Preliminary Phytochemical Screening and Antibacterial Activity of Leaves ofPassiflora foetida Linn.

Introduction

Plant products have been utilised for a variety of reasons, including medicine, throughout human history. Higher plants produce a number of secondary metabolites as a natural defence against illness and infection. Plant-based medicines have a significant impact on the healthcare of many cultures, both ancient and modern. Ayurveda, an Indian holistic medical system, treats several ailments including cancer, mostly with plant-based medicines or formulations. Between 1981 and 2002, roughly 877 small molecule medicines were released globally, with 61% having their roots in natural sources. Ayurveda includes information on the medicinal use of up to 290 natural medications. Healthcare systems in traditional societies rely heavily on medicinal plants. Even though allopathic medicine is available in many nations, around 70–80% of the rural population in developing countries rely on traditional medicine for basic healthcare today.¹ However, effective attempts to better understand the molecular mechanisms of action of several natural compounds have sparked interest in their therapeutic usage in modern medical settings over the last two decades. As a result, the importance of natural goods in human treatment cannot be overstated. According to recent polls, one in every three Americans uses medical natural products on a regular basis, and one in every two cancer patients does as well.²

Thus, ethnobotanical studies are certainly of interest and importance. Numerous phytoconstituents and essential oils isolated from plants have been demonstrated to exhibit biological effects both in vitro and in vivo, supporting more investigations into the characterization of antimicrobial activity of these plants.³

Microorganisms have played an increasingly important role in the manufacturing of antibiotics and other medications for some major infectious illnesses throughout the last century. Advances in the description of the human genome, the genomes of pathogenic microorganisms and parasites, have allowed the structures of numerous proteins related with disease processes to be determined. The requirement for novel molecular diversity for screening is rising based on introduction of newer molecular targets based on these proteins. Bacterial infection is considered as one of the most important worldwide health concerns of the twenty-first century. Antimicrobial resistance settings have been unable to address this critical component of antibiotic usage.⁴

In underdeveloped nations, medicinal plants are playing crucial part in meeting fundamental health requirements. Active principles of many medications can be discovered in plants as secondary metabolites. The amazing contribution of plants to the pharmaceutical business was made possible by a huge number of phytochemical and biological investigations conducted worldwide.

Herbal treatments employed in folk medicine offer an interesting and mostly undiscovered source for the creation and development of potentially new medications, which might aid in overcoming the rising problem of resistance and the toxicity of presently available commercial antibiotics. It is consequently of considerable importance to conduct screening of medicinal plants in order to validate their usage in folk medicine.⁵

In an effort to expand the spectrum of antibacterial agents from natural resources, Passiflora foetida belonging to passifloracea family has been selected. Passiflora foetida, sometimes known as passion flower in English, is a member of the Passion genus and the foetida and passifloracea families.⁶ In the Indian literature, this plant has been described for the decoction of leaves and fruits to treat asthma and biliousness; leaves and root decoction is an emmenagogue, used in hysteria. ⁷ This plant is widely used by tribal people to treat various ailments including ringworm and other fungal skin infections. Passiflora foetida plant leaves are known to be an important source of secondary metabolites. In Brazil, the herb is used in the form of lotions or poultices for erysipelas and skin diseases with inflammation. ⁸

The present study envisaged to select an appropriate local plant with anti-bacterial property based on its folklore claim and to scientifically establish its antibacterial activity.

Materials and Methods

Plant Material:

Passiflora foetida (Passifloraceae) was collected from Arignar Anna Government Hospital Campus, Arumbakkam, Chennai. It was identified and authenticated by Mr. Arunachalam, Research officer (Botany), Captain Srinivasa Murti Research Institute of Ayurveda and Siddha Drug Development (CSMDRIA), Arumbakkam, Chennai. The leaves were dried in shade for two weeks, powdered and were taken up for the studies.

Physiochemical Constants:

Physiochemical constants were studied using shade dried powdered material of leaves of Passiflora foetida Linn., in accordance with WHO guidelines.^9,10 All the procedures were carried out in triplicates.

Preparation of Extracts:

The first step was the preparation of subsequent solvent extracts. The dried, coarsely powdered Passiflora foetida leaf (700 gm) was first extracted with n-hexane to remove the colored pigments and then extracted with petroleum ether (60º-80ºC) in Soxhlet apparatus for 24h and then accordingly with polarity. The marc obtained was dried and extracted with chloroform for 24h followed by ethyl alcohol for 24h.

The resultant marc was charged in an aspirator bottle and extracted with water by cold maceration method for seven days. The extract was evaporated and dried on a water bath and preserved in a vacuum desiccator for further screening purpose. The yield of the aqueous extracts was recorded and was further preceded for detailed phytochemical study and pharmacological screening.

Phytochemical Evaluation:

Shade dried powdered material of leaves of Passiflora foetida Linn., and its extracts were used for preliminary phytochemical studies.⁹

Study of Colour Changes Under UV Light:

Passiflora foetida leaves powder and its various extracts were studied for any color changes under ordinary light, short UV (254 nm) and long UV (356 nm) light.

In-Vitro Antibacterial Studies:

Antibacterial activity of the extracts was studied against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermis, and Klebsiella pneumoniae. The bacterial strains were sub cultured from nutrient agar slants in an Erlenmeyer’s flask containing 100.0 mL of nutrient broth and incubated at 37°C for 24 hours for preparing stock cultures. The extracts were dissolved in dimethyl sulfoxide (DMSO) to make 1000 g/mL stock solution. For the subsequent in vitro investigations, various concentrations were used. Mueller Hinton agar (38 gm) was taken and diluted in 1000 mL of distilled water, adjusted to pH 7.30, autoclaved at 121°C for 15 minutes at 15 psi pressure, and used for sensitivity testing. For antibacterial tests, amikacin was used as the standard reference medication.¹¹

Few bacterial strain colonies taken for the activity were selected from the agar slopes which was inoculated in a test tube using 4 mL peptone water. These tubes were incubated for 2-4 hours to form suspensions. The suspensions were then diluted, if necessary, with saline, to a density visually equivalent to that of standard prepared by adding 0.5 mL of 1% barium chloride to 99.5 mL of 1% sulfuric acid. These suspensions were used for inoculation.

The antibacterial activity was studied by two methods:

1. Minimum Inhibitory Concentration

2. Antibiotic Disc Diffusion Technique (Zone of Inhi bition)

Minimum inhibitory concentration:

Dilution procedures are helpful for finding the minimum inhibitory concentrations (MICs) of antimicrobial drugs and act as the reference standard for antimicrobial studies, against which disc diffusion method is calibrated. In novel drug comparative studies, MIC techniques are commonly employed. They are used in clinical labs to determine the capability of organisms that provide ambiguous findings in disc tests, for testing on organisms when disc tests may be inaccurate, and for clinical management. Microorganisms are examined for their capacity to create observable growth on a succession of agar plates in dilution experiments (agar dilution). The MIC is the lowest concentration of an antimicrobial agent that can prevent observable microorganism growth.

The samples were aseptically introduced into sterilized petri dishes to achieve final concentrations ranging from 50 µg/mL, 75 µg/mL, 100 µg /mL, 200 µg /mL, 300 µg /mL, and 400 µg /mL, and the quantity of 15 mL was made using MH medium and the petri dishes were spun until the agar began to set. The plates were infected with a loopful of cultures at the designated areas with different test solutions of varying dilutions. For 24 hours, these plates were kept at 37°C. The existence or lack of organism growth was used to determine the outcomes.¹²

Antibiotic Disc Diffusion Technique:

Whatman filter paper No.1 discs of 6 mm diameter were prepared and sterilized in a hot air oven at 160°C for 1 hour. After that, the discs were impregnated with the samples and the solvent Dimethyl sulfoxide (DMSO).

Amikacin discs were used as a standard. Each amikacin disc contained 30 g of the medication. The microbial strains were then inoculated on a petri dish on MH agar medium by streaking the plate with a sterile swab. Care was taken to ensure that the culture was evenly distributed on the plate. After allowing the seeded plates to dry, the amikacin, test solutions, and DMSO discs were introduced in medium plates and kept at 4°C for 30 minutes to allow drug perfusion. The plate was incubated for 24 hours at 37°C. By using a micropipette, different concentrations of the plant extract solutions (50 µg/mL, 75 µg/mL, 100 µg /mL, 200 µg /mL, 300 µg /mL, and 400 µg /mL of plant extracts mixed with 5% of DMSO) and 5% DMSO were carefully added to the respective wells in the plate media and the test was performed in triplicates. After that, the zone of inhibition was determined.¹³

Results

Plant Material

Passiflora foetida (Passifloraceae) was collected from Arignar Anna Government Hospital Campus Arumbakkam, Chennai. The leaves were dried, powdered and were taken up for the physicochemical screening given in Table 1.

Preparation of Extracts

Petroleum ether, chloroform, ethanol and aqueous extracts were prepared successively from the leaves of Passiflora foetida and the percentage yield was determined (Table 2). The ethanolic extract showed more percentage yield compared to other extracts.

Phytochemical Evaluation

The powder and extracts were subjected to phytochemical screening. The powder from leaves with various chemical reagents revealed the chemical constituents of alkaloids, flavonoids and phenolic compounds. (Table 3)

Study of Colour Changes Under UV Light

The leaf powder and extracts showed fluorescence at 254 and 366 nm. This indicates presence of chromophore in the plant.

In-Vitro Antibacterial Studies

Determination of Minimum Inhibitory Concentration

The petroleum ether, chloroform, ethanol and aqueous extract of the leaves of Passiflora foetida Linn., were subjected to anti-bacterial screening against various microorganisms such as. Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus epidermis and Pseudomonas aeruginosa. Growth of all cultures was seen in the solvent control and in positive control.

The results are given in Figure 1 to 4. Though all the extracts showed anti-bacterial activity the chloroform and ethanol extract exhibited a more potent activity.

Zone of Inhibition

All the extracts exhibited strong to moderate effect against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, and Enterococcus faecalis.

Maximum zone of inhibition values were seen with chloroform and ethanol extracts against Enterococcus faecalis. The results for 400 µg/mL of different plant extracts are shown in Figures 5 to 8.

Discussion

The present study permitted the evaluation of some biological properties of Passiflora foetida.

The physiochemical constants like loss on drying (9.2±0.42% w/w), total ash (10.5±0.56% w/w), water soluble ash (4.2±0.37% w/w), acid insoluble ash (0.08±0.54% w/w), ethanol soluble extractive (2.3±0.82% w/w), water soluble extractive (4.1±0.53% w/w), petroleum ether soluble extractive (5.4±0.32% w/w), crude fibre content (30±0.43% w/w) were determined. Petroleum ether, chloroform, ethanol extract and water extract were prepared successively from the leaves of Passiflora foetida and the percentage yield was determined (3.62, 1.77, 4.56, 3.84% w/w respectively). Groups of phytochemical compounds commonly associated with combating microbial resistance and having antimicrobial activity in medicinal plants are flavonoids, alkaloids, tannins, triterpenoids, essential oils, saponins, glycosides, and phenols.¹⁴ Even though at this point in time it is difficult to judge the mechanism of actions of the bioactivity of the extract of Passiflora foetida leaves, it is possible to say that the plant has antibacterial activity based on the chemical detection methods in the phytochemical screening of various extracts. Preliminary phytochemical analysis of the leaf extracts of Passiflora foetida revealed the presence of glycosides, phenolics, and flavonoids, alkaloids. So, these and other phytochemical constituents of the plant extract may contribute to its antibacterial activity. Among the tested bacteria in this study, Enterococcus faecalis (30 mm) was susceptible compared to the other tested bacteria within the concentration of 400 µg/mL of plant extract of chloroform. As depicted in Table 7, the moderate susceptible bacterium at 400 µg/ mL was Enterococcus faecalis (7.67 mm) followed by Staphylococcus epidermis, Escherichia coli, S. aureus and Pseudomonas aeuroginosa with a mean of zone of inhibition diameters 30 mm, 28 mm, 19 mm, 15 mm and 11 mm, respectively. Based on the values of mean zone of inhibition, antibacterial activity was dependent on the concentrations of the plant extracts used. At the concentrations of 50 and 75 µg /mL of plant extract, no inhibition was observed with S. aureus, whereas minimal zones of inhibition were observed with other bacteria. The zones of inhibition observed at 400 µg/mL concentration of plant extract were significantly different when compared to other concentrations of plant extracts.

Conclusion

Passiflora foetida Linn. extract possesses broadspectrum antibacterial action against Gram-positive and Gram-negative bacteria. The broad-spectrum antibacterial activity of the plant extract was presumably due to the identified alkaloids, flavonoids, and phenolic compounds. As a result, its use as a health treatment in traditional medicine is confirmed. The bioactive ingredient of this plant can thus be used in the creation of antibacterial agents for the treatment of various bacterial infections such as gonorrhoea, pneumonia, eye infections, typhoid, early abortion, and staphylococcus infections, among others.

Conflicts of Interest

None