., Sharma, M.L., Kaul, A., Khajuria, A., 1986. Immunomodulating agents of plant origin. I: Preliminary screening. Journal of Ethnopharmacology 18, 133–141. Basaran, A.A., Ceritoglu, I., Undeger, U., Basaran, N., 1997. Immunomodulatory activities of some Turkish medicinal plants. Phytotherapy Research 11, 609–611. Benacerraf, B., 1978. A hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and B lymphocytes. Journal of Immunology 120, 1809–1812. Biozzi, G., Benacerraf, B., Halpern, B.N., 1953. Quantitative study of the granulopectic activity of the reticulo-endothelial system I: the effect of ingredient present in India ink and of substances affecting blood clotting in vivo on fate of carbon particles administration intravenously in rats mice and rabbits. British Journal of Experimental Pathology 34, 426–457. Byrne, L.T., Sasse, J.M., Skelton, B.W., Suksamrarn, A., White, A.H., 1987. The minor iridoid glycosides of Barleria lupulina: isolation crystal structure and plant growth-inhibiting properties of 6-O-acetylshanzhiside methyl ester. Australian Journal of Chemistry 40, 785–794. Charak Samhita, 1949. Translator. Shree Gulabkunverba Ayurvedic Society, Jamnagar, India. Chen, J.L., Blanc, P., Stoddart, C.A., Bogan, M., Rozhon, E.J., Parkinson, N., Ye, Z., Cooper, R., Balick, M., Nanakorn, W., Kernan, M.R., 1998. New iridoids from the medicinal plant Barleria prionitis with potent activity against respiratory syncytial virus. Journal of Natural Products 61, 1295–1297. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1966. Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi. CPCSEA, 2003. CPCSEA. Guidelines for laboratory animal facility – committee for the purpose of control and supervision of experiments on animals. Indian Journal of Pharmacology 35, 257–274.

Damtoft, S., Jensen, S.R., Nielsen, B.J., 1982. Structural revision of barlerin and acetylbarlerin. Tetrahedron Letters 23, 4155–4156. Doherty, N.S., 1981. Selective effects of immunosuppressive agents against the delayed hypersensitivity response and humoral response to sheep red blood cells in mice. Agents and Actions 11, 237–242. Fathalla, M.H.H., El-Halawany, A.M., El Gayed, S.H., Sattar, E.A., 2009. Iridoid glycosides from Barleria trispinosa. Natural Product Research 23, 903–908. Ferrante, A., Thong, Y.H., 1980. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human blood by the Hypaque–Ficoll method. Journal of ImmunologicaI Methods 36, 109–117. Ghule, B.V., Murugananthan, G., Nakhat, P.D., Yeole, P.G., 2006. Immunostimulant effects of Capparis zeylanica Linn. leaves. Journal of Ethnopharmacology 108, 311–315. Harborne, J.B., 1984. Phytochemical Methods – a Guide to Modern Techniques of Plant Analysis, Second ed. Chapmann and Hall, New York. Hay, F.C., Westwood, O.M.R., 2002. Practical Immunology, fourth ed. Blackwell Science Ltd., London. Hudson, L., Hay, P., 1980. Chapter 5: Antibody interaction with antigen. In: Thomas, A. (Ed.), Practical Immunology. Blackwell, Edinburgh, pp. 73–92. Khare, C.P., 2004. Encyclopedia of Indian Medicinal Plants – Rationale Western Therapy, Ayurvedic and other Traditional Usage Botany. Springer-Verlag, Berlin, Heidelberg. Kosmulalage, K.S., Zahid, S., Udenigwe, C.C., Akhtar, S., Athar, A., Samarasekera, R., 2007. Glutathione S-transferase, acetylcholinesterase inhibitory and antibacterial activities of chemical constituents of Barleria prionitis. Zeitschrift für Naturforschung 62, 580–586. Lagrange, P.H., Mackaness, G.B., Miller, T.E., 1974. Influence of dose and route of antigen injection on the immunological induction of T cells. Journal of Experimental Medicine 139, 528. Lehrer, R.I., Cline, M.J., 1969. Interaction of Candida albicans with human leukocytes and serum. Journal of Bacteriology 98, 996–1004. Miller, L.E., Ludke, H.R., Peacock, J.E., Tomar, R.H., 1991. Manual of Laboratory Immunology, second ed. Lea and Febiger, Philadelphia, London. Nadkarni, K.M., 1954. Indian Materia Medica, vol. I., third ed. Popular Prakashan, Bombay. OECD, 1996. Organization for economic cooperation and development. OECD Guidelines for Testing of Chemicals. Guideline 423, Acute Oral Toxicity–Acute Toxic Class Method, Adopted, March 22. Park, B.H., Fikrig, S.M., Smithwick, E.M., 1968. Infection and nitroblue tetrazolium reduction by neutrophils. Lancet 2, 532–534. Patwardhan, B., Kalbag, D., Patki, P.S., Nagsampagi, B.A., 1991. Search of immunomodulatory agents – a review. Indian Drugs 28, 249–254. Puri, A., Saxena, R., Saxena, R.P., Saxena, K.C., 1993. Immunostimulant agents from Andrographis paniculata. Journal of Natural Products 56, 995–999. Schmid, L., Brune, K., 1974. Assessment of phagocytic and antimicrobial activity of human granulocytes. Infection and Immunity 10, 1120–1123. Sell, S., 1980. Immunology, Immunopathology and Immunity, third ed. Harper and Row, MD, USA. Sing, B., Bani, D.K., Gupta, D.K., Chandan, B.K., Kaul, A., 2003. Antiinflammatory activity of ‘TAF’ – an active fraction from the plant Barleria prionitis Linn. Journal of Ethnopharmacology 85, 187–193. Sing, B., Chandan, B.K., Prabhakar, A., Taneja, S.C., Sing, J., Quzi, G.N., 2005. Chemistry and hepatoprotective activity of an active fraction from Barleria prionitis Linn in experimental animals. Phytotherapy Research 19, 391–404. Smith, C.W., Marlin, S.D., Rothlein, R., Toman, C., Anderson, D.C., 1989. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. Journal of Clinical Investigation 83, 2008–2017. Srikumar, R., Parthasarathy, N.J., Devi, R.S., 2005. Immunomodulatory activity of triphala on neutrophil functions. Biological and Pharmaceutical Bulletin 28, 1398–1403. Suri, J.L., Banerjee, S.K., Taneja, S.C., Chandra, S., Anand, A.S., Prabhakar, A., Jaggi, B.S., Saxena, A.K., Chandan, B.K., Handa, S.S., Swami, S., 2003. Synergistic composition of bioactive fraction isolated from Barleria prionitis Linn. and a method of treatment for hepatotoxicity, immune-deficiency and fatigue and a process thereof. US Patent Appl. Publication 20030181397. Taneja, S.C., Tiwari, H.P., 1975. Structures of two new iridoids from Barleria prionitis Linn. Tetrahedron Letters 24, 1995–1998. The Aurvedic Pharmacopoeia of India, 1999. Government of India, Ministry of Health and Family Welfare, Department of Indian Systems of Medicine and Homeopathy, New Delhi.

B.V. Ghule, P.G. Yeole / Journal of Ethnopharmacology 141 (2012) 424–431 Trease, G.E., Evans, W.C., 2008. Pharmacognosy, fifteenth ed. WB Saunders, Balliere, Tindall, London. Wagner, H., 1984. Economic and medicinal plant research. In: Hikino, H., Farnsworth, N.R. (Eds.), vol. I. Academic Press, London, pp. 113–153. Wagner, H., Bladt, S., 1996. Plant Drug Analysis – a Thin Layer Chromatography Atlas, second ed. Springer-Verlag, Berlin.

431

Wilkinson, P.C., 1978. Neutrophil adhesion test. In: Vane, J.K., Ferreria, S.H. (Eds.), Handbook of Experimental Pharmacology. , first ed. Springer Verlag, Berlin, p. 109. Ziauddin, M., Phansalkar, N., Patki, P., Diwanay, S., Patwardhan, B., 1996. Studies on the immunomodulatory effects of Ashwagandha. Journal of Ethnopharmacology 50, 69–76.