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Journal of Ethnopharmacology 123 (2009) 177–180

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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thnopharmacological communication

n vitro and in vivo antimicrobial activities of seeds ofaesalpinia bonduc (Lin.) Roxb.

asleem Arif a, T.K. Mandala, Naresh Kumara, J.D. Bhosalea, Archana Holea,.L. Sharmac, M.M. Padhib, G.S. Lavekarb, Rajesh Dabura,∗

Regional Research Institute (Ay), (CCRAS), Nehru Garden, Kothrud, Pune, MS 411038, IndiaCentral Council for Research in Ayurveda and Siddha, Opp ‘D’ Block, Janakpuri, New Delhi, IndiaInstitute of Genomics and Integrative Biology, DU Campus, Mall Road, Delhi 110007, India

r t i c l e i n f o

rticle history:eceived 20 October 2007eceived in revised form 24 December 2008ccepted 13 February 2009vailable online 9 March 2009

eywords:aesalpinia bonducystic fibrosisntimicrobialung infection

a b s t r a c t

Aim of the study: Caesalpinia bonduc (Lin.) Roxb. is a known drug in Ayurveda to treat various diseasesspecifically tumors, cysts and cystic fibrosis (CF). The aim of this study was to assess in vitro as well as invivo antimicrobial activity of Caesalpinia bonduc seeds.Materials and methods: The in vitro antimicrobial activities of seed coat and seed kernel extracts wereinvestigated by microbroth dilution assay. In vivo activities of hydro-alcoholic extracts were investigated inrat models of chronic Pseudomonas aeruginosa pneumonia mimicking that in patients with cystic fibrosis.Results: Various extracts of plant seeds exhibited in vitro antimicrobial activities in a range of22–350 �g/ml. The extracts also showed activity against methicillin resistant (MR) Staphylococcus aureusand ampicillin resistant (AR) Pseudomonas aeruginosa as in the sensitive strains. In rat model of chronicPseudomonas aeruginosa pneumonia, hydro-alcoholic extracts of Caesalpinia bonduc seed kernel (CBSK)

and Caesalpinia bonduc seed coat (CBSC) were injected subcutaneously in the test groups of animals. Thecontrol groups were treated with cortisone and saline. Two weeks after challenge with Pseudomonasaeruginosa, the CBSK treated animals showed a significant bacterial clearance from the lungs (P < 0.04)and less severe incidence of lung abscess (P < 0.05).Conclusion: Results showed that Caesalpinia bonduc may have the potential to be promising natural

ms o

medicine, with other forinfections.

. Introduction

The mortality rate due to infectious diseases has become moreidespread in recent years due to the emergence of antibiotics

esistance strains of micro-organisms (WHO, 1999). Ayurvedic sys-em of medicine describes the use of Caesalpinia bonduc seeds toreat tumors, cyst (known as granthi and arbud) and cystic fibrosis.he seeds of the plant are traditionally used for the treatment ofever, inflammation and liver disorders. In addition, various partsf this plant have been reported to possess multiple therapeuticroperties like antipyretic, antidiuretic, anthelmintic, antibacterial,nticonvulsant, antiviral, antiasthmatic, antiamebic, and antiestro-

enic activities (Neogi and Nayak, 1958; Dhar et al., 1968; Gayarajat al., 1978; Adesina, 1995). Recently hepatoprotective and antiox-dant properties of Caesalpinia bonduc were reported (Gupta et al.,003).

∗ Corresponding author. Tel.: +91 20 25383138/25380326; fax: +91 20 25386715.E-mail address: rajeshdabur@yahoo.com (R. Dabur).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.02.040

f treatments, for CF patients with chronic Pseudomonas aeruginosa lung

© 2009 Elsevier Ireland Ltd. All rights reserved.

The chemical constituents of the plant include flavonoids, triter-penoids, diterpenoids, and steroids (Purushothaman et al., 1982;Peter et al., 1997; Lyder et al., 1998). Several reports on flavonoids,triterpenoids and steroids showed that these molecules have themultiple biological effects due to their antioxidant and free radicalscavenging abilities. Recently, antibacterial and antifungal activi-ties of diterpene bondenlide from the plant were reported by Siminet al. (2000). Knowing the facts that seed of this plant is used inAyurvedic system of medicine to treat cystic fibrosis for which Pseu-domonas aeruginosa is one of the causative factor, the present workwas carried out to evaluate the antimicrobial activity of the seedsof Caesalpinia bonduc (Lin.) Roxb.

2. Materials and methods

2.1. Materials

The media and chemicals were obtained from indicated com-mercial sources. Nutrient broth, nutrient agar, ampicillin andmethicillin (Hi-media), cortisone (Cipla Ltd.), and solvents (E.Merck).

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.2. Plant material

The seeds of Caesalpinia bonduc (Lin.) Roxb. were collected fromhe plants grown in Nehru Garden, Kothrud, Pune. Plant was iden-ified at Regional Research Institute (Ay), Nehru Garden, Kothrud,une, where voucher sample (Voucher No. 248) was deposited. Theature seeds were used to prepare different extracts.

.3. Preparation of extracts

The seed coats and seed kernels were separated and powdered.he powdered materials of seeds were subjected to cold extractionuccessively with petroleum ether, chloroform, acetone, methanolnd water to obtain respective fractions (Dabur et al., 2004). 20%lcoholic extracts were also prepared from seed kernel and seedoat. The process of extraction with each solvent was repeatedhrice. Solvents were evaporated under reduced pressure usingotavapour R-114 (Buchi) and dry fractions were stored at 4 ◦C tillse.

.4. Microorganisms

Clinical isolates of the microorganisms were used along with thetandard strains. Quality control strains of Salmonella typhi MTCCB33, Escherichia coli MTCCB 1662, Pseudomonas aeruginosa MTCCB41, Staphylococcus aureus MTCCB 737, Bacillus cereus MTCCB 1272,acillus thuringenesis MTCCB1824, Proteus mirabilis MTCCB 564,lebsiella pneumoniae MTCCB 109 and Vibro cholera MTCCB 458ere purchased from Institute of Microbial Technology, Chandi-

arh, India and used in each test as recommended by the Nationalommittee for Clinical Laboratories Standards (NCCLS). Addition-lly, three methiclillin-resistant strains of Staphylococcus aureus andhree ampicillin resistant strains of Pseudomonas aeruginosa werencluded in the study.

.5. Antibacterial screening

Antibacterial activities of the various extracts of Caesalpinia bon-uc seeds were determined by the micro broth dilution assay asescribed by Buwa and Van Staden (2006). Stock solutions of differ-nt fractions were prepared by dissolving 3.5 mg of extract in 1.0 mlf solvent. Stock solutions of water extracts were prepared in water.thanol and acetone extracts were prepared in ethanol. Chloroformnd petroleum ether extracts were prepared in DMSO. Further dilu-ions of the fractions were made in the nutrient broth. Extracts werelter sterilized using membrane of 0.45 �m pore size. Proper con-rols were kept for each experiment. The bacterial strains used asnocula were grown at 37 ◦C to get OD 0.6 at 600 nm. Colony formingnits (CFU) were counted by using serial plate dilution method andacterial counts were adjusted to 1 × 105 to 1 × 106 CFU/ml for sus-eptibility testing. The lowest concentration of each extract, whichnhibited any visual growth, was considered to be the minimumnhibitory concentration (MIC).

.6. Immobilization of P. aeruginosa in alginate beads

Pseudomonas aeruginosa MTCCB 741, which stably maintains aucoid phenotype was used for in vivo experiments. 1 ml of the

seudomonas aeruginosa bacterial culture was mixed with 9.0 ml ofeaweed alginate and the mixture was forced once with air throughcannula into a solution of 0.1 M CaCl2 in 0.1 M Tris–HCl buffer (pH.0). The suspension was adjusted to yield 108 CFU/ml, and the yieldas confirmed by colony counts.

acology 123 (2009) 177–180

2.7. Treatment protocol

All experiments were performed with prior approval of the Insti-tutional Animal Ethics Committee. Thirty-six female Albino rats(Wistar strain) of 7-week age, having body weight of 110–130 gwere taken and divided into four groups, each comprising of nineanimals. The dosages in group 1 and 2 animals were determined onthe basis of dosages used for treatment of cystic fibrosis patients inAyurveda (Sharma, 1999, 2000).

• Group 1: The hydro-alcoholic extract of seed kernel was solublizedin phosphate buffer saline and filtered through a membrane of2 �m pore size (Waters). The drug was injected subcutaneouslyat a dose of 25.0 mg/kg body weight once a day for 10 days.

• Group 2: Hydro-alcoholic extract of seed coat was solublized inPBS and filtered as in case of seed kernel. Drug was injected sub-cutaneously at a dose of 25.0 mg/kg body weight once a day for10 days.

• Group 3: Hydrocortisone was used as a control drug in the study.Cortisone was injected subcutaneously as a dose of 25.0 mg/kgbody weight once a day for 10 days. The dose was determinedon the basis of the dosage used for treatment of cystic fibro-sis patients with Pseudomonas aeruginosa infections (Eigen et al.,1995).

• Group 4: This was the control group where sterile PBS was injectedsubcutaneously as a dose of 1.0 ml/kg of body weight once a dayfor 10 days.

• The treatments of animals with drug in all groups were started onthe same day as challenge with Pseudomonas aeruginosa alginatebeads.

2.8. Challenge procedures

The animals were anesthetized subcutaneously with a 1:1 mix-ture of etomidate and midazolam at a dose of 1.5 ml/kg body weightat the time of challenge and were tracheotomized. Animals werechallenged intratracheally with 0.1 ml of Pseudomonas aeruginosa(108 CFU/ml) in alginate beads (Johansen et al., 1994). The incisionswere sutured with silk thread and the wounds healed without com-plications. Fourteen days after challenge, all rats were sacrificed byusing 20% pentobarbital at 3.0 ml/kg of body weight.

2.9. Bacteriology

The lungs of the animals in the groups were prepared for thequantitative bacteriological examination as described previously(Johansen et al., 1994). Three milliliters of phosphate-bufferedsaline was added to each set of lung tissue samples and homog-enized. The total volume of each lung homogenate was 4.5 ml. Atotal of 0.1 ml of the homogenate was plated for bacterial culture todetermine the numbers of CFU.

2.10. Macroscopic lung pathology

The lungs were removed from the thoracic cavity and observedin situ. The lungs were scored and divided into three grades accord-ing to pathological changes, grade 1, normal lungs; grade 2, swollenlungs with small atelectases; grade 3, swollen lungs with pleuraladhesions and hemorrhages (Johansen et al., 1994, 1996).

3. Results and discussion

The yield of petroleum ether, chloroform, acetone, methanol,water and hydro-alcoholic extracts of CBSC was found to be 6.3,0.092, 0.116, 1.26, 4.08 and 5.01 percent respectively. On the other

T. Arif et al. / Journal of Ethnopharmacology 123 (2009) 177–180 179

Table 1Antimicrobial activity of seed extracts by microbroth dilution assay.

Micro-organisms Extracts MIC (�g/ml)

Water extract Methanol extract Acetone extract Chloroform extract Pet ether extract Amp Met

Pseudomonas aeruginosa MTCCB 741 CBSC 88 22 – – 175 2.5 NDCBSK 350 175 – 44 –

Pseudomonas aeruginosa 135 (AR) CBSC 175 22 – – 350 – NDCBSK 350 175 – 175 –

Pseudomonas aeruginosa 1124 (AR) CBSC 88 22 – – 175 – NDCBSK 175 175 – – –

Pseudomonas aeruginosa 325 (AR) CBSC 88 22 – – 175 – NDCBSK 175 175 – – -

Staphylococcus aureus MTCCB 737 CBSC 175 44 – – 88 ND 2.5CBSK 88 – – – 175

Staphylococcus aureus 187 (MR) CBSC 878 22 – 88 175 ND –CBSK 44 – 44 44 350

Staphylococcus aureus 349 (MR) CBSC 175 22 – – 88 ND –CBSK 88 – – – 88

Staphylococcus aureus 674 (MR) CBSC 88 88 – 175 – ND –CBSK 350 175 – – 350

E. coli MTCCB 82 CBSC – 88 88 44 88 2.5 NDCBSK 88 – 175 175 175

Proteus mirabilis MTCCB 564 CBSC 22 88 175 350 175 5.0 NDCBSK – – 88 88 -

Klebsiella pneumoniae MTCCB 109 CBSC 350 88 175 350 175 2.5 NDCBSK – 350 88 175 88

Salmonella typhi MTCCB 733 CBSC 22 44 – 88 44 2.5 NDCBSK 88 – 350 88 175

Vibrio cholera MTCCB 458 CBSC 88 88 175 88 350 2.5 NDCBSK 175 88 175 175 22

B. thuringiensis MTCCB 1824 CBSC 175 175 22 44 88 5.0 NDCBSK 175 175 22 – 175

B. cereus MTCCB 1272 CBSC – 22 22 350 22 2.5 ND

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and yield of CBSK extracts with the same solvents were recordedo be 4.1, 5.01, 0.94, 3.83, 5.45 and 7.32 percent respectively.

Seed coat extracts exhibited better in vitro antimicrobial activ-ty than seed kernel extracts. Methanol and water extracts of CBSChowed maximum activity against all the bacteria in the rangef 22.0–350 �g/ml, used in the studies except E. coli (Table 1).etroleum ether extract also exhibited broad range activity at theigher concentrations. Chloroform and acetone extracts exhib-

ted antimicrobial activity against all the bacteria used in thetudies except Staphylococcus aureus and Pseudomonas aeruginosa.BSK extracts exhibited variable activity at higher concentra-ions than CBSC (Table 1). It was observed that the extractsf Caesalpinia bonduc were effective against the methicillin andmpicillin resistant strains of Staphylococcus aureus and Pseu-omonas aeruginosa. The antibacterial activity of methanolic extractf seeds was previously reported by Saeed and Sabir (2001).ighest in vitro activity was observed in water and methanol

xtracts, therefore hydro-alcoholic extract were used for in vivotudies.

During in vivo study, no deaths were recorded in any group. Pseu-omonas aeruginosa was cultured from the lungs of most surviving

able 2acroscopic pathology, abscess incidence and median numbers of CFU of Pseudomonas ain each group.

reatment group Median (range) bacterial count (CFU)/lung No. (%) of rats with <10

ontrol 2.4 × 102 (1.3 × 101–5.6 × 104) 2 (22.2)BSK 1.9 × 101 (0–2.4 × 103) 6 (66.6)BSC 8.0 × 101 (1.5 × 101 –2.7 × 104) 4 (44.4)ortisone 5.6 × 103 (0.4 × 102–6.7 × 105) 2 (22.2)

wo-tailed P values showed significant difference (P < 0.05) in CBSK treated group as com

ND ND ND

duc seed kernel; CBSC: Caesalpinia bonduc seed coat; MR: methicillin resistant; AR:

rats, two weeks after challenge (Table 2). CBSK extract reducedbacterial load significantly in the rat models as compared to the con-trols (P < 0.046) and 6 out of 9 rats in the CBSK-treated group werefound to be infected with less than 100 CFU. CBSC treated groupsalso showed some improvement but difference was not significantas compared to control groups.

Abscesses, atelectases, hemorrhages and fibrinous adhesion tothe thoracic wall or diaphragm were found in all groups after twoweeks challenge. However, the lung pathology in the CBSK treatedgroup was found to be significantly milder as compared to controland the cortisone-treated groups. Caesalpinia bonduc seeds, there-fore, might exhibit promising therapeutic value against pneumoniacaused by Pseudomonas aeruginosa (Table 2).

Studies showed that subcutaneous administration of cortisonehas no effect on Pseudomonas aeruginosa pneumonia. On the otherhand the CBSK treated group showed significant improvementin lung pathology. Therefore, Caesalpinia bonduc appears to be

promising to treat chronic lung infections in the CF patients inconjunction with other treatments. Further studies to clarify themechanism and immune reactions involved in the rat model arewarranted.

eruginosa in rat lungs after 14 days intratracheal challenge. Number of animals was

0 CFU No. of rats with lung pathology score Lung abscess incidence (%)

Grade 1 Grade 2 Grade 3

1 2 6 66.65 3 1 11.13 3 3 33.30 2 7 77.7

pared to control.

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cknowledgment

The authors are thankful to Central Council for Research inyurveda and Siddha (CCRAS) for providing facilities to conducthese studies.

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