Antibacterial activity of Acalypha indica and Albizzia lebbeck on Pseudomonas aeruginosa -An Invitro study

Auxilia Hemamalini Tilak et al. / Journal of Pharmacy Research 2012,5(1),69-71

Research Article ISSN: 0974-6943

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Antibacterial activity of Acalypha indica and Albizzia lebbeck on Pseudomonas aeruginosa -An Invitro study Auxilia Hemamalini Tilak * 1 & Lakshmi.T 2 Faculty of Microbiology,Saveetha Dental college & Hospitals,Chennai, Tamil Nadu, India 2 Faculty of Pharmacology,Saveetha Dental college & Hospitals,Chennai, Tamil Nadu, India 1

Received on:20-09-2011; Revised on: 15-10-2011; Accepted on:10-12-2011 ABSTRACT In view of increasing resistance to existing antimicrobial agents, botanicals are being noticed as very important source for discovery of new agents for treating various ailments related to bacterial infections. Acalypha indica and Albizia lebbeck are known plants in India which posses wide range of pharmacological activities. Butanol, ethanol and methanol leaf extract of Acalypha indica and Albizia lebbeck were tested for antibacterial activity by discdiffusion technique and broth dilution method(MIC & MBC)against Pseudomonas aeruginos, a gram negative, aerobic rod found commonly in soil and water. All the extracts exhibited antibacterial activity against the tested bacterial strain .Butanol extract showed maximum activity. The butanolic extract of A. lebbeck was more potent than A. indica and had a MBC of 0.1975mg. The ethanolic and methanolic extracts of both the leaf extracts had identical MIC of .312mg and 1.25mg.

Key words: Acalypha indica , Albizia lebbeck , Pseudomonas aeruginosa ,antibacterial activity, disc diffusion, MIC&MBC. INTRODUCTION Pseudomonas aeruginosa is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. It is a free-living bacterium, commonly found in soil and water. Pseudomonas aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance.1 Pseudomonas aeruginosa is an opportunistic pathogen, meaning that it exploits some break in the host defenses to initiate an infection. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner. It causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed.2 P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin (from “pyocyaneus”) refers to “blue pus”, which is a characteristic of suppurative infections caused by Pseudomonas aeruginosa.It is notorious for its resistance to antibiotics and is, therefore, a particularly dangerous and dreaded pathogen. The bacterium is naturally resistant to many antibiotics due to the permeabiliity barrier afforded by its Gram-negative outer membrane. 3 Only a few antibiotics are effective against Pseudomonas aeruginosa which include fluoroquinolones, gentamicin and imipenem, and even these antibiotics are not effective against all strains. Albizia lebbeck (Family: Mimosaceae) is an deciduous tree which contains tannins, saponins, cardiac glycosides, flavanoids. The root is used in hemicrania. The bark is bitter, cooling, alexiteric, antihelminthic, cures diseases of the blood, leucoderma, itching, piles, bronchitis and used in rat bite. The bark is good for opthalmia. The flowers are given for asthma and for snakebite. All parts of the plant are recommended for the treatment of snake-bite. It is reported to possess nootropic 4, 5, anxiolytic 6,anticonvulsant 6, 7, antifertility

*Corresponding author. Dr. Auxilia Hemamalini Tilak , Professor & Head,Department of Microbiology, Saveetha Dental College & Hospitals, Chennai, Tamil Nadu, India

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and antidiarrhoeal 9 effects. Different phytochemicals have been isolated from beans which include albigenin — a triterpene 10 and albigenic acid —a triterpenoid sapogenin. 11 Albiziahexoside– a bioactive saponin is isolated from bark. 12 Acalypha indica known as kuppaimeni in tamil is an annual shrub seen in Indian gardens, backyards of houses and waste place throughout the plains of India. Parts used are Leaves, root, stalks (young shoots) and flowers. The major phytochemical constituents are Alkaloids “acalypus” and “acalyphine.” Pharmacological actions include Cathartic, Antihelminthic, expectorant, emetic and hypnotic.13 It also possesses post-coital antifertility activity.14 Acalypha is an irritant to the gastro-intestinal mucous membrane. It is used as a substitute for ipecacuanha. This drug has also been used as a laxative and anthelmintic.15 It has good antimicrobial activity,16 wound healing activity,17 Acalypha indica is widely used in Ayurveda18 and homeopathic medicine19. Leaves possess laxative properties; “are used as a substitute for senega” in the form of powder or decoction. Powder of dry leaves is used in bed sores. According to Siddha Materia Medica 20 the leaf powder when given in the dose of 950 mg to 1300 mgs, cures repiratory diseases. The leaf juice when mixed with neem oil and applied to the inner part of children’s tongue with the help of quill, induces vomiting and acts as expectorant.21 Pseudomonas aeruginosa is a bacteria known for it’s resistance to antibiotics and there arise situations in clinical practice where the strains become resistant to all known antibiotics.It is with this thought that the possibility of herbal extracts with antipseudomonal activity has been explored. Hence In the present study, an attempt has been made to enrich the knowledge of antibacterial activity of Acalypha indica, Albizia lebbeck plant extract against Pseudomonas aeuroginosa. MATERIALS AND METHODS Clinical history A 40 year old male reports to the hospital with history of a fever 102 F, pulse 100/min, BP 100/50 and an infected burns wound on his right leg. His random blood sugar was 208mg. He had been previously treated with amoxicillin clavulininc acid combination. After putting an intravenous line and IV fluids, a swab was taken from the wound and a dose of Cefotaxim 1gm and garamycin 80mg were administered parenterally. Pseudomonas aeruginosa was isolated in pure culture from the pus sample

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Auxilia Hemamalini Tilak et al. / Journal of Pharmacy Research 2012,5(1),69-71 Test organism The clinical isolate was was compared with the standard strain ATCC 27853 strain and subcultured on nutrient agar, incubated overnight at 37°C and stored in the refrigerator at 4°C until further use

Table 2 Acalypha indica Zone of Inhibition Extract (mm)

The antibiogram of the clinical isolate The clinical isolate was sensitive to Cefotaxime, Ceftriaxone, Ceftazidime, Meropenem, Piperacillin, gentamicin, Amikasin and Ciprofloxacin as tested by the disc diffusion method. Their zones of inhibition was recorded in mm

Butanol Ethanol Methanol

Antibacterial assay Diffusion Method20 Antimicrobial activity was carried out using disc diffusion method(1). Petri plates were prepared with 20 ml of sterile Mueller Hinton Agar (MHA) (HIMEDIA, Mumbai, India). A broth culture of the test organism was prepared in nutrient broth and incubated for 4-6 hours at 37°C. The broth culture was matched with .5 Macfarlands turbidity standard. This corresponds to a bacterial concentration of 108 A lawn culture of the test organism was made on Mueller Hinton agar and allowed to dry for 10 min. The filter paper discs were made to hold 100µl containing 500µg of the crude extract. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvents. The plates were incubated for 24 h at 37°C. Zone of inhibition was recorded in millimeters and the experiment was done in triplicate.

Albizzia lebbek Extract Zone of inhibition (mm) Butanol Ethanol Methanol

RESULTS AND DISCUSSION Butanol, ethyl acetate and methanol extracts of Acalypha indica leaves and Albizzia lebbeck leaves showed significant zone of inhibition against Pseudomonas aeruginosa and matched the zones of inhibition of the well documented antipseudomonal antibiotics . The zones of inhibition of theantipseudomonal antibiotics is given in Table 1. and that of the herbal extracts in Table 2 Table 1.The zones of inhibition of theantipseudomonal antibiotics Antibiotic

Zone Of Inhibition(mm)

Ciprofloxacin Meropenem Am ikasin Piperacillin Gentamicin Cefotaxime Ceftriaxone Ceftazidime

33 28 18 22 25 27 23 28

40 11 10

The zone of inhibition of the butanolic extract of A. lebbeck at a concentration of 500µg was 40mm and showed the largest zone of inhibition among the extracts tested. The butanolic extracts of the leaves of both A. indica and A. lebbeck were more potent than the ethanolic and methanolic extracts. Their MIC was the same as the MBC of all the extracts tested and is given in Table 3 Table 3. MIC AND MIC Acalypha indica MIC AND MIC Acalypha indica

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Dilution Method For determination of minimum inhibitory concentration (MIC) and minimum Bactericidal concentration (MBC), the broth macrodilution technique was employed. 1ml of the crude leaf extracts of the two herbs were serially diluted in nutrient broth (Hi-media, Mumbai) to give a final concentration of 1.25mg to 9.875 µg, incubated for 4-6 hours at 37°Cand observed for turbidity. Two tubes were free of test extract and served as broth control solvent control.After incubation, approximately 10 µl was transferred with a bacteriological loop onto Muller Hinton agar. The inoculated plates were incubated overnight under aerobic conditions at 37°C. MIC was defined as the lowest concentration or the highest dilution of the extract that shows no turbidity that corresponds to the highest dilution that allows no more than 20% bacterial growth, and MBC is the highest dilution or the lowest concentration of the extract which gave nil growth.

26 11 10

BE EE ME

1.25mg .625mg

.312mg

.156mg

.078mg

.039mg

.01975mg .009875mg

NG NG NG

NG NG G

NG G G

NG G G

NG G G

G G G

NG NG G

G G G

MIC AND MIC Albizzia lebbeck

BE EE ME

1.25mg

.625mg

.312mg

.156mg

.078mg

.039mg

.01975mg .009875mg

NG NG NG

NG NG G

NG NG G

NG G G

NG G G

NG G G

NG G G

G G G

BE-Butanolic extract,EE- Ethanolic extract,ME-Methanolic eextract G-Growth,NG-No growth

Among the two, the leaf extract of A. lebbeck was more potent than A. indica and had a MIC of 0.1975mg. The ethanolic and methanolic extracts of both A. indica and A. lebbeck had identical MIC of .312mg and 1.25mg. In our study all the leaf extracts showed variable levels of inhibitory activity against Pseudomonas aeruginosa. The maximum level of antipseudomonal activity was showed by the butanolic extract of A. lebbeck. Also the butanolic extracts of both A. indica and A. lebbeck exhibited bactericidal activity at much lower concentrations than the ethanolic and methanolic extracts. Among the two butanolic extracts, the butanolic extract of A. lebbeck was the most potent with an MIC of .01975mg. In general the methanolic extracts were the least potent. Further characterization of the active components and purification is warranted to put these extracts to clinical use. They will be great benefit in cases of multidrug resistant Pseudomonas when all known antibiotics have failed. REFERENCES 1. Todar’s Text book of Bacteriology-Pseudomonas aeruginosa, Page 14 2. Chintawar SD, Somani RS, Kasture Veena S, Kasture SB, Nootropic activity of Albizia lebbeck in mice,Journal of Ethnopharmacology, 2002, 81,299-305.

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Une HD, Sarveiya VP, Pal SC, Kasture VS, Kasture SB, Nootropic and anxiolytic activity of saponins of Albizia lebbeck leaves, Pharmacology Biochemistry and Behavior 2001,69, 439-444. 4. Kasture VS, Chopade CT, Deshmukh VK, Anticonvulsive activity of Albizia lebbeck, Hibiscus rosa sinesis and Butea monosperma in experimental animals, Journal of Ethnopharmacology, 2000, 71, 6575. 5. Kasture VS, Kasture SB, Pal SC, Anticonvulsant activity of Albizia lebbeck leaves. Indian J of Exp Biol,1996, 34, 78-80. 6. Gupta RS, Kachhawa JB, Chaudhary R, Antifertility effects of methanol pod extract of Albizia lebbeck (L.) Benth in male rats, Asian J Androl, 2004, 6,155-159. 7. Besra SE, Gomes A, Chaudhury L, Vedasiromoni JR, Ganguly DK, Antidiarrhoeal activity of seed extract of Albizia lebbeck Benth, Phytotherapy Research, 2002, 16, 529-533. 8. Barua AK, Raman SP, Triterpenoids—X: The constitution of albigenic acid—a new triterpenoid sapogenin from Albizia lebbeck benth Tetrahedron 1959, 1, 19. 9. Barua AK, Raman SP, Triterpenoids-XII: The constitutionof albigenin—a new triterpene from Albizialebbeck benth, Tetrahedron 1962, 18, 155-159. 10. Ueda M, Tokunaga T, Okazaki M, Satan NU, Ueda K, Yamamura S. Albizia hexoside: a potential source of bioactivesaponin from the

leaves of Albizia lebbeck, Nat. Prod. Res, 2003, 17, 329-335. 11. www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd= Retrieve&db= PubMed&list_uids=10617059 12. PMID: 10617059 [PubMed - indexed for MEDLINE] 13. Council of the Pharmaceutical Society of Great Britain, 1911. 14. Indian Journal of Pharmaceutical Sciences. 2000 Sep-Oct.; 62(5): 347-50 15. PMID: 11801388 [PubMed - indexed for MEDLINE] 16. http://www.abchomeopathy.com/r.php/Acal 17. Indian Materia Medica by Dr. K.M. Nadkarni, Volume I, pages: 1719, Publisher: Bombay Popular Prakashan, reprinted: 2000. 18. Materia Medica (Vegetable section), Volume I, by Dr. Murugesa Muthaliar, pages: 359, publisher, Tamilnadu Siddha Medical Council, Chennai. Fourth edition 1988. 19. Thomas M.Walter , Review of Acalypha indica, Linn in Traditional Siddha Medicine, http://openmed.nic.in/2001/01/ Microsoft_ Word__Acalypha.pdf 20. Kirby MDK, Sherris JC, Turck M. Antibiotic susceptibility testing by standard single disc diffusion method. Am J Clin Pathol 1966; 45: 493-496. 21. National committee for clinical laboratory standards 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, PA.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 1.January 2012

69-71