Key words

Passiflorasubpeltata, Phytochemical screening, Secondary metabolites, Antibacterial activity.

Introduction

Nature provides everything for the wellbeing of human being. For achieving this nature protects man from various diseases. She stores her magical power for curing diseases in plants and various organic and inorganic compounds. Of these plants play a major role. The only thing is that we have to identify and apply them in the proper way. WHO estimates that about 80% of the global population still relies on plant based medicines for primary health care, and a major part of this therapy involves the use of plant extract or their active principles. This estimate still shows an increase day to day.

Health-care, which was a part of the traditional culture of the people, has become a professional in the modern industrial world. Synthetic drugs created by the affluent and influential pharmaceutical industries have given rise to effects which are more dangerous than the diseases they claim to cure. The world’s attention has again turned to traditional medical systems. The medicinal properties of a crude drug depend on the presence of one or more chemical constituents of physiological importance. They may be glycosides, alkaloids, tannin, resins or enzymes.

Plants are rich reservoir of antimicrobials. A single plant is known to contain several active principles of biological significance. Plant base antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobials. Biomolecules of plant origin appear to be one of the alternatives for the control of these antibiotic resistant human pathogens. The last two decades have witnessed increased investigation on plants as a source of human disease management due to the genetic variability of microorganisms against the antibiotics. The development of multidrug resistance in the pathogenic bacteria parasites has created major clinical problems in the treatment of infectious diseases. In recent times, there has been increasing interest in the study of bioactive compounds from peels, seeds, leaves, flowers and stem bark due to their antioxidative, antimicrobial and other health promoting properties [1].

In this study we are reporting the results of preliminary phytochemical screening and antibacterial activity of different solvent extracts of leaves and fruits of P.subpeltata(Fam: Passifloraceae) in order to orient future investigations towards the finding of new potent and antiinfectious compounds.

The passion flowers or passion vines (Passiflora) have a genus of about 400 species of flowering plants and the largest in the family of Passifloraceae[2][3]. Plants from the genus Passiflorahave been used in traditional medicine by many cultures [4]. The genus Passifloramay be suitable for the screening of bioactive molecules, since ethnobotanical use, chemotaxonomic information, and observation of the interaction of the plants with their environment have been suggested as selection criteria for potential sources of natural molecules of pharmacological relevance [5][6]. Flavonoids, glycosides, alkaloids, phenolic compounds and volatile constituents have been reported as the major phyto-constituents of the Passifloraspecies [7].

P.subpeltata(Syn. P. calcarata) or white passion flower, is a type of climbing vine native to Brazil, Mexico, Central America and more northern parts of South America but a weed in New South Wales, Australia. Known to be growing wild in California. It is also found as an escaped ornamental to Zimbabwe and Africa. In India it is distributed in southeren states mainly Karnataka (Chikmagalur, Coorg, Hassan, Mysore dist.), Kerala (Kottayam, Palakkad dist.) and Tamil Nadu (Dindigul, Tiruvannamalaidist). This pale green climber has tendrils. Leaves grow up to 10 cm long and are pale green and usually three-lobed. The lobes have tips more or less rounded. Between one and three glands are scattered along the leaf stalk. Two leaf-like stipules occur where the leaf stalk meets the stem. Flowers are white, tinged with green and about 5 cm across. The fruit is oval-shaped and bluishgreen in color, a berry, about 4 cm long, spread by birds, animal, water and humans. White passion flower will grow over other vegetation and smother wanted plants and shrubs. Their fruits and leaves are edible. In Western Ghats of India, P. subpeltataleaves are widely consumed as a green leafy vegetable [8]. In traditional medicine, the leaves and fruits are used in the treatment of jaundice. A coumarin derivative, cyanogenic glycosides- barterin have been isolated in the P. subpeltataand the compound was found to possess sedative, anti-plasmodic and antimicrobial properties [9][10]. Saravanan and Parimelazhaga reported that the seeds of P. subpeltatahave promising therapeutic value for natural antioxidant and antimicrobial properties [11]. They also reported that the acetone extract of P. subpeltataleaves acquire substantial antioxidant, analgesic, anti-inflammatory and antipyretic properties. No reports are available revealing the antibacterial potential of P. subpeltataleaves and fruits. Hence the present study was undertaken.

Materials and Methods

Collection of plant materials

P.subpeltataplants were collected from Aigoor village (Somwarpettaluk, Kodagu district, Karnataka state), identified using standard flora [12] and authenticated at the Taxonomy Section of Department of studies in botany, University of Mysore, Mysore. Fresh leaves and fruits of P.subpeltata were used for the preparation of aqueous and different organic solvent extracts.

Preparation of Solvent extracts

The leaves and fruits of P.subpeltata were separately washed thoroughly 2-3 times with running water and once with sterile water, chopped into small pieces, shade dried, coarse powdered in a mechanical grinder, sieved and used for extraction. The dried leaf and fruit materials (50gm) were extracted separately with petroleum ether, chloroform, ethyl acetate, and methanol in the increasing order of their polarity by using Soxhlet apparatus [13]. The extract was decanted, filtered with Whatman No. 1 filter paper, concentrated and preserved at 5oC in air tight bottle until further use. All the extracts were subjected to antibacterial activity assay and preliminary phytochemical analysis.

Test microorganisms

The test microorganisms used were Escherichia coli, Klebsiella pneumonia, Enterobacteraerogenes (gramnegative), Staphylococcus aureus, and Enterobacterfaecalis (Gram- positive). All these microorganisms were obtained from Herbal Drug Technology Laboratory, Department of studies in Microbiology, University of Mysore, Mysore, Karnataka.

Determination of antimicrobial activity

Antibacterial activity of the aqueous and organic extracts of the plant sample was evaluated by the paper disc diffusion method [14]. The bacterial cultures were adjusted to 0.5McFarland turbidiometric standard and inoculated onto Nutrient agar plates (diameter: 15 cm) and incubated at 37 o C for 18h.Sterile filter paper discs (diameter 6 mm) impregnated with 100 μl of extract dilutions reconstituted in minimum amount of solvent at concentrations of 20 to 100 mg/ml were applied over the culture plates. Paper discs impregnated with 20 μl of a solution of 10 mg/ml of streptomycin as standard antimicrobial was used for comparison. Filter paper discs dipped into sterile distilled water and allowed to dry were used as control. The plates were then incubated at 37oC for 24 h. Antibacterial activity was determined by measurement of zone of inhibition around each paper disc. For each extract three replicate trials were conducted against the test organism.

Table 1

Results of Antibacterial activity of different solvent extracts of leaves and fruits of P. subpeltata by paper disc diffusion method (zone of inhibition in mm)

Streptomycin PE CL EA MT AQ
LEAVES
E.coli 18±1.414 9 ± 1.414 12 ± 2.121 8 ± 2.121 15 ± 6.212 11 ± 1.414
K.pneumoniae 18 ± 1.414 10 ± 2.828 10 ± 1.414 8 ± 6.414 13 ± 4.121 8 ± 1.414
E.aerogenes 20 ± 2.828 13 ± 1.414 8 ± 1.414 12 ± 2.828 12 ± 6.282 12 ± 2.828
S.aureus 22 ± 2.828 8 ± 1.414 14 ± 6.212 10 ± 2.828 12 ± 2.121 9 ± 2.828
E. faecalis 20 ± 1.414 15 ± 1.414 10.5 ± 2.121 11 ± 1.212 13 ± 1.212 8 ± 1.414
FRUIT
E.coli 18±1.414 8 ± 2.121 6.4 ± 1.212 13 ± 1.414 14 ± 1.412 12± 1.414
K.pneumoniae 18 ± 1.414 11 ± 6.212 9 ± 1.414 14± 1.414 15 ± 2.121 8 ± 1.414
E.aerogenes 20 ± 2.828 15 ± 2.212 12 ± 1.414 8 ± 1.414 13 ± 2.121 11 ± 2.828
S.aureus 22 ± 2.828 12 ± 2.121 6 ± 2.828 12 ± 2.828 8± 2.828 6 ± 1.414
E. faecalis 20 ± 1.414 12± 2.121 5 ± 2.828 6 ± 2.212 16 ± 2.121 13 ± 2.828

PE- Petroleum ether CL- Chloroform, EA- Ethyl acetate, MT- Methanol, AQ- Aqueous

Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) of the methanol and ethyl acetate extracts was estimated for the test organisms in triplicates, method described by Doughariet al [15]. To 0.5 ml of varying concentrations of the extracts (20.0, 18.0, 15.0, 10.0, 8.0, 5.0, 1.0 and 0.5 mg/ml), 2 ml of nutrient broth was added and then a loopful of the test organism previously diluted to 0.5 McFarland turbidiometric standard was introduced to the tubes. The procedure was repeated on the test organisms using the standard antibiotic -Streptomycin. A tube containing nutrient broth only was seeded with the test organism as described above to serve as control. All the broth cultures were then incubated at 37 oC for 24 h. After incubation the tubes were then examined for microbial growth by observing for turbidity. The lowest concentration showing no turbidity in the tube was considered as the MIC.

Table 2

Minimum inhibitory concentration (in µg/ml) of ethyl acetate and methanol extracts of leaves and fruits of P.subpeltata against selected bacteria

MIC(µg/mL)
Extract E.coli K. pneumoniae E.aerogenes S.aureus E. faecalis
Leaves Ethly acetate 2.5 3 2.5 2.5 2.5
Methanol 2 2.5 2 2 2
Fruits Ethyl acetate 2.5 2.5 3.5 3.5 3
Methanol 2 2.5 2 2 2
Control Streptomycin 2 1.5 3 2.5 3.5

Preliminary phytochemical screening

The preliminary phytochemical tests were conducted to find the presence of the active chemical constituents such as alkaloids, glycosides, flavonoids, triterpenes, sterols and tanninsfollowing method adapted by Harbone[16] and Edeogaet al[17].

Alkaloids

The extract was dissolved in 1N Hcl, filtered and the filtrate was used for alkaloid test.

i. Mayer’s test (Potassium mercuric iodide solution). The filtrate was treated with a few drops of Mayer’s reagent and observed for the presence of yellow precipitation.

ii. Dragendroff’s test (Potassium bismuth iodide solution). The filtrate was treated with a few drops of Dragendroff’s reagent and observed for the presence of orange precipitation.

iii. Wagner’s test(solution of iodine in Potassium iodide). The filtrate was treated with a few drops of Wagner’s reagent and observed for the presence of reddish brown precipitation.

Terpenoids and sterols

i. Salkowski’s test. The extract was treated with few drops of chloroform and con. Sulphuric acid, shaken well, allowed to stand, appearance of golden yellow colourin the lower layer indicated the presence of triterpenoids and red colour in the lower layer indicated sterols.

ii. Libermann-Burchard test. The extract was treated with a few drops of chloroform and acetic anhydride and mixed well. 1 ml of con. Sulphuric acid was added from the sides of the test tube. Appearance of red ring at the junction of two layers indicated the presence of triterpenoids. Appearance of a brown ring at the junction of two layers and the upper layer turned green indicated the presence of sterols.

Flavonoids

i. Shinoda test. Extract was dissolved in methanol and to this a small piece of magnesium ribbon was added. 1 ml of con. Hcl was added from the sides of the test tube. Formation of magenta colour indicated the presence of flavonoids.

ii. Lead acetate test. Extract was treated with a few drops of 10% lead acetate. formation of yellow precipitate indicated the presence of flavonoids.

iii. Ferric chloride test. Extract was treated with a few drops of 1% neutral ferric chloride solution. Formation of blackish green colour indicated the presence of flavonoids.

Glycosides

i. KellarKillani test. The extract was mixed with a few drops of glacial acetic acid, boiled for a minute and cooled. To this solution, 2 drop of ferric chloride solution and then 1 ml of con. Sulphuric acid was added from the sides of the test tube. Formation of reddish brown ring at the junction of two layers indicated the presence of glycosides.

ii. Molisch’s test. The extract was mixed with 1 ml of Molisch’s reagent and to which 1 ml of con. Sulphuric acid was added from the sides of the test tube. Formation of reddish violet ring at the junction of two layers indicated the presence of glycosides.

Tannins

i. Gelatin test. To the extract, 1% gelatin containing 10% sodium chloride was added. Formation of White precipitate indicated the presence of tannins.

ii. Ferric chloride test. The extract was mixed with few drops of 1% neutral ferric chloride solution. Formation of blue-green or brownish colour indicated the presence of tannins.

Saponins

i. Foam test. The extract was shaken vigorously with little quantity of distilled water. The formation and persistence of honey comb like foam indicated the presence of saponins.

ii. Haemolysis test. The extract was mixed with a drop of blood and observed under the microscope. Occurrence of haemolysis (destruction of RBCs) indicated the presence of saponins.

Table 3:Qualitative preliminary phytochemical analysis of leaf and fruit extracts of P.subpeltata
Phytocontituents Tests PE CL EA MN AQ
Alkaloids Dragendroff’s test
Wagner’s test
Mayer’s test
Glycosides Keller Killani test ++ ++
Molisch’s test ++ ++
Flavonoids Shinoda test ++ ++ ++
Ferric chloride test ++ ++ ++
Lead acetate test ++ ++ ++
Triterpenes Libermann-Burchard test
Salkowski’s test
Sterols Libermann-Burchard test ++ ++ ++ ++
Salkowski’s test ++ ++ ++ ++
Tannins Gelatin test ++ ++ ++
Ferric chloride test ++ ++ ++
Saponins Foam test ++ ++
Haemolysis test ++ ++

++ Present, — Absent

PE- Petroleum ether, CL- Chloroform, EA-Ethyl acetate, MN- Methanol, AQ- Aqueous

Results and Discussion

Antibacterial activity

Antibacterial activity of the all solvent extracts is presented in Table 1. All the extracts showed broad spectrum activity against the bacterial strains used. The ethyl acetate and methanol extract showed significant antibacterial activity as compared to chloroform and petroleum ether extracts of leaves. The methanol extract of leaves showed highest inhibitory activity against E. coli (15 ± 6.212 mm), K.pneumoniae (13 ± 4.121mm). The Pet.ether extract of leaves showed highest inhibitory activity againstE.aerogenes(13 ± 1.414) and E. faecalis(15 ± 1.414). The chloroform extract showed highest inhibition against S.aureus(14 ± 6.212).

The ethyl acetate and methanol extract of fruits showed significant antibacterial activity. The methanol extract of leaves showed highest inhibitory activity against E. coli (14 ± 1.412 mm), K.pneumoniae (15 ± 2.121mm) and E.faecalis (16 ± 2.121mm). The Pet.ether extract of leaves showed highest inhibitory activity againstE.aerogenes(15 ± 2.212) and ethyl acetate extract against S.aureus(12 ± 2.828).

Streptomycin (standard antibacterial drug ) have shown inhibitory activity ranged from 18 ± 1.414 mm to 22 ± 2..828 mm at a concentration of 20 μl of a solution of 10 mg/ml. The sensitive test result, showed that the extracts were less potent than the standard antibiotic-streptomycin, used in the study. Generally, the reduced efficacy of the extracts, relative to the standard antibiotic, used in the study may be due to the fact that, they are still crude and require further purification.

The result of MIC assay of methanol and ethyl acetate extracts is shown in Table 2. Both extracts showed broadspectrum activity against the bacterial strains used. Methanol extract of both leaves and fruits exhibited the highest antibacterial efficacy against S.aureus, E.faecalis and E.aerogenes each at 2.0µg/ml concentration when compare to Streptomycin with MIC values of 2.5µg/ml, 3.5µg/ml and 3.0µg/ml respectively. The ethyl acetate extracts of leaves showed a good bactericidal activity at 2.5µg/ml against E.aerogenesand E.faecaliswhen compare to standard antibiotic with MIC values of 3.0 µg/ml and 3.5µg/ml respectively. In this study, E.aerogenes, S.aureus, and E.faecaliswere found to be sensitive to methanol and ethyl acetate extract of both leaves and fruits of P.subpeltata.

Preliminary phytochemical analysis

Information on chemical constituents of plants helps for the discovery of novel drugs.The qualitative phytochemical analysis of leaves and fruits of P.subpeltatais depicted inTable 1. It is clear from the experimental data presented that the substances like sterols, glycosides, flavonoids, and tannins are medicinally active components of leaves and fruits of P. subpeltata. These bioactive components are naturally occurring in most plant materials, known to be bactericidal and fungicidal, thus conferring the antimicrobial property to plants [18]. All solvent extracts confirmed the presence of sterols. The ethyl acetate and methanol extract confirmed the presence of glycosides, flavonoids, sterols, tannins and saponins. But alkaloids and triterpenes were absent in all solvent extracts.

Flavonoids are hydroxylated phenolic substances. The biological functions of flavonoids include: protection against allergies, inflammation, free radicals, platelet aggregation, microbes, ulcers, hepatotoxin, viruses and tumours [19][20]. Their activity is probably due to their ability to complex with bacterial cell walls. More lipophilic flavonoids may also disrupt microbial membranes [21]. They exhibit activity against a wide range of gram positive bacteria as well as fungi [22]. Flavonoids are found in ethyl acetate, methanol and aqueous extracts.

Phenolic compounds are reported to act as antimicrobial activities [23]. Tannin is a general descriptive name for a group of polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution. They are found in almost every plant part: bark,wood,leaves, fruits and roots [24]. Many human physiological activities, such as stimulation of phagocytic cell, host mediated tumor activity and wide range of anti-infective actions, have been assigned to tannins [25]. Their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope, transport-proteins etc.[26]Ethyl acetate and methanol extracts of P.subpeltata showed highest phenolic content. The presence of the phenolic compounds in these studied samples proved that they have antimicrobial activity.

The results show that antibacterial potency of P.subpeltata may be due to the presence of some active principles like flavonoids, sterols, cardiac glycosides, tannins and saponins. This result agrees with the report of Trease and Evans [27].

Conclusion

P.subpeltata, a weed, contains medicinally useful phytochemicals, such as flavonoids, sterols, tannins, saponins, and cardiac glycosides. These substances are antibacterial and could be extracted for bacterial management, pharmaceutical exploits, research in microbiology, biotechnology and general medicine. Bioactive substances from P.subpeltataplantcould be employed in the formulation of antibacterial agents for the treatment of various bacterial infections.Themethanol leaf extract of P.subpeltatacan be used as the active constituent of antibacterial agents. The presence of antibacterial activity in P.subpeltataplant extracts give support to their traditional use for treating conditions associated with microorganisms in humans and consequently seems to fight against multi-resistant microbes.

Acknowledgement

Grateful thanks goes to the Department of Studies in Botany, University of Mysore for providing the facilities for research work. The authors also thank the Management and Principal of AVK College, Hassan, for their invaluable constant support during the research work.