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1 IihvlIiWlIflllV I In Vitro Study of Pomegranate Used for the Treatment of Diarrhea: Anti-bacterial Activity Malyn Chulasiri B.S. Pharm., Ph.D.* Orasa Suthienkul B.Sc., M.S.** Abstract Pomegranate plant (Punica granatum L.), a medicinal herb which has been used to treat diarrhea in Thai traditional medicine, was examined for its effect against enteropathogenic bacteria in vitro. Results from the study revealed that the boiled-water extract of dried Pomegranate peels could inhibit the growth of many enteropathogenic bacterial strains especially Shigello. The antibacterial activity of this extract was further demonstrated to be bactericidal..... ~. ~ I -4 1 clci 91 IV q IV 'i ' vq l'jinljij fll1ftfllll lnhl6~yl~"6-3yliflu1'\jthfluqylfiylhfllnflllli1fl6;);)11~11-3'\j6-3yluyl)j: """" qylfifll1vlllh'lf6uuflyl11u Ina'U 'OtU;;" ilnn f191lltj,m, 111f11"llH~;h1'Yl[n f1m:;llli"ll"lthl'~. **f1m:;ffllil1mff"lj"1'g{~1 3J'V111'Yl[Jl~mJ~"~, v ' "'It.'" V "'" 711/111 T:1F1~'1Jl'lJ8lUl::lJ1~TIJVfl'lJ'ff 2529; 3: , nuihj (Punica granatum L.) lii'uyt'llfllj'u'vt'~'~':::u,n'u9iu1tjlfllj'u'vt1'y1tji1f11ljl'tl, ti.., l'.. ~ q, IV ti _I.at "... '", 'II'fl1l1, flil 'O'OU:::' 1~ Ht1'01flflU ftfl1l1fl1 ~'UvtU11f11'f1flVl'Ul91lJ '01fllut1ilflH t1i1'1h"uil~vt'llvl~flt1l1 ",'" "". Jii q.e:t ""...ca ".:. tjufj~fl1?1;nil!"uil~i'iiillluflyli'tj'i1 t11tjfl1tjvt'u'l,y1ii1'ufl11'11 \J!"Uil~ 1, flt1 '0'01':::' 1~1V1tJ l11vtl:::iltjhtj~ Shigella.. ti.ii.e!'i" I.Ii 1 1It1:::qYlfj9l1'UI'IIilYlPtn'OvtUIU'U'II'UVI~1Y11t11tJl'IIil 'fl INTRODUCfION In spite of the modern and advancement of technology in medicine, diarrheal disease is still a major world health problem. Generally, the proper management with fluid and electrolytes is the most important aspect in the treatment of diarrheal patients.! In many cases, however, the applications of other categories of the treatment to shorten the duration of *Department of Microbiology, Faculty of Pharmacy, Mahidol Univer sity **Department of Microbiology, Faculty ofpublic Health, Mahidol University clinical symptom are needed. These include the correction of nutritional deficit and the using of antidiarrheal agents. Four main groups of the latter are known. They are stool thickening agent, antimotility agent, antisecretory agent and antimicrobial agent. Among developing countries where some specific bacterial infections are common, the use of antimicrobials in the anagement of acute diarrhea has been found to be effective, although such chemotherapy is not recommended in the well-developed countries. 2 According to the suggestion of WH0 3 concerning the application of antimicrobial therapy in bacteria causing diarrhea, only cholera and shigella cases are specified to be treated by antimicrobials. However, in malnourished or severely debilitated patients, 72

2 J Infect Dis Antimicrob Agents in patients with depressed immunity or severe symptom, these agents are required although they are infected with other enteric bacterial infections. 2,4 In the notion of easy access, availability as well as in economic considerations, it is accepted that medicinal herbs which are since centuries well known, c~ot be separated from Primary Health Care. In the study of the utilization of medicinal herbs in Thailand, Muangman et al 5 revealed that more than 50 per cent of the total respondents still used herbal medicine. The users were found to have a lot of trust in these drugs and claimed for high rate of cure in various common complaints such as fever, headache, diarrhea. However, these claims were their subjective feelings which needed strict scientific testing. Pomegranate plant (Punica granatum L.) is one of medicinal herbs popularly employed in Thai folklore medicine. Its fruitpeel has been used in the treatment of diarrhea. 6 In a preliminary study, the crude aqueous extract obtained from blending the fresh Pomegranate peels with water were found to be effective against enteropathogenic bacteria. 7 The antibacterial compound(s) in this extract was further demonstrated to be tolerant to drying, heat stable and bactericidal. The present work was designed to investigate whether the boiled-water extract derived by boiling dried peels with water would exhibit the same effect. MATERIALS AND METHODS Plant Material and Extraction Fruits of Pomegranate were purchased from local markets in Bangkok. They were washed and the peels were removed. These peels were dried under sunligat. The dried peels were boiled with water (in a ratio of 1:10, w/v) for 15 min. After cooled to room temperature, the boiled mixture was filtered and the filtrate was evaporated on a hot water bath (90 C) in an evaporating dish until it was dry. The dried residue obtained was designated here as the "boiled-water extract". The extract was redissolved in hot water before employed in any tests. Bacterial Culture Twenty-five enteropathogenic bacterial strains isolated from human diarrheal stools were used in this investigation. These included five isolates each of Aeromonas, Escherichia, Salmonella, Shigella and Vibrio. Their species, strains and suppliers are listed in Table 1. E. coli ATCC and Staphylococcus aureus ATCC were control strains employed in the study. All strains were maintained at room temperature in sealed tubes of "Tryptic Soy semisolid medium" (20 g of Tryptic Soy llgar [Difco Laboratories, Detroit, Mich.] in 1 liter of water). Vol. 3 No.2 Apr. - Jun Antibacterial Sensitivity Test Disc diffusion test The method as described by WH0 8 was used. Briefly, each bacterial strain was grown in Mueller Hinton broth (MHB; Difco) and incubated at 37 C for 18 h. The cultures were diluted in same broth to match the BaS0 4 standard (prepared by adding 0.5 ml of M BaCl 2 to 99.5 ml of 0.18 M H 2 S04 ). Each inoculum was distributed evenly on Mueller Hinton agar (MHA; Difco) plates. Discs (6.5 mm) containing the extract (5000 JJg) and control discs (discs with only water and ampicillin sensitivity discs, 10 JJg) which had been previously put at 37 C overnight were applied to the surface of the seeded plates. All plates were incubated at 37 C for h and zones of inhibition were then measured. The bacteria were interpreted to be sensitive to the extract if zones of inhibition appeared to be :> 10 mm in diameter. Agar dilution test Doubling dilutions of the extract were incorporated into MHA to provide final concentrations ranged from 39 JJg/ml to 5000 JJg/ml. The ampicillin control plates were similarly done to give final concentrations ranged from 0.78 JJg/ml to 100 JJg/ml. The cultures which grew for 18 hat 37 C in MHB were diluted further in the same broth. A 10-4 or 10-5 dilution of the inoculum was inoculated onto the extract, ampicillin and extract-free plates. All plate was incubated at 37 C for h. The minimal inhibitory concentration (MIC) was the lowest concentration of the extract or drug that inhibited growth. The presence of a single colony was ignored. Broth dilution test Serial 2-fold dilutions of the extract were made in 1 ml of MHB. The range of the extract concentrations was similar to those used in the agar dilution test. The preparation of the test organisms was also similar except that a 10-3 dilution of the overnight culture was employed. One-twentieth ml of the diluted inoculum was inoculated into all tubes including a control tube containing no extract. Following incubation at 37 C for h, the tubes were examined for growth as indicated by turbidity and the MIC determined. Tubes containing no visible growth were subcultured by inoculating 0.05 ml of broth onto MHA plates. The plates were incubated at 37 C for h. The minimal bactericidal concentration (MBC) of the extract was the lowest concentration of the agent from which fewer than 10 colonies grew. RESULTS The quantities of the boiled-water extract of dried Pomegranate peels were shown in Table 2. Yields obtained were approximately 20%. When the effect of the extract in inhibiting enteropathogenic

3 74 lid 3 uvv~ 2 W.O. -ii.o Table 1 list of enteropathogenic bacteria studied Species Organism Strain Received from A. hydrophila E. coli, enterotoxigenic S. anatum S. derby S. Krefeld S. typhi S. jlexneri S. dysenteriae V. cholerae, El Tor, Inaba Ogawa non 0-1 V. mimicus V. parahaemolyticus 215 BC BC BC 1 439BC BI 1 (STH 001)** 2 (XAC 101) 1 (ST 201) 2 (LT 301) 2 (STLT 401) 2 ST404 3 ST215 3 ST Y Y Y Y Y Y Y Ct Ct AD 69 4 W60 4 DB , Armed Forees Research Institute of Medical Science, Bangkok; 2, International Centre for Dianheal Disease Research, Bangladesh, Dacca; 3, Rajvithi Hospital, Bangkok; 4, Bamrasnaradura Hospital, Nonthaburi Originalstrain not known, but the strain designated here was as shown in parentheses Table 2 Yields of the boiled-water extract of Pomegranate Sample Wt of dried peels (g) Wt of extract (g) (22.0%) (19.7%) (21.2%) (18.5%) Total (19.6%) bacterial growth was studied by disc diffussion. method, it was found that this extract inhibited the growth of all strains (5/5) of Aeromonas and Shigella, 1 strain (1/5) of Vibrio, and non (0/5) of Escherichia and Salmonella (Table 3). In agar dilution test, the MICs of the extract for the sensitive and resistant strains tested were" 156 and >- 312 /Jg/ml, respectively. For control strains, E. coli ATCC was resistant to the extract, while S. aureus ATCC was sensitive. These two strains had MICs of the extract of 2500 and" 39 1Jg/ml, respectively. From Table 3, it can be observed that the order of sensitive bacteria to the extract was Shigella> Aeromonas > Vibrio> Salmonella> Escherichia. In comparison with ampicillin, a different picture was obtained. As shown in same Table, Escherichia and Shigella were most sensitive and resistant to ampicillin, respectively. In case of Vibrio, V. parahaemolyticub was sensitive to the extract but resistai:1t to this antibiotic. When broth dilution method was employed for

4 J Infect Dis Antimicrob A2ents Vol. 3 No.2 Apr. - Jun Table 3 Evaluation of inhibitory effect against enteropathogenic bacteria of the boiled-water extract of Pomegranate by disc diffusion and agar dilution tests Disc diffusion Agar dilution Organism zone of inhibition (mm) MIC {s.lg/ml)t Extract Ampicillin Extract Ampicillin SOOO/Jg 10/Jg A. hydrophilia215 BC BC BC BC BI E. coli (STH 001) (XAC 101) (ST 201) (LT 301) (STLT 401) < 0.78 S. anatum ST S. derby ST S. krefeld ST > 100 S. typhi Y Y < 0.78 S. [lexneri Y > 100 Y Y > 100 Y <39 > 100 S. dysenteriae Y < 0.78 V. cholerae Ct Ct AD V. mimicus W V. parahaemolyticus DB < E. coli ATCC S. aureus ATCC <39 < 0.78 *See Table 1 **Average of 3 discs tested on MHA ttesu were done in triplicate detecting activity, results in Table 4 reveal that the MICs of the boiled-water extract of Pomegranate detected by this method were higher than those detected by agar dilution test. However, the MICs of the extract for inhibition the growth of the tested bacteria as determined by both techniques were correlated. Shown in same Table are the results of in vitro MBC test. It can be seen that the ratios of MBC/MIC of the extract for most strains tested were about 1-2. DISCUSSION Our previous investigations revealed that the crude aqueous extract, which 0 btained from blending the fresh Pomegranate peels with water, exerted the effect against enteropathogenic bacteria. 7 In the present study such activity was also demonstrated when the boiled-water extract of Pomegranate, which obtained from boiling dried peels with water, was em

5 76 Table 4 Comparison of MIC and MBC of the boiled-water extract of Pomegranate against enteropathogenic bacteria performed by broth dilution method* Organism A. hydrophilia 215 BC 135 BC 385 BI E. coli (STH 001) (ST 201) (STLT 401) S. anatum ST 404 S. krefeld ST 142 S. typhi Y S. flexneri Y Y S. dysenteriae Y V. cholerae Ct 784 AD 69 V. parahaemolyticus DB120 E. coli ATCC S. aureus ATCC Telts were done in triplicate MIC (pg/ml) MBC (pgfml) MBC/MIC >5000 > ployed. Considering the diameter of the zone of inhibition determined by disc diffussion method, the MIC by agar and broth dilution methods and number of sensitive strain found, the order of sensitive bacteria rapid emergence of antibiotic resistant Shigella strains has posed a problem on the selection of antibiotics used. A search for new antimicrobial agents for the treatment of shigellosis is needed. It seemed to be lucky to discover that the boiled-water extract of to the boiled-water extract of Pomegranate was Pomegranate had promising antibacterial effect on Shigella > Aeromonas > Vibrio > Salmonella > Shigella strains tested in vitro, and also possessed Escherichia (Tables 3 and 4). This extract also produced inhibitory effect on S. aureus ATCC which was used as the control strain. Moreover, the boiled-water extract of Pomegranate could inhibit some other gram-positive bacteria, e.g., Bacillus subtilis, Sarcina lutea (Authors, unpublished data), suggested a relatively broad spectrum antibacterial activity of the extract. In comparison with ampicillin, the different inhibitory some good antibacterial properties. As shown in the test, the extract was not effective against enterotoxigenic E. coli strains tested and E. coli ATCC Normal E. coli flora of the intestine were assumed not to be affected either. Because of no inhibitory activity on E. coli, the extract was expected to cause a little or no change in intestinal flora. It is apparent that many commonly used antibiotics can destroy resident bacteria, particularly effect was observed. This antibiotic was E. coli, in the intestine. As a results, the change in shown to be most effective against Escherichia while bacterial flora of the gut occurs, and hence the least effective against Shigella. Recommended by normal function of the gastrointestinal tract is usually WH0 3, ampicillin is the drug of choice used in the affected. Also, the present study revealed that the treatment of shigella diarrhea. However, many reports ratios of MBC/MIC of the boiled-water extract of reveal that shigella organism is easily resistant Pomegranate were about 1-2. It was therefore pre to the antibiotics commonly employed in the treatment.2,~12 The findings of ampi~illin resistant Shigel was bactericidal, and this is well accepted that bactesumed that the antibacterial activity of the extract la strains in this study were therefore undoubtful. The ricidal antibiotics are better than bacteriostatic ones.

6 J Infect Dis Antimicrob Agents Vol 3 No.2 Apr. -Jun Because of the properties described above, it is hoped that the boiled-water extract of Pomegranate which can be easily prepared would be effectively used in the household for the treatment of bacterial diarrhea especially that coused by Shigella. However, there is a need to explore some other properties required for such treatment before this extract is recommended for public use. ACKNOWLEDGEMENT This work was supported in part by grant RES/ 3/2527/10 from the ASEAN Training Centre for Primary Health Care Development Project. We are indebted to Dr. S. Vibulbandhitkij, Bamrasnaradura Hospital, Nonthaburi; Miss N. Surapetana, Rajvithi Hospital, Bangkok; Dr. P. Echeverria, AFRJM, Bangkok and Dr. M.1. Huq, JCDDR, Dacca, for generous gifts of the clinical bacterial strains used in this study. REFERENCES 1. World Health Organization. Treatment and prevention of dehydration in diarrheal diseases: a guide for use at the primary level. Geneva: WHO, Leksomboon U. Antimicrobial therapy. In: Seminar on diarrheal disease of children in Southeast Asia in the context of primary health care, 9-12 Nov Bangkok: Faculty of Tropical Medicine Mahidol University, 1981.!!. World Health Organization. A manual for the treatment of acute diarrhea. Geneva: WHO, Pathnacharoen S, Vibulbandhitkij S, Wamkitcharoen S, Varavithya W. Accuracy of clinical diagnosis of bacterial diarrhea in adults. Remathibodi Med J 1984; 7: 18!! Muangman D, Klasunthorn R, Hiranrak A, et al. A report on the nationwide survey in the utilization of medicinal herbs in Thailand. Bangkok: Faculty of Public Health, Mahidol University, 198!!. 6. Muanwongseyat P. Herbal medicine manual 2nd ed. Bangkok: Medical Media Press, 198!!. 7. Chulasiri MU, Suthienkul 0, Pavalo C, WongkrajarkN. Herbal extracts for diarrheal treatment: antibacterial activity in vitro. J Pub Hlth 1986; 16:21-!!5. 8. World Health Organization. WHO expert committee on biological standardization. Geneva: who, Akiba T. Mechanisms of develoj;lment of resistance in Shigella. 15th Gen MeetJap Med Ass 1959; 5: Farrar WE Jr, Eidson M. Antibiotic resistance in Shigella mediated by R factors. J Infect Dis 1971; 127: Leksomboon U, Echeverria P, Sovongse C, et al. Viruses and bacteria in pediatric diarrhea in Thailand: study of multiple antibio tic-resistant enteric pathogens. AmJ Trop Hyg 1981;!!0: Nakaya R, Nakamura A, Murata Y. Resistance transfer agents in Shigella. Biochim Biophys Res Commun 1960;!!:654-9.