Ethnobotanical Leaflets 12: 1085-89. 2008.

 

 

Antibacterial Activity of Punica granatum L. against Gastro Intestinal Tract Infection Causing Organisms

 

B.V. Pradeep1*, M.K. Manojbabu2 and M. Palaniswamy1

 

1Department of Microbiology, Karpagam University, Coimbatore – 641 021, India

2Department of Microbiology, P. G. P. College of Arts and Science, Namakkal – 637 206, India

*Corresponding author: bvpradeep123@rediffmail.com

 

Issued 01 December 2008

 

Abstract

 

            The pericarp of Punica granatum Linn. has been commonly employed as a crude drug in Indian traditional medicine for the treatment of diarrhoea as well as for use as an astringent, antihelminthic, asphrodisacs, laxative, diuretic, stomachic, cardiotonic and refrigerant. Antibacterial activity of P. granatum pericarp extracts was evaluated against ten Gastro Intestinal Tract (GIT) infection causing bacterial strains using paper disc agar diffusion method. The result indicated that the extracts obtained from P. granatum pericarp exhibited antimicrobial activity against all organisms except the crude extract used against Pseudomonas aeruginosa. The methanol extract has exhibited maximum antibacterial activity against Salmonella typhimurium, Salmonella typhi and Shigella dysenteriae Serotype 1. Methanol extract shows significant activity against tested bacterial strains when compared to other extracts used in the study. Our findings suggest that an appropriate bioactive compound(s) may be developed from P. granatum pericarp as complementary alternative medicine for the treatment of GIT infection causing bacterial strains.

 

Key words: Medicinal plant, Antibacterial activity; Punica granatum; Gastrointestinal Tract Infections

 

Introduction

 

            Ever since the dawn of civilization man has used plants for his food, shelter, and fodder for his animals. Plants were also identified for use to cure him from innumerable ailments which struck his physical being. They designated these plants as ‘medicinal plants’. In India, Ayurvedic system of medicine has existed for over four thousand years. From ancient literature it is evidence that the various parts of the plants were used in Siddha, Ayurvedha and Unani medicine for the treatment of disease of human beings (Palaniswamy et al., 2008).

            Punica granatum Linn (Pomegranate) belonging to family punicaceae, has long been esteemed as food and medicine, and is a diet in convalescence after diarrhoea (Nadkarni, 2000). It is used in Siddha, Ayurvedha and Unani medicine especially for the treatment of Gastro-Intestinal (GI) diseases.  Pomegranate is a fruit of great antiguity and is known to have been cultivated in the Middle East more than 5,000 years ago.  The plant is found all over India.  Pomegranate has been considered important since prehistoric times as an agency of longevity (Ram, 1998).  The fruit is good for dysentery, diarrhoea and gastralgia (Warrier, et al., 2002). Hindoo physicians use the rind of the fruit and flowers, combined with aromatics, such as cloves, cinnamon, coriander, pepper etc as bowel astringent in diarrhoea (Blatter, et al., 2001). In addition to its ancient historical uses, pomegranate is used in several systems of medicine for a variety of ailments. In Ayurvedic medicine the pomegranate is considered “a pharmacy unto itself” and is used as an antiparasitic agent, a “blood tonic,” and to heal aphthae, diarrhea, and ulcers (Jurenka, 2008).

            The fresh rind of the fruit contains: wax,0.8; resin, 4.5; mannitol, 1.8; non-crystallized sugars, 2.7; gums, 3.2; inulin,1.0; mucilage, 0.6; tannin, 10.4; gallic acid, 4.0; and calcium oxalate, 4.0%. Pectin occur to the extent of 2-4 % (Ram, 1998). Pomegranate peel combined with optimum level of aromatic such as cloves is a most useful remedy in chronic dysentery as well as diarrhoea. The rind is an antihelmintic and an astringent and useful in treating diarrhoea, dysentry and gastralgia (Prashanth, et al., 2001). Commonly used as febrifuge and part of the diet in convalescence after diarrhoea. Wet and dry fruit is good for heart, stomach and enhances the production of hemoglobin.  It is a good diuretic agent and gives strength.  Pulp is a good anti-diarrhoeal agent (Chaterjee and Pakrashi, 1991; Hussain, et al., 1992). Duraipandiyan et al., (2006) reported that dried fruit coat is grounded and mixed with water and taken internally to treat stomachache and diarrhoea. Extract of different parts of the fruit exhibited antibacterial activity.  Extracts of the whole fruit were highly active against Micrococcus pyogens, S. aureus, E.coli, and Pseudomonas aeruginosa. They were also very effective against intestinal pathogenic bacilli such as Salmonella paradysenteriae III-Z, S. typhi, S. monetevideo, S. scholtmuelleri and Shigella paradysentriae B.H. Alcoholic extracts of the fruit rind and root bark showed activity against Micrococcus pyogens 60% (Ram, 1998).

            Ingestion of pathogens can cause many different infections. These may be confined to the GIT or initiated in the gut before spreading to other parts of the body.  A syndrome characterized by GI symptoms including nausea, vomiting, diarrhoea and abdominal discomfort. Worldwide, diarrhoea diseases are the second leading cause of death; about 25 million enteric infections occur each year.  These infections cause significant morbidity and death, particularly in elderly people and children younger than age 5.  It has been estimated that 4 to 6 million children die each year from diarrhoea, particularly in developing countries in Asia and Africa. Even in developed countries, significant morbidity occurs as a result of diarrhoea illness, although acute diarrhoeal syndromes are usually self-limited, some persons with infectious diarrhoea will require diagnostic studies and treatment. The last decade has seen a resurgence of global interest in medicinal plants as therapeutic agents.

            This traditional treatment approach is of much significance in the world especially in India due to the endemic presence of infective gastrointestinal diseases, which are the major causes of infant and adult mortality. Knowing the activity of P.granatum a study has been carried out to know its antibacterial activity, which has been reported in this paper.

 

 

Materials and Methods

 

Plant Material

 

            The pericarp of the ripened and unripened fruit of P. granatum Linn. was selected for this study based on their traditional practices by Indians. Fresh fruits both ripened and unripened were collected from the local market, Namakkal, Tamil Nadu, India. Taxonomic identification of the plant was established.

 

Preparation of crude extracts

 

            Pericarp of ripened and unripened fruit was collected and washed with sterile distilled water. Samples were crushed into parts and squeezed to remove the crude extract. The crude extracts were filtered through sterile musculine cloth into vials.

 

Preparation of methanol and acetone extract

 

            The pericarp of ripened fruit was dried under shade and stored into fine powder using electric blender. 50g of dried powder sample was taken and extracted by soxhlet apparatus using methanol and acetone separately. The solvents were removed under reduced pressure in a rotary evaporator until they become completely dry. The residues were stored at 4°C for further use.

 

Antimicrobial screening

 

            The crude, methanol and acetone extracts of the pericarp of P. granatum was screened against a total of ten bacterial strains. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, S. paratyphi A, S. paratyphi B, S.  typhimurium, Shigella dysenteriae Serotype 1, Shigella flexneri Serotype 2 and Vibrio cholerae were obtained from the Christian Medical College and Hospital , Vellore, Tamil Nadu, India.

 

Determination of antibacterial activity

 

            The disc diffusion method (Voraruthikunchai et al., 2005) was used to screen the antibacterial activity. In-vitro antibacterial assay was screened by using Mueller Hinton Agar (MHA) obtained from HiMedia, Mumbai, India. The MHA plates were prepared by pouring 15ml of molten media into sterile petridishes. The Plates were allowed to solidify for 10 minutes and 0.1% inoculum suspension was swabbed uniformly and the inoculum was allowed to dry for 5 minutes. Sterile paper disc (6mm) were soaked with 10ml of extract residue diluted into corresponding extraction solvents, so that each disc was impregnated with 2.5mg of residue and dried at 37°C overnight. The loaded disc was placed on the surface of medium and the compound was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37°C for 24 hrs. Antibiotic discs containing Ofloxacin, Ciprofloxacin and Tetracycline (5-30µg) were used as controls. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone formed around the discs.

 

Results and Discussion

 

            The development of drug resistance in human pathogens against commonly used antibiotics has necessitated a search for new antimicrobial substances from other sources including plants and microbes (Erdogrul, 2002). The results on antimicrobial screening of the crude extracts of the P.granatum are shown in table 1. The antibacterial activity of the crude extract of both ripened and unripened pericarp extracts resulted in clear inhibition zones of atleast 10mm for all the strains tested except Pseudomonas aeruginosa strain. This antibacterial activity may be indicative of the presence of metabolic toxins or broad spectrum antibiotic compounds. This is in agreement with previous reports by the several researchers (Prasanth et al., 2001; Machado et al., 2002; Voravuthikunchai et al., 2005). Methanol extracts exhibited a higher degree of antimicrobial activity as compared with acetone extracts. Both methanol and acetone extracts of P.granatum pericarp showed high degree of antibacterial activity tested against GIT infection causing bacterial species which may be due to interesting novel secondary metabolites. Prasanth et al., 2001, reported that, different extracts of P.granatum fruit showed some antibacterial activity against Proteus vulgaris and Bacillus subtilis. Voravuthikunchai et al., (2004) reported that P.granatum contains large amount of tannins (25%) and the antibacterial activity may be indicating the presence of some secondary metabolites. The ethanolic extract of P.granatum showed some antibacterial activity against E.coli (Voravuthikunchai et al., 2005) and S.aureus (Machado et al., 2002).

 

Table 1.  Antibacterial activity of extract of P. granatum pericarp (concentration 2.5 mg / disc, inhibition zone in mm).

 

 Test organism

Unripened crude extract

Ripened crude extract

Methanol extract

Acetone extract

Ciprofloxacin (30mcg)

Ofloxacin (5mcg)

Tetracycline (30mcg)

Staphylococcus aureus

21

12

21

16

22

20

20

Escherichia coli

16

20

21

13

26

29

24

Pseudomonas aeruginosa

05

05

14

16

15

20

24

Salmonella typhi

18

22

21

22

20

20

23

Salmonella paratyphi A

10

16

15

15

18

18

19

Salmonella paratyphi B

16

12

18

14

15

18

20

Salmonella typhimurium

19

21

25

17

17

19

20

Shigella dysenteriae Serotype 1

21

20

22

12

20

20

25

Shigella flexneri Serotype 2

19

20

25

12

17

19

21

Vibrio cholerae

22

25

20

14

20

18

21

 

Conclusion

 

            The antibacterial activity of crude extract of unripened fruit of P. granatum is reported for the first time. Further phytochemical elucidations are required to determine the nature of compound(s) responsible for the antibacterial effects. This study is generally considered an effective approach in the discovery of new antibacterial agents from P. granatum.

 

Acknowledgements

 

The authors BVP and MP are sincerely grateful to the Management, Karpagam University, Coimbatore, Tamil Nadu, India for encouragement and support.

 

References

 

Blatter, E., Cains, J.F. and Mhaskar, K.S. Indian Medicinal Plants 2nd ed. Uttranchal, India: Oriental Enterprises, 2001, Vol 5, p1506-1513.

 

Chatterjee A, and Pakrashi S.C. The treaties of Indian Medicinal Plants. New Delhi, India: CSIR, 1991, p190-192.

 

Duraipandiyan, V., Ayyanar, M. and S. Ignacimuthu. (2006) Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complementary and Alternative Medicine, 6:35 doi:10.1186/1472-6882-6-35

 

Erdogrul, O.T. Antibacterial activities of some plant extracts used in folk medicine. Pharmaceutical Biology. 2002, 40: 269-273.

 

Hussain, A., Virmani, O.P. and Popli, S.P. Dictionary of Indian Medicinal Plants. Lucknow, India: CIMAP, 1992:384.

Jurenka, J.M.T. Therapeutic Applications of Pomegranate (Punica granatum L.): A Review. Alternative Medicine Review. 2008, 13:128-144.

 

Machado, T.B,. Leal, C.R., Amerl, A, C. F., Santos, K.R.N., silva, M.G. and Kuster, R.M. Antimicrobial ellagitannin of Punica granatum fruits. Journal of Brazilian Chemical Society, 2002, 13; 606-610

 

Nadkarni, A.K. Dr. K.M. Nadkarni’s Indian Materia Medica, 3rd ed. Mumbai, India: Popular Prakashan Private Limited, 2000: Vol. I, p1031-1035.

 

Palaniswamy, M, Pradeep, B.V., Sathya, R. and J. Angayarkanni 2008. In vitro anti-plasmodial activity of Trigonella foenumgraecum L. eCAM 2008; doi:10.1093/ecam/nen030; 1 - 5.

 

Prashanth, D., Asha, M.K., Amit, A. Antibacterial activity of Punica granatum. Fitoterapia, 2001, 72; 171-173.

 

Ram, A.  The Wealth of India, A Dictionary of Indian Raw materials and Industrial products, Raw material. 1998, Vol VIII: Pe – Re. National institute of scientific communication, Council of Industrial and Scientific Research, New Delhi.

 

Voravuthikunchai, S., Lortheeranuwat,A., Jeeju,W., Sririrak, T., Phongpaichit, S. and Supawita, T. Effective medicinal plants against Enterohaemorrhagic Escherichia coli O157: H7. Journal of Ethanopharmacology. 2004, Vol. 94, p49-54.

 

Voravuthikunchai, S.P., Sririrak, T., Limsuwan, S., Supawita, T., Iida, T. and T. Honda. Inhibitory effect of active compounds from Punica granatum Pericarp on verocytotoxin production by Enterohemorrhagic Escherichia coli O157: H7. Journal of Health Science, 2005, Vol. 51, p590-596.

 

Warrier, P.K., Nambiar, V.P.K., Ramankutty, C. Indian Medicinal Plants a compedium of 500 species. Hyderabad, India: Orient Longman Private Limited, Hyderabad, 2002: Vol 4, p396-402.