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Journal of Ethnopharmacology 125 (2009) 252–256

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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mmunomodulatory activities of the ethanolic extract ofaesalpinia bonducella seeds

hruti Shuklaa,∗, Archana Mehtaa, Jinu Johna, Pradeep Mehtaa,uresh Prasad Vyasb, Savita Shuklac

Faculty of Life Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar 470003, Madhya Pradesh, IndiaDepartment of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar 470003, Madhya Pradesh, IndiaP.M.B. Gujrati Science College, Devi Ahilya University, Indore 452-001, India

r t i c l e i n f o

rticle history:eceived 6 April 2008eceived in revised form 16 May 2009ccepted 7 July 2009vailable online 14 July 2009

eywords:aesalpinia bonducella seedsmmunostimulation

a b s t r a c t

Caesalpinia bonducella FLEMING (Caesalpiniaceae) is a plant well known for its medicinal value in IndianAyurveda. However, to prove its efficiency for the clinical utilization, more experimental data will bebeneficial.Aims of the study: The present study involved the investigation of immunomodulatory activities of ethano-lic extract of Caesalpinia bonducella seeds.Materials and methods: Neutrophil adhesion test, haemagglutinating antibody (HA) titre, delayed-typehypersensitivity (DTH) response, phagocytic activity and cyclophosphamide-induced myelosuppressionwere determined by in vivo experiments.

eutrophil adhesionA titreTH responsehagocytic activity

Results: The evaluation of immunomodulatory potential by oral administration of ethanolic seed extractof Caesalpinia bonducella (200–500 mg/kg) evoked a significant increase in percent neutrophil adhesionto nylon fibers as well as a dose-dependent increase in antibody titre values, and potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells. Also it prevented myelosuppression incyclophosphamide drug treated rats and good response towards phagocytosis in carbon clearance assay.Conclusions: The results obtained in this study indicate that Caesalpinia bonducella possesses potential

ity a

immunomodulatory activ

. Introduction

Modulation of immune responses to alleviate the diseases haseen of interest for many years and the concept of ‘Rasayana’ isased on related principles (Patwardhan et al., 1990). Rasayana,

isted as a class in the texts of traditional Indian medicine litera-ure, consists of a number of plants reputed to promote physicalnd mental health, improve defence mechanisms of the bodynd enhance longevity. Besides, a number of medicinal plants asasayanas have been claimed to possess immunomodulatory activ-

ties. Some of the Rasayana drugs as immunomodulatory agentsuch as Withania somnifera, Tinospora cordifolia, Asparagus race-osus and Mangifera indica (Dahanukar and Thatte, 1997; Dhuley,

997; Davis and Kuttan, 2000; Makare et al., 2001) are well knownor their traditional uses. Furthermore, medicinal plants used formmunomodulation can provide potential alternatives to conven-ional chemotherapies for a variety of diseases, especially when the

∗ Corresponding author at: Department of Botany, Dr. H.S. Gour University, Sagar70003, Madhya Pradesh, India. Tel.: +91 9893 460620; fax: +91 7582 223236.

E-mail address: s.microb@yahoo.com (S. Shukla).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.07.002

nd has therapeutic potential for the prevention of autoimmune diseases.© 2009 Elsevier Ireland Ltd. All rights reserved.

host defense mechanism has to be activated under the conditionsof impaired immune response. The use of plant products in theindigenous system of medicines as immunomodulators, indeed,can modulate the body’s immune system, as a variety of plantderivatives such as polysaccharides, lectins, peptides, flavonoidsand tannins have been reported to modulate the immune systemin various in vivo models (Shivaprasad et al., 2006).

Caesalpinia bonducella F., commonly known as Nata Karanja, aprickly shrub found throughout the hotter parts of India, Myan-mar and Sri Lanka, has grey, hard, globular shaped seeds with asmooth shining surface. Seeds consist of a thick, brittle shell witha yellowish white bitter fatty kernel (Nadkarni, 1954). Caesalpiniabonducella is reported to have multiple therapeutic properties likeantipyretic, antidiuretic, anthelmintic and antibacterial (Neogi andNayak, 1958), anti-anaphylactic and antidiarrheal (Iyengar andPendse, 1965), antiviral (Dhar et al., 1968), antiasthmatic (Gayarajaet al., 1978), anti-amoebic and anti-estrogenic (Raghunathan and

Mitra, 1982). Further, it has also been revealed that Caesalpiniabonducella has been traditionally used for the treatment of tumor,inflammation and liver disorders (Kritkar and Basu, 1984). Besides,the aqueous solution of the outer shell of the seeds of Caesalpiniabonducella has also been used traditionally by the tribal people

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f Andaman and Nicober Islands for the relief of the symptomsf diabetes mellitus. Blood sugar lowering activity of Caesalpiniaonducella has been primarily evaluated with significant results inabbit (Rao et al., 1994) and rat models (Biswas et al., 1997; Sharmat al., 1997).

However, there is no scientific report available in the litera-ure on the immunomodulatory activity of Caesalpinia bonducellaeed extract. Therefore, the present study was undertaken to assesshe immunomodulating activities of the ethanolic extract derivedrom the seeds of Caesalpinia bonducella in relation with its folklore

edicinal properties.

. Materials and methods

.1. Plant material

The seeds of Caesalpinia bonducella were collected in March006 from Sagar District, Madhya Pradesh, India. Further taxo-omic identification was conducted by Professor Pradeep Mehtat the Department of Botany, Dr. H.S. Gour University, Sagar, MP,ndia. A voucher specimen has been deposited in the herbariumt the Laboratory of Ecology under the voucher specimen numberBot/H/2692).

.2. Preparation of the extract

The air-dried seeds of Caesalpinia bonducella (50 g) werextracted with 500 ml of ethanol by using soxhlet apparatus. Therude extract was filtered, and evaporated under reduced pressureo give a viscous dark mass with a percentage yield of 3.0% (w/w).

.3. Drugs

Accurately weighed quantities of the ethanol extract were sus-ended in 1% sodium carboxy methylcellulose (SCMC) to prepareuitable forms of the dosages. Cyclophosphamide was used as atandard immunosuppressant.

.4. Preliminary phytochemical screening

To identify the essential constituents of the ethanolic extractf Caesalpinia bonducella seeds such as alkaloids, terpenesnd steroids, saponins, flavonoids, polysaccharides and tan-ins, a preliminary phytochemical screening was carried outsing various test methods of Draggendorff’s and Mayer’s test,iebermann–Burchard test, foam formation test, lead acetate test,olisch’s and Fehling’s test and ferric chloride test, respectively

Trease and Evans, 1983).

.5. Experimental animals

Animal use protocol was approved by the Dr. Hari Singhour University, Sagar, MP, India (Animal Eths Comm/IE/98/Reg No79/01/ab/CPCSEA) and was in accordance with International Stan-ard on the care and use of experimental animals (CCAC, 1993).wiss albino rats of either sex weighing between 100 and 125 g weresed for the experiment of this study. Animals were housed undertandard conditions of temperature (25 ◦C), 12 h/12 h light/darkycles and fed with standard pellet diet and tap water.

.6. Antigen

Fresh blood was collected from sheep sacrificed in the locallaughter house. Sheep red blood cells (SRBCs) were washed threeimes in large volumes of pyrogen free 0.9% normal saline and

macology 125 (2009) 252–256 253

adjusted to a concentration of 0.5 × 109 cells/ml for immunizationand challenge.

2.7. Toxicity assay

Caesalpinia bonducella dried ethanolic seed extract was dis-solved in water and administered orally to different groups of ratsin dosages ranging from 100 to 1000 mg/kg for the LD50 study usingthe modified method (Ghosh, 1971). There was no lethality in anyof the groups after 7 days of treatment.

2.8. Neutrophil adhesion test

Wilkinson (1978) method was employed for neutrophil adhe-sion test. Rats of group I, were served as control and received10 ml/kg normal saline, whereas groups II, III, IV and V werepre-treated with different concentrations of ethanolic extract ofCaesalpinia bonducella seeds (200–500 mg/kg, oral). On day 14of drug treatment, blood samples were collected by puncturingretro-orbital plexus into heparinized vials and analyzed for totalleukocyte cell (TLC) and differential leukocyte cell (DLC) counts.After initial counts, blood samples were incubated with nylon fibersfor 15 min at 37 ◦C. The incubated blood samples were again ana-lyzed for TLC and DLC, respectively to give neutrophil index of bloodsamples. The percent neutrophil adhesion was calculated by thefollowing formula:

Neutrophil adhesion (%) = NIu − NItNIu

× 100

where NIu is the neutrophil index of untreated blood samples andNIt is the neutrophil index of treated blood samples.

2.9. Haemagglutinating antibody (HA) titre

Puri et al. (1993) described the method for haemagglutinatingantibody titre. The animals were immunized by injecting 0.1 mlof SRBCs suspension containing 0.5 × 109 cells intraperitoneally onday 0. Blood samples were collected in micro-centrifuge tubes fromindividual animal by retro-orbital puncture on day 7. The bloodsamples were centrifuged and serum was obtained. Antibody lev-els were determined by the hemagglutination technique. Equalvolumes of individual serum samples of each group were pooled.Twofold serial dilutions of pooled serum samples made in 25 �l vol-ume of normal saline in microtitration plates was added to 25 �l of1% suspension of SRBCs in saline. After mixing, the plates were incu-bated at 37 ◦C for 1 h and examined for haemagglutination undermicroscope. The reciprocal of the highest dilution of the test serumagglutination was taken as the antibody titre.

2.10. Delayed-type hypersensitivity (DTH) response

The rats were challenged by injection of 0.5 × 109 cells SRBCsin right hind foot pad. Foot thickness was measured after +24and +48 h of this challenge. The differences obtained for pre- andpostchallenge foot thicknesses were taken for the measurementof DTH and were expressed in mm. The extract was administeredorally on day 0 and continued till day 7 of challenge (Shivaprasadet al., 2006).

2.11. Phagocytic response

The method was described by Cheng et al. (2005). The animalswere treated from day 0 to day 7 with different concentrations of theextract. On day 7, all the animals of the entire groups received thetreatment of an intravenous injection of (0.3 ml per 30 g) Indian ink

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ispersion (pre-warmed at 37 ◦C). 50 �l of blood samples was col-ected from each animal by retro-orbital bleeding at an interval of 2nd 10 min after the injection of ink dispersion. Blood samples weredded to 4 ml of 0.1% sodium carbonate solution to lyse the erythro-ytes. Absorbance of the samples was measured at 675 nm usingpectrophotometer. After 10 min of blood collection animals wereacrificed and the livers and spleens were collected and weighed.

Rate of carbon clearance (K) and phagocytic index (˛) were cal-ulated by using following formula:

ate of carbon clearance (K) = log OD2 − log OD10

T2 − T1

hagocytic index (˛) = K1/3 × body wt of animalliver wt+spleen wt

here OD2 is the log absorbance of blood at 2 min; OD10 is logbsorbance of blood at 10 min; T2 is the last time point of bloodollection; T1 is the first time point of blood collection.

Rate of carbon clearance and phagocytic index of treated groupnimals were compared with the control group animals.

.12. Cyclophosphamide-induced myelosuppression

Ziauddin et al. (1996) method was employed foryclophosphamide-induced myelosuppression. Albino rats wereivided into six groups designated as: control (I) and treatmentII–X) groups, each group containing six rats. The control groupeceived saline solution. Group II was administered with onlyyclophosphamide at the dose of 30 mg/kg, i.p. while groups III, IV,, VI, VII, VIII, IX and X rats received cyclophosphamide with variedoncentrations of ethanolic seed extract of Caesalpinia bonducella200–500 mg/kg, p.o.) for 10 days. On day 11, blood samples wereollected from the retro-orbital plexus of individual animals andnalyzed for haematological and serological parameters.

.13. Statistical analysis

Data were expressed as the mean standard deviation (S.D.) of theeans and statistical analysis was carried out employing one-wayNOVA. Differences between the data were considered significantt P < 0.05.

. Results

.1. Phytochemical screening

The preliminary phytochemical screening of Caesalpinia bondu-ella seeds revealed the presence of alkaloids, saponins, terpenoids,henolics, flavonoids and polysaccharides as essential phytochem-

cal constituents of the ethanolic seed extract of Caesalpiniaonducella.

able 1ffect of ethanolic extract of Caesalpinia bonducella seed on neutrophil adhesion test.

roup Dose (mg/kg) TLC (103/mm3) [A] Neutrophil % [B]

UB FTB UB FTB

Control 6.60 ± 0.11 6.36 ± 0.15 48.33 ± 1.96 45.I 200 7.56 ± 0.44 7.25 ± 0.48 57.66 ± 4.96 52II 300 7.26 ± 0.30 6.83 ± 0.38 54.66 ± 4.63 47.V 400 8.11 ± 0.53 7.51 ± 0.32 60.0 ± 6.32 50.

500 8.30 ± 1.67 7.70 ± 0.32 63.0 ± 1.67 49.

alues are mean ± S.D., n = 6.* P < 0.05 significant; UB: untreated blood; FTB: fiber treated blood.

macology 125 (2009) 252–256

3.2. Neutrophil adhesion test

The % neutrophil adhesion rate in control group animals wasnoted to be 8.82 ± 1.71, whereas, in ethanolic extract-treated groupsit was found with increased pattern as compared to their respec-tive control groups. No significant increase in neutrophil adhesionwas observed when ethanolic extract was administered at a dose of200 mg/kg. However, the dose concentrations ranging from 300 to500 mg/kg revealed significant increase in neutrophil adhesion ascompared to control, suggesting possible immunostimulant actionof the herbal formulation (Table 1).

3.3. Delayed-type hypersensitivity (DTH) reactions

The cell-mediated immune response of ethanolic extract wasassessed by DTH reaction, i.e. foot pad reaction. As shown in Table 2,the ethanolic extract produced a significant, dose-related increasein DTH reactivity in rats. Increase in DTH reaction in rats in responseto cell dependent antigen revealed the stimulatory effect of ethano-lic extract on T cells (Table 2).

3.4. Haemagglutinating antibody (HA) titre

The HA titre was used to assess humoral immune response.The humoral antibody titre value was found to be 104.23 ± 9.73.Administration of ethanolic seed extract of Caesalpinia bonducellaproduced a dose-dependent increase in the HA titre after 1 h incu-bation with SRBCs (Table 2). Administration of higher dose, i.e.300, 400 and 500 mg/kg, respectively produced significant increasein HA titre as evident from haemagglutination after incubation ofserum with SRBCs.

3.5. Phagocytic response

The faster removal of carbon particles has been correlated withthe enhanced phagocytic activity. The phagocytic activity of thereticulo-endothelium system was measured by the removal of car-bon particles from the blood circulation. Oral administration ofethanolic seed extract of Caesalpinia bonducella (200, 300, 400 and500 mg/kg) for 7 days, and 10 min prior to carbon injection exhib-ited a dose-related increase in the clearance rate of carbon bythe cells of the RES. Ethanolic extract showed phagocytic indexas 4.72 ± 0.66, 5.15 ± 0.27, 6.19 ± 0.45 and 8.13 ± 0.39 with doses of(200, 300, 400 and 500 mg/kg body wt), respectively. The phago-cytic index of control (group I) was 3.12 ± 0.49 (Table 3).

3.6. Cyclophosphamide-induced myelosuppression

Cyclophosphamide at the dose of 30 mg/kg, i.p. caused a signifi-cant reduction in the haemoglobin, RBCs, WBCs and platelets count.Combined treatment of cyclophosphamide and ethanolic extract ofCaesalpinia bonducella (200–500 mg/kg, p.o.) resulted in a restora-tion of bone marrow activity as compared with cyclophosphamide

Neutrophil index [A × B] Neutrophil adhesion (%)

UB FTB

66 ± 1.50 319.13 ± 17.25 290.66 ± 16.43 8.82 ± 1.71.0 ± 4.19 437.83 ± 60.05 378.23 ± 52.21 13.60 ± 1.53

66 ± 3.20 398.13 ± 50.05 326.73 ± 41.92 18.09 ± 0.73*

66 ± 6.65 489.16 ± 74.98 383.43 ± 60.09 21.57 ± 2.17*

66 ± 1.36 523.06 ± 22.55 382.46 ± 20.11 26.96 ± 1.12*

S. Shukla et al. / Journal of Ethnopharmacology 125 (2009) 252–256 255

Table 2Effect of ethanolic seed extract of Caesalpinia bonducella on HA titre and DTH response using SRBCs as an antigen in rats.

Group Treatment Dose (mg/kg) DTH response (mm) 24 h DTH response (mm) 48 h HA titre

I Control (10 ml/kg vehicle control) – 0.18 ± 0.010 0.10 ± 0.08 22.23 ± 0.33II Ethanolic extract of Caesalpinia bonducella 200 0.42 ± 0.004 0.34 ± 0.02 33.11 ± 0.068III Ethanolic extract of Caesalpinia bonducella 300 0.49 ± 0.05* 0.40 ± 0.14* 64.44 ± 5.53*

IV Ethanolic extract of Caesalpinia bonducella 400 0.61 ± 0.05* 0.45 ± 0.03* 93.03 ± 4.00*

V Ethanolic extract of Caesalpinia bonducella 500 0.74 ± 0.03* 0.51 ± 0.20* 104.23 + 9.73*

* Values are mean ± S.D., n = 6, P < 0.05 significant.

Fig. 1. Effects of ethanolic seed extract of Caesalpinia bonducella on blood cells of rats treated with cyclophosphamide for 10 days.Group I: control (no treatment), group II: cyclophosphamide-treated group; group III: treatment with ethanolic extract (200 mg/kg); group IV: cyclophosphamide (30 mg/kg,i.p.) and ethanolic extract (200 mg/kg) treated group; group V: treatment with ethanolic etreated group; group VII: treatment with ethanolic extract (400 mg/kg); group VIII: cyclopwith ethanolic extract (500 mg/kg); group X: cyclophosphamide and ethanolic extract (50Values are mean ± S.D., n = 6; P < 0.05 significant.

Table 3Effect of administration of Caesalpinia bonducella ethanolic seed extract on phago-cytic index.

Group Treatment Dose(mg/kg)

Phagocyticindex

I Control (10 ml/kg vehicle control) – 3.12 ± 0.49II Ethanolic extract of Caesalpinia bonducella 200 4.72 ± 0.66III Ethanolic extract of Caesalpinia bonducella 300 5.15 ± 0.27*

IV

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V Ethanolic extract of Caesalpinia bonducella 400 6.19 ± 0.45*

Ethanolic extract of Caesalpinia bonducella 500 8.13 ± 0.39*

* Values are mean ± S.D., n = 6, P < 0.05 significant.

reatment alone. Significant reduction in white blood cell count wasbserved in animals treated with cyclophosphamide alone (groupI) as compared to the control group (group I). The ethanolic seedxtract of Caesalpinia bonducella increased the levels of WBC in aose-dependent manner as compared to the group treated withyclophosphamide and ethanolic extract together. It was observedhat ethanolic extract at the doses of 300, 400 and 500 mg/kg,espectively restored the levels of WBC back to normal (Fig. 1).

. Discussion

The results of scientific evidences obtained in this study sup-ort the traditional claim of Caesalpinia bonducella for medicinalurposes. In the present study, the immunomodulatory activitiesf ethanolic seed extract of Caesalpinia bonducella, an importantlant of indigenous system of Indian medicine were explored.

Modulation of the immune response through stimulation or

uppression may help in maintaining a disease-free state. Herbalgents that activate host defense mechanisms in the presence ofn impaired immune responsiveness can provide supportive ther-py to conventional chemotherapy (Wagner, 1984). The resultsbtained in the present study indicate that Caesalpinia bonducella

xtract (300 mg/kg); group VI: cyclophosphamide and ethanolic extract (300 mg/kg)hosphamide and ethanolic extract (400 mg/kg) treated group; group IX: treatment0 mg/kg) treated group.

is a potent immunostimulant, stimulating both specific and non-specific immune mechanisms.

The neutrophil, an end cell unable to divide and with lim-ited capacity for protein synthesis is, nevertheless, capable ofa wide range of responses, in particular chemotaxis, phagocy-tosis, exocytosis and both intracellular and extracellular killing(Dale and Foreman, 1984). In the present study, ethanolic seedextract of Caesalpinia bonducella (300–500 mg/kg, p.o.) evoked asignificant increase in percent neutrophils. This may potentiallyhelp in increasing immunity of body against microbial infections(Benacerraf, 1978).

Antibody molecules, a product of B lymphocytes and plasmacells, are central to humoral immune responses, IgG and IgM arethe major immunoglobulins which are involved in the complementactivation, opsonization, neutralization of toxins, etc. (Miller, 1991).The augmentation of the humoral immune response to SRBCs byCaesalpinia bonducella, as evidenced by increase in the antibodytitre in mice (Table 2) indicated the enhanced responsiveness ofT and B lymphocyte subsets, involved in the antibody synthesis(Benacerraf, 1978). The high values of haemagglutinating antibodytitre obtained in the case of ethanolic seed extract of Caesalpiniabonducella have indicated that immunostimulation was achievedthrough humoral immunity.

Cell-mediated immunity (CMI) involves effectors mechanismscarried out by T lymphocytes and their products (lymphokines).CMI responses are critical to defense against infectious organ-isms, infection of foreign grafts, tumor immunity and delayed-typehypersensitivity reactions (Miller, 1991). Therefore, increase in DTHreaction in rats in response to T cell dependent antigen revealed the

stimulatory effect of ethanolic seed extracts of Caesalpinia bondu-cella on T cells (Table 2).

Phagocytosis is the process by which certain body cells, collec-tively known as phagocytes, ingests and removes microorganisms,malignant cells, inorganic particles and tissue debris (Miller, 1991).

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aesalpinia bonducella derived ethanolic seed extract appearedo enhance the phagocytic function by exhibiting a dose-relatedncrease in clearance rate of carbon by the cells of the reticulo-ndothelium system (Table 3).

Besides, the administration of ethanolic seed extract of Cae-alpinia bonducella significantly ameliorated the total WBCs count,BCs count, haemoglobin and platelets count and also restored theyelosuppressive effects induced by cyclophosphamide (Fig. 1).high degree of cell proliferation renders the bone marrow a

ensitive target particularly to cytotoxic drugs. In fact, bone mar-ow is the organ most affected during any immunosuppressionherapy with this class of drugs. Loss of stem cells and inabil-ty of the bone marrow to regenerate new blood cells resultsn thrombocytopenia and leucopenia (Agrawal and Singh, 1999).dministration of the ethanolic extract of Caesalpinia bonducellaeeds was found to increase the total WBC count, which was low-red by cyclophosphamide, a cytotoxic drug, indicating that the testrug can stimulate the bone marrow activity.

. Conclusion

The present investigation suggests that ethanolic extract derivedrom Caesalpinia bonducella seeds may stimulate both cellular andumoral immune responses. The extract not only potentiate non-pecific immune response, but also improve humoral as well asell-mediated immunity effectively. The effectiveness of extract-reated animals in overcoming the side effects of drug-inducedyellosuppression provides sufficient evidences for balancing

nd adaptogenic efficacy of the ethanolic extract of Caesalpiniaonducella seeds. Thus, from the results obtained, it can be con-luded that Caesalpinia bonducella has therapeutic potential andould be served as an effective immunomodulatory candidate.urther studies on the mechanism of action of Caesalpinia bon-ucella derivatives, in order to establish its therapeutic potentialor the prevention of autoimmune diseases are planned in theaboratory.

cknowledgement

Authors are grateful to Head, Department of Botany and Phar-aceutical Sciences, Dr. H.S. Gour University, Sagar, MP, India, for

roviding laboratory facilities and Madhya Pradesh Science andechnology, Bhopal, MP, India for providing financial assistance.

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